Cancer cell metastasis

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October 2015 • Volume 1 • Issue 1 • Pages 1–78

Advances in Modern Oncology Research

www.advmodoncolres.coom

Contents EDITORIAL 1

A forward from the editor Omar Abdel-Rahman

REVIEW 2

Cancer cell metastasis; perspectives from the focal adhesions

8

Emerging molecular-targeted therapies—the challenging case of endometrial cancer

18

Hepatocellular carcinoma (HCC); aetiological considerations

20

Indoles as anti-cancer agents

Lefteris C Zacharia, Vasiliki Gkretsi Ines Vasconcelos

Omar Abdel-Rahman

Mardia T. El Sayed, Nehal A. Hamdy, Dalia A. Osman, Khadiga M. Ahmed

CASE REPORT 36

Primary diffuse large B-cell lymphoma of the endometrium

Yuqing Zou, Hongmin Zhao, Jianhua Gao, Hong Zhu, Yong Li, Jianhua Zheng

ORIGINAL RESEARCH ARTICLE 41

Role of immunoexpression of cyclin D1, D3, retinoblastoma (Rb) mutant and clinical risk factors on complete moles as risk factors of per-sistent moles

Yudi M. Hidayat, Sofie R. Krisnadi, Supriadi Gandamihardja, Mieke H. Satari, Bethy S. Hernowo, Leri Septiani, Ahmad Faried

48

Antiproliferative effects of cembranoid derivatives obtained from Red Sea soft coral Sarcophyton glaucum on human colorectal cancer cell lines

Seif-Eldin N. Ayyad, Ahmed Abdel-Lateff, Khalid O. Alfooty, Hager Alorfi, Tamer M. Abdelghany, Ashraf N.E. Hamed

56

Osteopontin level correlates negatively with tumor shrinkage in neoadjuvantchemoradiation of locally advanced rectal cancer

Mirna Primasari, Soehartati A. Gondhowiardjo, DiahRini Handjari, Sahat Matondang, Adang Bachtiar Kantaatmadja

62

Follicular lymphoma in situ in intra-abdominal lymphadenectomies—a study of five cases: revisiting the entity Rinsey R Kurian, Thiagarajah Balamurugan, Bruno Ping, Louise Hendry, Stephen Whitaker, Izhar Bagwan

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Morphological modulation of human fibrosarcoma HT-1080 cells by hydroxybenzoate compounds during apoptosis Jassem G. Mahdi, Ahmed J. Mahdi, Eamon J. G. Mahdi, Asma Abdulsatar, Ali J. Mahdi, Abigail J. Manning, Chris J. Pepper

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Author Guidelines Advances in Modern Oncology Research

EDITORIAL

A foreword from the editor Omar Abdel-Rahman Clinical Oncology Department, Faculty of Medicine. Ain Shams University, Cairo, Egypt. Editor-in-Chief of Advances in Modern Oncology Research

Dear colleagues, On behalf of the editorial board, I am delighted to present to you the first issue of the journal “Advances in Modern Oncology Research (AMOR)”. AMOR is a multidisciplinary, open-access, peerreviewed journal that aims to present to you the latest developments in cancer research and to provide an information exchange platform for a broad audience of medical researchers with interest in cancer research as well as professionals working in the field. If you are an oncologist, pathologist, radiologist, surgeon, internist, basic science researcher, pharmacist or any other scholar with interest in cancerrelated research, you are more than welcome to contribute your work to AMOR. Our journal accepts original research articles, review articles, case reports, perspectives and opinion papers whether it is in a clinical or preclinical cancer-related discipline. Our journal website, www.advmodoncolres.com serves as a platform for all aspects in Advances in Modern Oncology Research (AMOR). From author guidelines, you can learn about our open access policies, it contains all the information the authors will need when deciding whether to submit to the journal. The website is linked to Open Journal Systems (OJS), which is a journal management and publishing system which will facilitate the peerreview process.

We have a database with a great number of peer reviewers, in which the number is still increasing continuously. Our Editorial Board consists of 276 members from 51 countries. The Editorial Board members have diverse expertise in different oncology subspecialties including breast cancer, lung cancer, gynecological malignancies, gastro-intestinal cancers, head and neck cancers, colorectal cancer, genito-urinary cancers, brain tumors, lymphoma, etc. Strict peer review process can ensure high quality paper to be published in this journal. We promise you a neutral, rigorous, professional and fast editorial and peer review process that will ensure the rapid publication of your high quality article. We are waiting for your contribution! Omar Abdel-Rahman, PhD

Clinical Oncology Department Faculty of Medicine Ain Shams University

About the Editor-in-Chief: Dr. Omar Abdel-Rahman has graduated from Faculty of Medicine, Ain Shams University, Cairo in 2005; he has received his oncology training at Ain Shams University Hospitals and he is working currently as a lecturer of clinical oncology at Ain Shams University. He received his Masters and Doctorate degrees from Ain Shams University in 2009 and 2014 respectively.

Copyright © 2015 Abdel-Rahman O. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

doi: 10 18282/amor.vi.i1.55

1

doi: 10.18282/amor.v1.i1.6

REVIEW ARTICLE

Cancer cell metastasis; perspectives from the focal adhesions Lefteris C Zacharia1, Vasiliki Gkretsi2

*

1

Department of Life and Health Sciences, University of Nicosia, Cyprus Department of Biomedical Research and Technology, Institute for Research and Technology-Thessaly (I.RE.TE.TH), Centre for Research and Technology-Hellas (CE.R.T.H), Greece

2

Abstract: In almost all cancers, most patients die from metastatic disease and not from the actual primary tumor. That is why addressing the problem of metastasis is of utmost importance for the successful treatment and improved survival of cancer patients. Metastasis is a complex process that ultimately leads to cancer cells spreading from the tumor to distant sites of the body. During this process, cancer cells tend to lose contact with the extracellular matrix (ECM) and neighboring cells within the primary tumor, and are thus able to invade surrounding tissues. Hence, ECM and the ECM-associated adhesion proteins play a critical role in the metastatic process. This review will focus on recent literature regarding interesting and novel molecules at the cell-ECM adhesion sites, namely migfilin, mitogen-inducible gene-2 (Mig-2) and Ras suppressor-1 (RSU-1), that are also critically involved in cancer cell metastasis, emphasizing on data from experiments performed in vitro in breast cancer and hepatocellular carcinoma cell lines as well as human breast cancer tissue samples. Keywords: apoptosis; breast cancer; cell-matrix adhesions; Fascin-1; hepatocellular carcinoma; invasion; metastasis; migfilin; PINCH-1; PUMA; Ras suppressor-1; VASP Citation: Zacharia LC, Gkretsi V. Cancer cell metastasis; perspectives from the focal adhesions. Adv Mod Oncol Res 2015; 1(1): 2–7; http://dx.doi.org/10.18282/amor.v1.i1.6. *Correspondence to: Vasiliki Gkretsi, Centre for Research and Technology-Hellas (CE.R.T.H.), Greece, [email protected].

Received: 20th July 2015; Accepted: 17th August 2015; Published Online: 6th October 2015

I

n most cancer types, including breast cancer (BC) and hepatocellular carcinoma (HCC), a large percentage of patients die from metastatic disease[1,2] and not from complications related to the original primary tumor. That is why addressing the problem of metastasis is of utmost importance for the successful treatment and improved survival of cancer patients. Metastasis is a complex process that ultimately leads to cancer cell spreading through tissues in the whole body. During the metastatic process, as cancer cells accumulate mutations or other molecular signals, they become more malignant and tend to easily lose contact with the extracellular matrix (ECM) and neighboring cells within the primary tumor. Hence, they start to invade surrounding

tissues. Both cell-cell adhesion and cell-ECM adhesions get deregulated promoting cancer cell aggressiveness[3]. In fact, communication between cells and the ECM, and between neighboring cells is severely disrupted in more aggressive and invasive cells[4-6]. Hence, ECM, integrins and the ECM-associated adhesion proteins play a critical role in this process[4]. This review will focus on interesting and novel molecules at the cell-ECM adhesion sites that are also critically involved in cancer cell metastasis, based on data from experiments performed in vitro in BC and HCC cancer cell lines as well as human BC tissue samples. A diagrammatic representation of the interactions analyzed in this review is shown in Figure 1.

Copyright © 2015 Zacharia LC, et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Zacharia LC, et al.

Figure 1 Diagrammatic representation of the protein-protein interactions studied in the present review and their involvement in different aspects of cancer cell metastasis

The role of migfilin in cancer cell metastasis Several novel molecules at cell-ECM adhesion sites have been implicated in cancer cell progression and have been recently shown to be deregulated in cancer cell metastasis. Migfilin, (also known as filamin-binding LIM protein 1 or FBLP-1), is a novel LIM domain—containing protein present both at cell-ECM[7] and cell-cell adhesions[8]. It interacts with the cell-ECM adhesion protein mitogen-inducible gene 2 (Mig-2) [7] and it also binds to vasodilator-stimulated phosphoprotein (VASP)[9] which is known to regulate actin polymerization in lamellipodia[10-12], the cellular protrusions responsible for migration and invasion of the cell. Migfilin has been associated with various types of cancer although its role has not been extensively studied. Cytoplasmic migfilin was strongly associated with higher tumor grade in leiomyosarcomas[13] and inversely correlated with clinical metastasis in esophageal cancer cells[14]. Moreover, high migfilin expression was significantly correlated with tumor grade in glioma and poor prognosis[15]. Finally, work from our group has shown that migfilin was also significantly reduced in human BC samples compared to normal adjacent tissue[16], indicating an involvement of the protein in cancer progression. Along the same line, migfilin was also examined in advanced-stage serous ovarian carcinoma[17] and was found to have significantly lower expression in primary carcinomas and solid metastases compared to effusions[17]. However, although as mentioned above, recent studies have shown that migfilin’s expression is negatively correlated with clinical metastasis in esophageal cancer[14], we found no statistically correlation between migfilin’s expression and metastatic

doi: 10 18282/amor v1 i1 6

status or disease stage in human BC samples[16]. As metastasis is a fundamental biological behavior of HCC and the main cause of treatment failure[2], we also tested the in vitro role of migfilin in two liver cell lines that differ in terms of their metastatic potential; the non-invasive hepatoma cell line PLC/ PRF/5 (Alexander cells herein) and the highly invasive HCC HepG2 cell line[18]. Using an siRNA-mediated silencing approach to knock-down the migfilin gene from both cell lines, we studied the effect of gene silencing on basic signaling pathways and functional cellular properties related to the metastatic potential of the cells. We found that migfilin was elevated in the more invasive HepG2 cells compared to the less invasive Alexander cells both at the mRNA and protein level, indicating a possible contribution of the protein to the aggressive phenotype of HepG2 cells[18]. Moreover, gene silencing of migfilin led to upregulation of proteins involved in actin reorganization such as the two main forms of phosphorylated VASP (Ser157 and Ser239) in HepG2 cells, and Fascin-1 and Rho kinase (ROCK-1) in both cell lines. Increased expression of these molecules led to increased actin polymerization and stabilization, which thus resulted in less available monomeric actin for cell migration or invasion ultimately contributing to reduced cell migration[19,20]. Indeed, migfilin depletion led to reduced cell invasion, indicating that migfilin promotes the metastatic phenotype of HepG2 cells[18]. In fact, this is in accordance with previous studies showing migfilin to be crucial for cell migration in a variety of cell types (HeLa, HT-1080, and MDA-MB231 cells), where it was shown that its depletion impairs cell migration[9]. As migfilin has been previously shown to link the cell-matrix adhesions to the actin cytoskeleton[7], the migratory defect induced by the loss of migfilin is probably caused, at least in part, by the im-

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Cancer cell metastasis; perspectives from the focal adhesions

paired connection between cell-ECM adhesions and the actin cytoskeleton. A diagrammatic representation of the interactions mentioned above is shown in Figure 1. Notably, the fact that elimination of migfilin severely impaired HepG2 cell invasion, while at the same time increasing cell proliferation, both through elevation of ERK1/2 and phosphor-β-catenin[18], indicates that migfilin has a dual function in HCC cells activating different signaling pathways perhaps through interaction with different binding partners. This could provide an explanation as to why its role in cancer is not strictly defined but greatly depends upon the type of cancer[14,16,17,21]. Nevertheless, evidence suggests that migfilin is worth being assessed as a therapeutic target for cancer cell metastasis for a number of cancer types.

Mitogen-inducible gene-2 (Mig-2) in cancer cell metastasis Mitogen inducible gene-2 (Mig-2) is a cell-ECM adhesion protein localized at cell-ECM adhesion sites[22], and through its binding partner, migfilin[23], interacts with filamin A, thus connecting cell-ECM adhesions to the actin cytoskeleton (Figure 1)[7]. Recent studies have implicated Mig-2 along with migfilin in a variety of human cancers. Mig-2 expression was found increased in leiomyomas compared with normal myometrium while it was decreased in leiomyosarcomas[24]. It was also upregulated in gastric cancer and its expression had a significant positive correlation with metastasis and poor survival[25]. Mig-2 was also shown to be highly expressed in 90% of malignant mesothelioma tumors[26] as well as in almost 100% of human bladder cancers[27] and in the majority of chondrosarcomas[28]. In fact, it has been postulated that Mig-2 could function as a promising marker of tumor progression[25,28]. Finally, Mig-2 deregulation[29] could contribute to BC malignancy and progression. Previous work from our research group[16] has actually shown that Mig-2 expression was significantly reduced in human BC tissues compared to normal adjacent tissue indicating that BC has lost Mig-2 expression and thereby has less invasion inhibitory mechanisms in action, as Mig-2 has been implicated in inhibition of cell invasion[30].

Ras suppressor-1 (RSU-1) in metastasis Mig-2 has been also shown in C. elegans to interact with integrin-linked kinase (ILK)[31] thus being connected to particularly interesting new cysteine-histidine rich pro-

4

tein-1 (PINCH-1) and parvin, forming a stable ternary protein complex at cell-ECM adhesion sites[32]. Although many studies have been performed on the role of ILK in cancer, even proposing that it is a potential anti-cancer therapeutic target[33,34], little is known with regard to the ILK-interacting partners. PINCH-1 in particular, functions as an adaptor protein at the cell-ECM adhesion sites playing an important role in promoting cell survival and apoptosis resistance[35], while also modulating cell shape and spreading[36]. Interestingly, PINCH-1 interacts with Ras suppressor-1 (RSU-1) at the cell-ECM adhesion sites[37,38], increasing the sites’ complexity. RSU-1 was originally identified as a suppressor of Ras-dependent oncogenic transformation[39]. Although, its connection to cancer is obvious due to its linkage with Ras oncogene, little is known regarding its involvement in the disease. It has been shown that RSU-1 is involved in the inhibition of anchorage independent growth of BC cells[40,41]. Moreover, in a recent study by our group it was shown that RSU-1 is upregulated in HepG2 highly invasive HCC cells compared to the more benign, non-invasive Alexander hepatoma cells, which indicates a possible involvement of RSU-1 in HCC pathogenesis[42]. Moreover, siRNA-mediated gene silencing of RSU-1 expression in both HCC cell lines resulted in increased proliferation, reduced cell adhesion and cell invasion in the aggressive HepG2 cells[42], suggesting that RSU-1 enhances adhesion and invasion in the aggressive HepG2 cells. Interestingly, in another recent study by our group, RSU-1 was investigated in BC cell lines that differ in terms of their metastatic potential (namely, non-invasive MCF-7 cells and highly invasive MDA-MB-231 cells) as well as in a set of thirty two[32] human BC samples from patients with or without lymph node metastasis[43]. In this study, the findings from the experiments performed in the cell lines were further supported by the findings in the human samples validating the hypothesis that RSU-1 plays a vital role in BC metastasis. More specifically, RSU-1 was found to be upregulated in the more aggressive MDA-MB-231 cells when compared to the less transformed MCF-7 cells, connecting RSU-1 with a more aggressive BC phenotype. Furthermore, it was shown that depletion of RSU-1 leads to upregulation of its binding partner PINCH-1, indicating that RSU-1 likely acts as a negative regulator of pro-survival ECM-adhesion protein PINCH-1. This was also in accordance with the fact that silencing of RSU-1 enhanced cell proliferation, which is not surprising, as RSU-1 was originally characterized as a suppressor of Ras-dependent oncogenic transformation[39]. Interestingly, RSU-1 depletion significantly reduced the population of apop-

doi: 10.18282/amor.v1.i1.6

Zacharia LC, et al.

totic cells indicating that RSU-1 actually promotes apoptosis. Consistent with this change, RSU-1 depletion, from both cell lines, also led to downregulation of the mRNA expression of the pro-apoptotic gene p53 upregulated modulator of apoptosis (PUMA). More importantly, the findings in the two BC cell lines were further validated in 32 human BC samples from patients with invasive BC with or without lymph node metastasis and each sample was accompanied by and compared to its corresponding normal adjacent tissue, eliminating any external factors and variations that might interfere with the results. Interestingly, the data showed that RSU-1 mRNA expression was significantly increased in BC tissues when compared to normal adjacent tissue. Moreover, RSU-1 expression was significantly reduced in non-metastatic samples and significantly elevated in metastatic samples, clearly stating a different role of RSU-1 depending on the aggressiveness of the tumor, that could potentially lead to the use of RSU-1 as a novel biomarker of metastasis[43]. In accordance with the cell line data that suggested RSU-1 to be a negative regulator of PINCH-1, PINCH-1 expression showed dramatic reduction in BC samples both at the mRNA and protein level with statistically significant reduction in metastatic samples, in which RSU-1 was found to be elevated. Moreover, the assessment of the pro-apoptotic PUMA expression also confirmed findings in the cell lines showing an increase of PUMA expression in BC and metastatic samples in particular and a positive statistically significant correlation with RSU-1 expression in the same samples[43]. Therefore, RSU-1 should definitely be evaluated further as a potential metastasis biomarker in BC tissue samples. Moreover, new therapeutic approaches that reinforce apoptosis through PUMA should also be assessed in comparison to current available therapies in patients with BC metastasis.

Cell adhesion and endothelial cell growth in metastasis Moreover, deregulation of focal adhesion proteins combined with an imbalance between pro- and anti-angiogenic signaling in cells[44] also affects tumor vasculature, which is another critical factor in tumor growth and metastasis. More specifically, the endothelial cell lining in tumor vessels consists of less junction proteins when compared to the endothelial cell lining in normal vessels leaving large numbers of fenestrae and intercellular openings, being accompanied, at the same time by a disorganized ECM and basement membrane[45].

doi: 10.18282/amor.v1.i1.6

As a result, tumor vessels become “leaky” facilitating the spreading of circulating tumor cells throughout the whole body. To this regard, pro-angiogenic factors (e.g. vascular endothelial growth factor, VEGF, platelet derived growth factor, PDGF) and their receptors are upregulated in most tumors while focal adhesion proteins are mainly downregulated resulting in loose cell-ECM connections and the formation of immature vessels with structural abnormalities. Interestingly, there has been no connection between either of the molecules under review (namely, Mig-2, migfilin, or RSU-1) and tumor angiogenesis, leaving the field open for future investigations.

Conclusion In the current review, we have summarized the recent data on novel and interesting proteins found at the cell-ECM adhesion sites of cells, namely migfilin, Mig-2 and RSU-1, and their involvement in cancer cell metastasis emphasizing on results from BC and HCC studies. We conclude that these cell-ECM adhesion proteins tend to function as adaptor proteins forming multiple protein-protein interactions at the cell-ECM adhesion sites, thus conferring different effects on different cancer cell types. However, in most cases, and as shown in Figure 1, they promote cell adhesion, cell invasion and apoptosis, all of which are important aspects of cancer cell metastasis. Therefore, more research is needed involving more human samples in order to evaluate their use as potential biomarkers of metastasis and perhaps, even potential targets for anticancer therapy.

Conflict of interest The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

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REVIEW ARTICLE

Emerging molecular-targeted therapies—the challenging case of endometrial cancer Ines Vasconcelos Berlin Oncology Center, Belin, Germany

Abstract: Endometrial cancer newly affects an estimated 54,870 women in the United States, being responsible for an estimated 10,170 deaths in 2015. It has demonstrated to harbor a complex carcinogenesis process, with limited treatment options for advanced or persistent disease. Identification and targeting of genetic alterations that lead to progressive disease and therapy resistance is not only challenging, but also often does not correlate with a clinical benefit. Targeted maintenance therapies in endometrial cancer have been largely disappointing. Nonetheless, targeted personalized treatment should be the main goal of treatment of advanced disease in the future. Due to the high variety of drugs being tested in early clinical trials, it is hard to keep pace with the latest developments and ongoing trials. This review aims to summarize the latest published and ongoing trials on targeted therapies in endometrial cancer. Keywords: endometrial cancer; targeted-therapy; PIK3/AKT/mTOR; ErbB; VEFG Citation: Vasconcelos I. Emerging molecular-targeted therapies—the challenging case of endometrial cancer. Adv Mod Oncol Res 2015; 1(1): 8–17; http://dx.doi.org/10.18282/amor.v1.i1.9. Correspondence to: Ines Vasconcelos, Berlin Oncology Center, [email protected].

Received: 21st July 2015; Accepted: 17th August 2015; Published Online: 6th October 2015

E

ndometrial cancer newly affects an estimated 54,870 women in the United States, being responsible for an estimated 10,170 deaths in 2015[1]. It has been classically classified as estrogen-dependent endometrioid carcinoma (type I, 80%) and estrogen-independent non-endometrioid carcinoma (type II, 20%), including serous, clear cell carcinoma, carcinosarcoma, mucinous adenocarcinoma, squamous cell carcinoma and mixed adenocarcinoma. Despite this classification, and the fact that each type correlates with characteristic genetic alterations, the PI3K/AKT/mTOR-PTEN pathway appears highly dysregulated in both type I and type II endometrial cancer types, but particularly in type I cancers (>90%). On the other hand, the most commonly described genomic changes in the rarer and more aggressive type II tumors are the amplification of the HER2 gene (17%–30%)[2] and mutation of the TP53 gene (~90%)[3]. Furthermore, the recent integrated genomic characterization of endometrial cancer was able to identify four categories:

POLE (polymerase epsilon gene) ultramutated, microsatellite instability (MSI) hypermutated, low copy number (CN) and high CN[4]. Endometrial cancer survival has not changed in the last decade. We have thoroughly explored the sequencing and dosing of standard cytotoxic drugs and have probably maximized, or nearly maximized, their benefits. The standard treatment of endometrial cancer remained similar in the last 20 years, with endocrine therapy and chemotherapy having shown only limited efficacy. Therefore we should be looking for potential targets for consolidation therapies, which will help patients remaining in remission. Novel antitumor agents are currently being evaluated in early clinical trials for endometrial cancer. This review summarizes the most recent early clinical trials evaluating the use of targeted agents in endometrial cancer. The main focus is trials with available results, although the most important ongoing early clinical trials will be as mentioned. Table 1 summarizes completed trials and their respective overall response

Copyright © 2015 Vasconcelos I. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

8

Vasconcelos I.

rates (ORR), while Table 2 summarizes currently ongoing trials. Table 1 Summarizes completed trials and their respective overall response rates (ORR) Drug

N

% ORR (CR + PR)

Table 2 Summarizes currently ongoing trials Drug

Trial number

PIK3/AKT/ mTOR inhibitors MK-2206

NCT01307631

PIK3/AKT/mTOR inhibitors XL-147 PF-05212384 PF-04691502 GDC-0980 AZD6244

64

6.0

38

39.5

Discontinued Results not published 54

NCT02476955 ARQ 092 NCT01473095 GSK2141895

NCT01935973

6.0

Everolimus Letrozol

38

32

alone

44

21

alone

35

21

Megestrol

71

14

alone

50

22

Bevacizumab

53

24.5

Chemotherapy

33

14

Chemotherapy-naive

27

4

Ridaforolimus

64

4

34

8.8

34

29

Erlotinib

34

12.5

Lapatinib

30

3.3

Trastuzumab

34

0

Cetuximab

23

5.0

FGFR2(mut)

22

5

FGFR2(WT)

31

16

49

7

19

0

Chemotherapy

15

73

Temsirolimus

53

24.5

56

13.5

Brivanib

46

13.8

Nintedanib

37

9.4

Sunitinib

34

18.1

NCT02397083

NCT01068249

Temsirolimus

Everolimus

NCT02188550

NCT01797523

NCT02228681

HER2 directed therapies Tyrosine kinase inhibitor NCT00977574

Monoclonal antibodies NCT01460979

VEGF directed therapies TKI258 (dovitinib)

Aflibercept Bevacizumab

alone

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Population and type of treatment combination

NCT01010126 Temsirolimus NCT00723255

NCT02093598

NCT01155258

NCT01065662

Recurrent or advanced endometrial cancer ARQ 092 in combination with carboplatin plus paclitaxel in selected solid tumors ARQ 092 in adult subjects with advanced solid tumors Trametinib with or without GSK2141795 in treating patients with recurrent or persistent endometrial cancer Everolimus and levonorgestrel IUD in the treatment of endometrial hyperplasia and/or early-stage endometrial cancer Letrozole and everolimus in patients with advanced or recurrent endometrial cancer Everolimus and letrozole in platinum resistant relapse or refractory or resistant endometrial cancer Everolimus, letrozole, and metformin in patients with advanced or recurrent endometrial cancer Everolimus and letrozole or tamoxifen/medroxyprogesterone acetate in advanced, recurrent, or persistent endometrial carcinoma. Paclitaxel, carboplatin, and bevacizumab or paclitaxel, carboplatin, and temsirolimus or ixabepilone, carboplatin, and bevacizumab to see how well they work in treating patients with stage III, stage IV, or recurrent endometrial cancer Activity, tolerability, safety of temsirolimus in women with advanced endometrial carcinoma Temsirolimus and bevacizumab in patients with advanced endometrial Bevacizumab and temsirolimus in treating patients with recurrent or persistent endometrial cancer POEM STUDY: a Phase IIa trial in endometrial carcinoma with temsirolimus Temsirolimus and vinorelbine ditartrate in patients with unresectable or metastatic solid tumors AZD2171 and temsirolimus in patients with advanced gynecological malignancies (Table 2 continued on next page)

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Emerging molecular-targeted therapies—the challenging case of endometrial cancer

(Table 2 Continued) Drug

Trial number

Population and type of treatment combination

Ridaforolimus

NCT01256268

Carboplatin/taxol/ridaforolimus in endometrial, ovarian and solid tumors

NCT01454479

Recurrent or persistent endometrial carcinoma (per invitation)

NCT01367002

Carboplatin/paclitaxel with and without trastuzumab in endometrial cancer

HER2 directed therapies Lapatinib Trastuzumab VEGF directed therapies NCT00977574

(see above under temsirolimus)

NCT01770171

Carboplatin-paclitaxel ± bevacizumab in advanced (stage III-IV) or recurrent endometrial cancer (MITOBEVAEND2)

NCT01010126

(see above under temsirolimus)

NCT02142803

TORC1/2 inhibitor MLN0128 and bevacizumab in treating patients with advanced solid tumors

Brivanib

NCT00888173

Brivanib in patients with recurrent or persistent endometrial cancer

Nintedanib

NCT01225887

Patients with recurrent or persistent endometrial cancer

Bevacizumab

inhibitors of the pan-class PI3K family of lipid kinases with potential antineoplastic activity. They inhibit class I PIK3 in either an ATP-competitive or ATP-independent manner, thereby inhibiting the production of the secondary messenger PIP3 and activation of the PI3K signaling pathway.

BKM-120 (Buparlisib) BKM-120 has shown a lack of activity in PI3K activated endometrial cancer. NCT01289041 evaluated the use of BKM-120 in endometrial cancer and included 70 patients, of which 49 showed PI3K activated pathway, one showed a complete response, and none showed a partial response. A median progression free survival (PFS) of 1.9 months was shown and an overall survival (OS) of 8.9 months was achieved for patients with PI3K-activated pathway undergoing treatment with BKM-120 (results available at clinicaltrials.gov, non-published). Pre-clinical models show that independent of PIK3CA gene mutation, BKM-120 mediated inhibition of the PI3K/AKT/mTOR pathway in endometrial tumors preclude tumor growth in a primary xenograft model. While a pattern of resistance did emerge, the effect appeared to be mitigated by the addition of conventional cytotoxic chemotherapy[6].

XL-147 (Pilaralisib)

Sunitinib

NCT00478426

Patients with recurrent or metastatic endometrial cancer

XL-147 showed minimal antitumor activity in advanced or recurrent endometrial carcinoma in a Phase II trial. XL-147 was administered to patients with endometrial carcinoma who progressed after first-line chemotherapy. Of the 67 enrolled patients, complete or partial tumor responses occurred in 4 patients (overall response rate (ORR) of 6.0%)[7].

Cabozantinib

NCT01935934

Patients with recurrent or metastatic endometrial cancer

AKT directed therapies

NCT02501096 E7090 NCT01111461

Lenvatinib (E7080) plus pembrolizumab in subjects with selected solid tumors Patients with advanced endometrial cancer and disease progression

The PI3K/AKT/mTOR pathway

AKT directed drugs are orally bioavailable inhibitors of AKT (protein kinase B) with potential antineoplastic activity. They bind to and inhibit the activity of AKT in either an ATP or a non-ATP competitive manner, resulting in the inhibition of the PI3K/AKT signaling pathway.

Mutations in the PI3K/AKT/mTOR pathway have been shown in the vast majority of type I endometrial carcinomas and in a likewise high-proportion of type II tumors. In fact, of all the cancer types studied by the Cancer Genome Atlas, endometrial cancer is the type that shows the highest incidence of mutations in this pathway. It is currently believed that PTEN loss is responsible for the pathogenesis of type I tumors, and mTOR is mainly involved in type II tumors[5].

NCT01307631 is the sole ongoing trial on MK-2206 in endometrial cancer and has finished accrual. It aims to assess the activity of MK-2206 in patients with recurrent or persistent endometrial cancer classified by PIK3CA mutation. Results are expected in January 2016.

PI3K directed therapies

AZD6244 (Selumetinib)

The below-described drugs are oral bioavailable specific

AZD6244 constitutes another drug that did not meet

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MK-2206

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Vasconcelos I.

pre-trial specifications for clinical efficacy. The sole Phase II study enrolled 54 patients and reported an ORR of 6%[8].

The results were reported online at ClinicalTrials.gov (www.clinicaltrials.gov), and the official report is eagerly awaited.

ARQ 092

GDC-0980

ARQ 092 in combination with carboplatin plus paclitaxel was tested in a Phase Ib open-label clinical trial in selected solid tumors, including endometrial cancer (NCT02476955). The trial started recruiting in June 2015. A second open-label, Phase I, dose escalation study of oral ARQ 092 administered to subjects with advanced solid tumors and recurrent malignant lymphoma is currently recruiting (NCT01473095).

GDC-0980 was evaluated in uterine serous cancer cell lines. GDC-0980 caused a strong differential growth inhibition in FISH+ cell lines when compared with FISH−. FISH+ cell lines harboring PIK3CA mutations were significantly more sensitive to GDC-0980 exposure when compared with cell lines harboring wild-type PIK3CA. Oncogenic PIK3CA mutations and c-erbB2 gene amplification may represent biomarkers to identify patients who may benefit most from the use of GDC-0980[9]. A study on the use of GDC-0980 was completed in 2013 (NCT01455493) but the results were not published.

GSK2141795 Trametinib with or without GSK2141795 is currently being tested in patients with recurrent or persistent endometrial cancer (NCT01935973). The Phase II clinical trial is currently recruiting.

mTOR directed therapies Newer drugs, such as PF-04691502, PF-05212384 and GDC-0989, target the phosphatidylinositol 3 kinase (PI3K) and mammalian target of rapamycin (mTOR) in the PI3K/mTOR signaling pathway, with potential antineoplastic activity. They inhibit both PI3K and mTOR kinases dually, which may result in a more potent apoptosis induction and growth inhibition of cancer cells overexpressing PI3K/mTOR. The other types of drugs targeting mTOR are rapalogs (rapamycin derivatives, including everolimus, temsirolimus and ridaforolimus). These are small molecule inhibitors, which bind to the cytosolic protein FK-binding protein 12 resulting in mTOR inhibition, by directly binding to mTOR complex 1 (mTORC1).

PF-04691502 The only Phase II trial investigating the role of PF-04691502 in endometrial cancer was discontinued (NCT01420081, information available online at www. clinicaltrials.gov, not published).

PF-05212384 This is the mTOR inhibitor which showed the most promising results, till date, in a Phase II trial, having enrolled 38 patients and reporting 39.5% of patients showing clinical benefit, 52.6% in the PI3K basal sub-group.

doi: 10.18282/amor.v1.i1.9

Everolimus (RAD-001) Everolimus has been thoroughly studied in patients with endometrial cancer, with several Phase II trials available and another few recruiting. The most recent clinical Phase II trial, analyzing the combination of everolimus with letrozol, published in March 2015, enrolled 38 patients, with a clinical benefit rate of 40% (14 of 35 patients) and a confirmed objective response rate of 32% (11 of 35 patients). In this trial, serous histology was the best predictor of lack of response. Patients with endometrioid histology and CTNNB1 showed the best response rates[10]. Analysis from clinical trial specimens determined that S6rp phosphorylation, loss of PTEN expression, and presence of KRAS mutations alone did not correlate with response to everolimus. However, positive pS6rp staining combined with KRAS mutation performed with 100% positive predictive value and specificity to predict nonresponse[11]. Another Phase II trial, enrolling 44 patients, showed at 20 weeks, a confirmed clinical benefit rate, defined as CR, PR or SD of ≥ 8 weeks in duration, of 21%[12]. The third Phase II trial enrolled 35 patients. Of the 28 patients available for response analysis, 6 (21%) showed a clinical benefit at 20 weeks of therapy[13]. There are also two smaller Phase I trials published[14,15]. A Phase II trial of everolimus in combination with letrozol has finished recruiting and results are expected in April 2016 (NCT01068249). A single arm trial of everolimus and letrozole in platinum resistant relapse or refractory or resistant endometrial cancer (NCT02188550) is currently recruiting. A trial on everolimus and levonorgestrel IUD in the treatment of

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Emerging molecular-targeted therapies—the challenging case of endometrial cancer

endometrial hyperplasia and/or early-stage endometrial cancer is ongoing but has not started accrual yet (NCT02397083). In another Phase II, single-arm, trial everolimus, letrozole, and metformin are being evaluated in patients with advanced or recurrent endometrial cancer. The trial is currently recruiting (NCT01797523). The Gynecologic Oncology Group (GOG) is also recruiting for a randomized Phase II trial of everolimus and letrozole or tamoxifen/medroxyprogesterone acetate in advanced, recurrent, or persistent endometrial carcinoma (NCT02228681).

Temsirolimus (CCI-779) Temsirolimus is the most extensively studied rapalog in the context of endometrial cancer (Table 1). The most recent Phase II trial examined the use of temsirolimus with or without megestrol acetate and tamoxifen for endometrial cancer: the combination arm was closed early because of an excess of venous thrombosis. Adding the combination of megestrol acetate and tamoxifen to temsirolimus therapy did not enhance activity and the combination was associated with an unacceptable rate of venous thrombosis[16]. Another Phase II trial evaluated the combination of bevacizumab and temsirolimus in the treatment of recurrent or persistent endometrial cancer but was associated with significant toxicity, with 3 treatment-associated patient deaths[17]. The first Phase II trial on temsirolimus evaluated the single-agent activity of temsirolimus in women with recurrent or metastatic chemotherapy-naive or chemotherapy-treated endometrial cancer. Temsirolimus showed encouraging singleagent activity in endometrial cancer, which was higher in chemotherapy-naive patients than in chemotherapytreated patients and independent of PTEN status[18]. There were further two Phase I trials evaluating temsirolimus with topotecan and with carboplatin-paclitaxel[19, 20]. A Phase II trial evaluating paclitaxel, carboplatin, and bevacizumab or paclitaxel, carboplatin, and temsirolimus or ixabepilone, carboplatin, and bevacizumab in patients with stage III, stage IV, or recurrent endometrial cancer has finished recruiting and results are eagerly expected (NCT00977574). Equally awaited are the results of the POEM study (NCT00659568). A number of trials evaluating temsirolimus in combination with other drugs are currently ongoing (Table 2).

Ridaforolimus (AP23573) Ridaforolimus is likewise a thoroughly studied rapalog. The latest Phase II trial enrolled 130 patients, of which

12

64 received ridaforolimus. Treatment discontinuation as a result of adverse events was 33%. Median PFS at the protocol prespecified interim analysis was 3.6 months for ridaforolimus and 1.9 months for the comparator. Oral ridaforolimus showed encouraging activity in advanced endometrial cancer but was associated with significant toxicity[21]. A second Phase II trial enrolled 34 patients end registered an ORR of 8.8%. No correlation was found between response and mutation status[22]. Another single-arm Phase II trial enrolled 45 patients. A clinical benefit was achieved in 29% of the patients and was generally well tolerated[23]. Apart from a trial evaluating carboplatin/taxol/ridaforolimus in endometrial, ovarian and solid tumors, which has finished accrual, there are no other ongoing trials. Other agents that target the PI3K/AKT/mTOR pathway are currently being developed, including the small molecule bisindolylmaleimide MKC-1 and suberoylanilide hydroxamic acid (SAHA). Their efficacy may later eventually be evaluated in endometrial cancer.

The epidermal growth factor receptor (EGFR or ErbB) family The EGFR or ErbB family consists of four distinct tyrosine kinases cell surface receptors (ErbB or HER 1-4) that are expressed in the normal endometrium and overexpressed in endometrial cancers, particularly type II cancers. When activated, ErbB dimerizes and induces signal transduction through the PI3K signaling pathway promoting carcinogenesis. As previously mentioned, it is one of the most frequent amplified genes in type II endometrial cancer. Two classes of targeted therapies exist: tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (MoAbs).

EGFR-specific tyrosine kinase inhibitors (TKIs) TKIs are small molecules to inhibit the cytoplasmic side of the EGFR tyrosine kinase domain, leading to EGFR inactivation. This leads to a halt in signaling the cascade of cells that rely on this pathway for growth, resulting in potential anti-tumor activity.

Gefitinib There were 2 Phase II trials evaluating the efficacy of gefitinib in recurrent metastatic endometrial cancer, however, they showed that it lacked sufficient efficacy to warrant further evaluation[24,25]. One of the trials enrolled doi: 10 18282/amor v1 i1 9

Vasconcelos I.

29 patients, of which one had a complete response, however, it was not associated with an ErbB mutation[24]. The other enrolled 24 patients and no clinical responses were observed[25]. There are currently no ongoing trials.

The vascular endothelial growth factor receptor (VEGFR) family

The results of the existing Phase II trial were not promising[26] with 4 partial responses out of 34 enrolled patients. There are currently no ongoing trials.

Vascular endothelial growth factor (VEGF) is an important signaling protein involved in angiogenesis. All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors on the cell surface, the vascular endothelial growth factor receptor (VEGFR).

Lapatinib

TKI258 (Dovitinib)

Lapatinib is a dual EGFR and ErbB2 inhibitor. A Phase I study of lapatinib plus ixabepilone as second-line treatment for patients with HER2 overexpressed recurrent or persistent endometrial carcinoma (NCT01454479) is enrolling patients by invitation. A Phase II trial exploring the use of lapatinib in women with persistent or recurrent previously treated endometrial cancer showed modest activity of this compound. However, a newly identified mutation in exon 18, E690K, occurred in a patient with a partial response and progression-free survival extending for the past 6 months, so further exploration of this mutation may establish the role, if any, of lapatinib in endometrial cancer[27].

TKI258 is an orally bioavailable lactate salt of a benzimidazole-quinolinone compound that strongly binds to several members of the receptor tyrosine kinase superfamily (RTK). It binds to the fibroblast growth factor receptor 1, 2 and 3 (FGFR-1, FGFR-2 and FGFR3), VEGF, platelet-derived growth factor receptor type 3 (PDGFR-3), FMS-like tyrosine kinase 3, stem cell factor receptor (c-Kit) and colony-stimulating factor receptor 1 (CSFR-1). It has been evaluated in the treatment of patients with FGFR2 mutated (mut) or wild-type (WT) advanced and/or metastatic endometrial cancer. Second-line dovitinib in FGFR2 (mut) and FGFR2 (WT) advanced or metastatic endometrial cancer had single-agent activity, but it did not reach the pre-specified study criteria. Of 248 patients with FGFR2 prescreening results, 27 (11%) had FGFR2 (mut) endometrial cancer, of which 22 patients FGFR2 (mut) and 31 patients FGFR2 (WT) were recruited. Seven (31.8%) FGFR2 (mut) patients and nine (29.0%) FGFR2 (WT) patients were progression-free at 18 weeks. On the basis of predefined criteria, neither group continued to stage two of the trial[29].

Erlotinib

Monoclonal antibodies Trastuzumab Trastuzumab binds to the extracellular segment of the HER2 receptor. Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle. It induces downregulation of HER2 leading to disruption of receptor dimerization and signaling through the downstream PI3K cascade. Trastuzumab as a single agent did not demonstrate activity against endometrial carcinomas with HER2 overexpression or HER2 amplification[28]. There is one ongoing trial (NCT01367002) evaluating the role of carboplatin/paclitaxel with and without trastuzumab in endometrial cancer.

Cetuximab Cetuximab is a chimeric (mouse/human) monoclonal antibody that binds to EGFR. However, if the KRAS protein is mutated, cetuximab has been found not to work. Cetuximab was equally inactive in endometrial cancer, showing an ORR of 5% in a Phase II trial including 23 patients. The results were not published but the abstract was presented at Society of Gynecological Oncology (SGO) in 2010. doi: 10 18282/amor v1 i1 9

Aflibercept Aflibercept binds to circulating VEGFs and acts like a “VEGF trap”, it is a soluble recombinant decoy VEGF receptor that is biologically engineered to bind to all forms of this growth factor. There are two published trials on endometrial cancer. One Phase II trial enrolled 49 patients, of which 7% had a partial response, while there were no complete responders. Of note, 10 patients (23%) met the PFS at 6 months endpoint without starting a subsequent therapy; the remaining 8 patients discontinued therapy for toxicity. Aflibercept met pre-trial activity parameters, but was associated with significant toxicity at this dose and schedule in this population[30]. The second Phase II trial enrolled 19 patients with endometrial carcinosarcoma and it showed only minor activity, with

13

Emerging molecular-targeted therapies—the challenging case of endometrial cancer

one patient presenting stable disease for more than 24 weeks but no objective response[31]. There are currently no ongoing trials.

Bevacizumab Bevacizumab has been the most extensively evaluated VEGFR targeted therapy in endometrial cancer. It is a monoclonal antibody directed at the cytokine (VEGF) and has showed very good activity in ovarian and metastatic breast cancer. There are three Phase II and two Phase I trials, which are published. The most recent Phase II trial evaluated the effect of adding bevacizumab to adjuvant paclitaxel and carboplatin and further as maintenance on PFS in advanced or recurrent endometrial carcinoma. It enrolled 15 patients on protocol when accrual to the study was discontinued due to the initiation of a national randomized Phase II trial. One patient suffered a bowel perforation, while five complete responses and six partial responses were seen for an overall response rate of 73%, so the results of the National Trial (NCT00977574) are eagerly awaited. The second Phase II trial evaluated the combination of bevacizumab with temsirolimus and enrolled 53 patients, resulting in three treatment-related patient deaths and clinical responses from 12 patients (24.5%). Although an active combination, it was associated with significant treatment associated toxicity[17]. The first Phase II trial on bevacizumab evaluated its efficacy in patients with persistent disease after cytotoxic treatment and enrolled 56 patients, with only seven patients (13.5%) showing clinical responses[32]. NCT01005329 evaluated intensity-modulated radiation therapy, cisplatin, and bevacizumab followed by carboplatin and paclitaxel enrolled 32 patients but reported an unacceptable percentage of treatment-related, grade 3+, non-hematologic adverse events of 23.3%, no results (available from www.clinicaltrials. gov). MITOBEVAEND2 (carboplatin-paclitaxel ± bevacizumab in stage III–IV or recurrent endometrial cancer, NCT01770171) is currently recruiting and results are eagerly awaited, as they may prove to be practice-changing. Temsirolimus and bevacizumab, in treating patients with advanced endometrial cancer (NCT01010126) is ongoing, but has finished accrual. The combination trial of the mTORC1/2 inhibitor, MLN0128 and bevacizumab in patients with advanced solid tumors (NCT02142803), is currently recruiting.

Brivanib (BMS-582664) Brivanib is an alaninate salt of a VEGFR-2 inhibitor and is hydrolyzed to an active moiety in vivo. It works as a

14

TKI, strongly binding to and inhibiting VEGFR-2, a tyrosine kinase receptor expressed almost exclusively on vascular endothelial cells. The only published Phase II single-arm trial enrolled 45 and reported 8 (18.6%) responses[33]. There is one ongoing trial that has finished accrual, (NCT00888173) evaluating single-agent brivanib in patients with recurrent or persistent endometrial cancer.

Nintedanib (BIBF 1120) Nintedanib is an orally bioavailable, indolinone-derived, RTK inhibitor. This multi-targeted TKI, selectively binds to and inhibits VEGFR, FGFR and PDGFR, which may result in a reduction of the tumor vasculature. There is one published Phase II trial, enrolling 37 patients, reporting three (9.4%) partial responses. Nintedanib lacked sufficient activity as a single agent to warrant enrollment to second stage[33]. There is one ongoing trial on Nintedanib in patients with recurrent or persistent endometrial cancer that has finished accrual (NCT01225887).

Lenvatinib (E7080) Lenvatinib is a synthetic, orally available, inhibitor of VEGFR2. Lenvatinib acts as a TKI, blocking the VEGFR2 TK, resulting in inhibition of the VEGF receptor signal transduction pathway, decreased vascular endothelial cell migration and proliferation, and vascular endothelial cell apoptosis. There are currently two undergoing trials on E7080 in endometrial cancer. NCT01111461 has finished accrual and NCT02501096 has not started accrual yet (Table 2).

Sunitinib Sunitinib (SU11248) is a small-molecule, multi-targeted receptor TKI. Its targets include all TK receptors for PDGFR and VEGFR. There is one published Phase II trial on sunitinib, enrolling 34 women, of which six (18.1%) had a partial response. Toxicity was seen frequently but was manageable[34]. There is one ongoing trial on sunitinib in patients with recurrent or metastatic endometrial cancer that has already finished accrual (NCT00478426).

Cabozantinib (XL184) Cabozantinib (INN) is a small molecule inhibitor of the TKs c-Met and VEGFR. There is one ongoing trial on cabozantinib-s-malate in the treatment of patients with recurrent or metastatic endometrial cancer, currently recruiting participants (NCT01935934). doi: 10 18282/amor v1 i1 9

Vasconcelos I.

Discussion Endometrial cancer has fallen behind, in terms of molecular characterization and development of molecular-targeted therapies, when compared to other disease sites. While in the adjuvant setting, a combination approach including chemotherapy and radiation therapy is likely to yield the best control rates for the patients, the current research focus should be maintenance therapy that helps patients to remain in remission. “Older” clinical trials have included women with type-I and type-II endometrial cancers, known to represent different disease entities. Unfortunately, only a few molecular-targeted agents have shown significant response rates in clinical trials in endometrial cancer. Many drugs that revealed antitumor activity in preclinical studies were unsuccessful when tested in the clinical setting. We have come to understand that identifying and targeting a mutation does not always result in a clinical response. Further challenging has been the unacceptable toxicity reported in some clinical trials, such as with aflibercept, temsirolimus and ridaforolimus. Mutation and loss of PTEN function with overexpression of mTOR signaling pathway are involved in the pathogenesis of endometrial cancer, with endometrial cancer being the cancer type that shows the highest incidence of mutations in this pathway. Therefore, the PIK3/AKT/mTOR pathway is a particularly attractive target in endometrial cancer. mTOR inhibitors have shown therapeutic efficacy alone or in combination therapy, regardless of the status of PTEN mutation. The results of the Phase II trial evaluating PF-05212384 are eagerly awaited, as are the results of the Phase II trial evaluating paclitaxel, carboplatin, and bevacizumab or paclitaxel, carboplatin, and temsirolimus or ixabepilone, carboplatin, and bevacizumab (NCT00977574). The dual blockade of mTOR and VEGF by combination with bevacizumab appears particularly attractive. Everolimus, PF-05212384 and temsirolimus showed promising ORR above 20%, either alone or in combination. Although ridaforolimus and temsirolimus show promising anti-tumor activity, they require caution due to a high- toxicity profile. HER2 amplification is present in a significant proportion of high-grade endometrial tumors at high risk of progression, recurrence, and decreased survival. While HER2-targeted therapies have been practice-changing in breast cancer treatment, HER2 directed therapies in endometrial cancer settings have been frustrating, to say the least. Clinical trials testing HER2-targeted therapies in endometrial cancer have shown minimal clinical benefit due resistance mechanisms that are poorly understood. doi: 10 18282/amor v1 i1 9

These therapies have failed to show adequate response rates in recurrent HER overexpressing endometrial cancer, suggesting that these tumors may possess resistance mechanisms. Attempts to block the VEGFR family have likewise shown good results in other cancer types, but efforts to block VEGF in endometrial cancer have obtained disappointing results. The timing and role of angiogenic agents remains unclear. Extrapolating from other cancer types, the combined use with chemotherapeutic agents at recurrence could be beneficial. Bevacizumab is the only agent that has consistently shown activity in endometrial cancer, particularly when used in combination with chemotherapy. Another interesting approach appears to be the combination with mTOR inhibitors such as temsirolimus (NCT00977574 and NCT01010126), whose trials results are eagerly awaited.

Conclusion Endometrial cancer has demonstrated to harbor a complex carcinogenesis process. Even with largely disappointing results obtained this far, targeted personalized treatment should be the main goal of treatment of advanced disease in the future. Better characterization of molecular subtypes is needed to optimize patient selection in future clinical trials.

Conflict of Interest The author declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

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cancer: a non-randomised, open-label, two-group, twostage, phase 2 study. Lancet Oncol 2015; 16(6): 686–694. 30. Coleman RL, Sill MW, Lankes HA, et al. A Phase II evaluation of aflibercept in the treatment of recurrent or persistent endometrial cancer: a Gynecologic Oncology Group study. Gynecol Oncol 2012; 127(3): 538–543. 31. Mackay HJ, Buckanovich RJ, Hirte H, et al. A Phase II study single agent of aflibercept (VEGF Trap) in patients with recurrent or metastatic gynecologic carcinosarcomas and uterine leiomyosarcoma. A trial of the Princess Margaret Hospital, Chicago and California Cancer Phase II Consortia. Gynecol Oncol 2012; 125(1): 136–140. 32. Aghajanian C, Sill MW, Darcy KM, et al. Phase II trial of

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bevacizumab in recurrent or persistent endometrial cancer: a Gynecologic Oncology Group study. J Clin Oncol 2011; 29(16): 2259–2265. 33. Dizon DS, Sill MW, Schilder JM, et al. A Phase II evaluation of nintedanib (BIBF-1120) in the treatment of recurrent or persistent endometrial cancer: an NRG Oncology/Gynecologic Oncology Group Study. Gynecol Oncol 2014; 135(3): 441–445. 34. Castonguay V, Lheureux S, Welch S, et al. A Phase II trial of sunitinib in women with metastatic or recurrent endometrial carcinoma: a study of the Princess Margaret, Chicago and California Consortia. Gynecol Oncol 2014; 134(2): 274–280.

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REVIEW ARTICLE

Hepatocellular carcinoma (HCC); aetiological considerations Omar Abdel-Rahman Clinical Oncology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Abstract: Hepatocellular carcinoma (HCC) is a commonly occurring cancer worldwide. The aetiology of HCC, often associated with different molecular carcinogenic pathways, differs geographically; with chronic viral hepatitis being the main cause in most localities. Different driving mutations resulting from distinct carcinogenic pathways potentially impact the choice of effective therapies for HCC. Keywords: HCC; aetiology; biology Citation: Abdel-Rahman O. Hepatocellular carcinoma (HCC); aetiological considerations. Adv Mod Oncol Res 2015; 1(1): 18–19; http://dx.doi.org/10.18282/amor.v1.i1.8. Correspondence to: Omar Abdel-Rahman, Ain Shams University, [email protected].

Received: 21st July 2015; Accepted: 21st August 2015; Published Online: 6th October 2015

H

epatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with approximately 600,000 new cases per year, and the third leading cause of cancer-related deaths[1,2]. According to data from GLOBOCAN 2012, wide geographic and socioeconomic differences exist among different ethnic groups of the world with 83% of the estimated 782,000 new cancer cases worldwide prevailing in less developed countries, and 50% in China alone[3]. In men, the regions of high incidence are Eastern and South-Eastern Asia with age-standardized rates (ASRs) of 31.9% and 22.2% respectively. Intermediate rates occur in Southern Europe (9.5%) and Northern America (9.3%) and the lowest rates are in Northern Europe (4.6%) and South-Central Asia (3.7%). In women, the rates are generally much lower than men with an overall male: female ratio of 2.5, the highest incidence being in Eastern Asia and Western Africa (10.2% and 8.1% respectively), the lowest in Northern Europe (1.9%) and Micronesia (1.6%). These geographical differences in HCC incidence have been largely ascribed to differences in risk factor epidemiology in different areas of the world[4,5].

Aetiology/biology correlations in HCC HCC usually develops in a liver already chronically damaged, often from cirrhosis. The aetiology of liver disease, and consequently that of HCC, differs geographically with consequent differences in the HCC genotype. In most areas, chronic viral hepatitis due to either hepatitis B virus (HBV) or hepatitis C virus (HCV) is the main cause of HCC[6].

HCV-related HCC Approximately 195,000 cases of liver cancer (31.1% of cases globally) are attributed to HCV, with northern and middle Africa being the areas of highest prevalence[7,8]. Additionally, HCV is the most common viral aetiology of HCC in the western population (Europe/North America)[9]. It is difficult to determine the likelihood of the development of HCC among HCV infected persons due to the paucity of adequate long term cohort studies. However, the best estimate is from 1% to 3% after 30 years[10]. Lastly, alcohol is an important factor in patients with HCV infection, with HCV reported in 4.6%–55.5% of alcoholics. Patients with HCV infection and who

Copyright © 2015 Abdel-Rahman O. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Omar Abdel-Rahman

have been alcohol abusers prove to develop more severe fibrosis and have higher rates of cirrhosis and HCC than non-drinkers[11].

2.

HBV-related HCC Approximately, 340,000 cases of liver cancer (54.4% of cases globally) are attribued to HBV, with the majority of these in Africa, Asia, and the Western Pacific region[12]. Risk is higher in those with HBeAg+, high ALT, high HBV DNA, cirrhosis and the elderly. Risk is markedly reduced with successful antiviral therapy[13]. Characteristically, HBV can cause HCC both in the presence and absence of cirrhosis due to its ability to integrate its DNA into host cells and act as a mutagenic agent, causing secondary chromosomal rearrangement and increasing genomic instability[14]. This is in contrast to HCV-related HCC which is found almost exclusively in patients with cirrhosis.

Alcoholic liver disease The development of cirrhosis from alcohol consumption increases the risk of HCC. It has been suggested that heavy alcohol consumption of >80 g/d ethanol for at least five years increases the risk of HCC by nearly fivefold[15]. This mechanism of developing HCC is particularly prevalent in many Western countries[16].

3.

4.

5.

6.

7. 8.

9.

Non-alcoholic steatohepatitis (NASH)

10.

NASH, in association with multiple components of the metabolic syndrome is thought to increase the risk for developing chronic liver disease, cirrhosis, and HCC[17].

11.

Conclusion

12.

Paying proper attention to the diverse aetiologies that may be associated with developing HCC has a profound significance in terms of prevention as well as treatment of this disease. Different aetiologies may be associated with different molecular carcinogenic pathways, and consequently different driving mutations that may impact the choice of effective therapies for this disease.

Conflict of interest The author declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

References 1.

Zaanan A, Williet N, Hebbar M, et al. Gemcitabine plus

doi: 10 18282/amor v1 i1 8

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oxaliplatin in advanced hepatocellular carcinoma: A large multicenter AGEO study. J Hepatol 2013; 58(1): 81–88. Abdel-Rahman O, Lamarca A, Valle JW, et al. Somatostatin receptor expression in hepatocellular carcinoma: prognostic and therapeutic considerations. Endocr Relat Cancer 2014; 21(6): R485–R493. Fact Sheets: All cancers (excluding non-melanoma skin cancer) estimated incidence, mortality and prevalence worldwide in 2012 [Internet]. 2014 [cited 1st April 2015]. Available from: http://goo.gl/JBcEHo. Altekruse SF, McGlynn KA, Dickie LA, et al. Hepatocellular carcinoma confirmation, treatment, and survival in surveillance, epidemiology, and end results registries, 1992-2008. Hepatology 2012; 55(2): 476–482. Abdel-Rahman O, Fouad M. Second line systemic therapy options for advanced hepatocellular carcinoma; a systematic review. Expert Rev Anticancer Ther 2014; 15(2): 165–182. Masuzaki R, Omata M. Screening program in high-risk populations. In: McMasters KM, Vauthey JN, editors. Hepatocellular carcinoma: targeted therapy and multidisciplinary care. New York: Springer; 2011. p. 55–68. Sanyal AJ, Yoon SK, Lencioni R. The etiology of hepatocellular carcinoma and consequences for treatment. Oncologist 2010; 15(Suppl 4): 14–22. Abdel-Rahman OM, Elsayed Z. Yttrium-90 microsphere radioembolisation for unresectable hepatocellular carcinoma (Protocol). Cochrane Database of Systematic Reviews 2014, Issue 9. Art. No.: CD011313. Deuffic S, Poynard T, Valleron AJ. Correlation between hepatitis C virus prevalence and hepatocellular carcinoma mortality in Europe. J Viral Hepat 1999; 6(5): 411–413. Goodgame B, Shaheen NJ, Galanko J, et al. The risk of end stage liver disease and hepatocellular carcinoma among persons infected with hepatitis C virus: publication bias? Am J Gastroenterol 2003; 98(11): 2535–2542. Singal AK, Anand BS. Mechanisms of synergy between alcohol and hepatitis C virus. J Clin Gastroenterol 2007; 41(8): 761–772. Venook AP, Papandreou C, Furuse J, et al. The incidence and epidemiology of hepatocellular carcinoma: a global and regional perspective. Oncologist 2010; 15 (Suppl 4): 5–13. Chang MH, You SL, Chen CJ, et al. Decreased incidence of hepatocellular carcinoma in hepatitis B vaccinees: a 20-year follow-up study. J Natl Cancer Inst 2009; 101(19): 1348–1355. But DY, Lai CL, Yuen MF. Natural history of hepatitis-related hepatocellular carcinoma. World J Gastroenterol 2008; 14(11): 1652–1656. Donato F, Tagger A, Gelatti U, et al. Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women. Am J Epidemiol 2002; 155(4): 323–331. Capocaccia R, Sant M, Berrino, et al. Hepatocellular carcinoma: trends of incidence and survival in Europe and the United States at the end of the 20th century. Am J Gastroenterol 2007; 102(8): 1661–1670. Bugianesi E. Non-alcoholic steatohepatitis and cancer. Clin Liver Dis 2007; 11(1): 191–207.

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doi: 10.18282/amor.v1.i1.12

REVIEW ARTICLE

Indoles as anti-cancer agents Mardia T. El Sayed1*, Nehal A. Hamdy1, Dalia A. Osman1, Khadiga M. Ahmed2 1

Applied Organic Chemistry Department, Chemical Industries Division, National Research Centre, Egypt Natural Products Department, Pharmaceutical Industries Division, National Research Centre, Cairo, Egypt

2

Abstract: Indoles are natural products which are well known for its anti-cancer activity due to its ability to induce cell death for many cancer cell lines. This review addresses indoles as natural products, mechanism of indoles, facilitated induction and recent studies with indoles and related compounds that were investigated via anti-cancer screening and that led to drug approval. Keywords: indoles; natural biosynthesis; cell death induction; screening; anti-cancer agents Citation: El Sayed MT, Hamdy NA, Osman DA, et al. Indoles as anti-cancer agents. Adv Mod Oncol Res 2015; 1(1): 20–35; http://dx.doi.org/10.18282/amor.v1.i1.12. *Correspondence to: Mardia T. El Sayed, Applied Organic Chemistry Department, Chemical Industries Division, National Research Centre, Egypt, [email protected].

Received: 30th July 2015; Accepted: 4th September 2015; Published Online: 6th October 2015

A

n indole is an aromatic heterocyclic composite which has its heterobicyclic configuration as a six-membered ring fused to a five-membered pyrrole ring. ‘Indole’ is the name given to all indole derivatives which have an indole ring system[1,2]. Indoles are obtained from coal pitch or a variety of plants and produced by the bacterial decay of tryptophan in the intestine. It has been synthesized by one of the oldest method that known as Fischer indole synthesis[2]. Indoles function as signal molecules in plants and animals. They also serve as a raw material, nucleus building blocks and an efficient group of numerous imperative biochemical molecules and compounds, such as alkaloids, indigoids, etc. Most of these important molecules and compounds originate, either fully or partly, from bio-oxidation of indoles.

Naturally biosynthesized indole products Indoles are natural compounds can be found in numerous types of plant. They are, however more predominantly found in cruciferous vegetables[2]. Cruciferous vegeta-

bles comprise of cauliflower, cabbage, turnip, broccoli and brussels sprouts (Figure 1). Indoles fit in a class of phytonutrient compounds (plant compounds with healthprotecting qualities) which have been systematically proven to profit the body in a number of imperative ways. Consuming of cruciferous vegetables has been associated with reduced of the risk of colon, breast and prostate cancers. Cruciferous vegetables are a rich source of many phytochemicals, including indole derivatives, dithiolethiones and isothiocyanates. Cruciferous vegetables are full of glucobrassicin (GB) which throughout metabolism, produce indole-3-carbinol (I3C), 3,3′diindolylmethane (DIM) and ascorbigen (ASC) (Figure 2). The anti-carcinogenic property of I3C and DIM was exhibited in human cancer cells. It appears that these indolic compounds may be efficient anti-cancer agents for several cancer cell lines[3-6]. Natural compounds found in fruits and vegetables are recognized to have anti-mutagenic and anti-carcinogenic properties. A high dietary intake of fruits and vegetables has proved to be beneficial against carcinogenesis. An inhibitory effect of indoles and cruciferous vegetables against tumorgenesis and cancer hazard has also been veri-

Copyright © 2015 El Sayed MT, et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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fied[7]. Epidemiological statistics reveal that populations that consume higher amounts of cruciferous vegetables have lower incidences of cancer or improved biochemical parameters, such as decreased oxidative pressure compared to controls. Cruciferous vegetables protect against cancer more successfully than fruits and other vegetables. The National Research Council, USA, has recommended consumption of cruciferous vegetables as a measure to diminish the commonness of cancer[8-11]. Bio-oxidation of indoles and respective enzymes in microorganisms and in plants has been well documented in the recent review by Yuan et al.[3] In this review, the pathways of indole bio-oxidation which lead to configuration of indigo and indirubin in plant and the perspectives of the study in indole bio-oxidation have been discussed (Scheme 1). Where E1 is indole oxygenase, E2 is indole oxidase, E3 is indole 2,3-dioxygenase, E4 is indican synthase, E5 is indoxyl-UDPG-glucosyltransferase, E6 is formylase, E7 is aldehyde oxidase, S is

Brussel Sprout

Kohlrabi

Broccoli

spontaneous reaction, P is plant tissues and organs, GT? is glucosyltransferase (unidentified) and GLU? is glucosidase (unidentified)[3]. The amino acid tryptophan is a biogenetic precursor of all indole alkaloids. The first step of synthesis is decarboxylation of tryptophan to form tryptamine. Dimethyltryptamine (DMT) is formed from tryptamine by methylation with the contribution of coenzyme of Sadenosyl methionine (SAM). Psilocin is produced from dimethyltryptamine by oxidation and is then phosphorylated into psilocybin. In the biosynthesis of serotonin, the intermediary product is not tryptamine but 5-hydroxytryptophan, which is sequentially decarboxylated to shape 5-hydroxytryptamine (serotonin) (Figure 3)[12-14]. Biosynthesis of β-carboline alkaloids takes place via formation of Schiff base from tryptamine and aldehyde (or keto acid) and consequent intramolecular Mannich reaction, where the carbon in position two of indole acts as a nucleophile (Figure 4). This is followed by the

Radish

Cauliflower

Figure 1 Cruciferous vegetables. [Source available from: http://www.fotosearch.com/photos-images/cruciferous-vegetables.html] OH S GB

O

OH OH OH

OH

N O SO 3

N N H H

Myrosonase H2O

D-glucose HSO4 N

-

I3C SCN

S H2O

N H H

NN HH

L-ascorbic acid H

OH

O HO

N H H

DIM

N N H H

H2 O O

O OH

N N H ASC H

Figure 2 Biosynthesis of indoles: 13C, DIM and ASC from GB

doi: 10.18282/amor.v1.i1.12

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Indoles as anti-cancer agents

aromaticity being restored via the elimination of a proton at the C two atom. The consequential tetrahydro-β-carIndigo plant Indigoproducing produc ing plant

Non-indigoproducing producing plant Non-indigo plant

O

HO N H

N H

HO

Hydroxy indo le

OH O

E4 /E5

N H

6-Hydroxyindole 6-Hydroxyindole

55-Hydroxyindole

boline frame steadily oxidizes to dihydro-β-carboline and β-carboline afterwards[16,20].

OH

HO

3-Oxindole 3-O xindole

NH

Indican Indican

P

P

O

OH

OH O2

P N N H H

N H H

Indole hydroxylase hydrunulase ? Indole

HO G T? GT?

N N H H

Indole

O2

O O

Glycosylation Gly cosy lation

Indole

O

G T?

OO22

OH

Hydrolysis Hydrolysis

N H

GLU ? GLU?

N H

O GT?

Indoxy l Indoxyl

HO IsatanBB Isatan

O

E6 ?

Isatine Isatine

CHO

OH

N H

N H Dioindole Dioindole

O

O-aminobenzaldehyde O- aminobe nzaldehyde

N H CO2H

O2 H N

O Indigo Indigo

NH

O

C 11H7O2 GT? O

NH

IsatanCC Isatan

O2 S NH 2

O

OH O

O2 S

OH

O

NHCHO N-form yla minobenzaldehyde benzaldehy de N-formylamino

NH

Isatan Isatan A A

OH

3 -Oxindole 3-Oxindole

OH

HO

O

O

HO

Indoxyl Indoxyl

E1 E2 E3 CHO

H C

O

HO

Precursors inInVivo P re cursors Vivo

S O

? N H Indirubin Indirubin

NH O

O N

Anthranil Anthranil (E2/E3) (E2/E3)

NH 2 Anthranilic a cid Anthranilic acid (E1/E3) (E1/E3)

Scheme (1): Bio oxidation of indole in higher plants.

Scheme 1 Bio-oxidation of indole in higher plants

Figure 3 Tryptophan as a biogenetic precursor of all indole alkaloids

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El Sayed MT, et al.

NH2

N

RCHO RCHO N H

N H Schiff schiff base base formation formation

NH

NH

R

tryptamine tryptamine

R N H terahydro-β-carboline terahydro-B-carboline

R

N H

Mannich Mannichreaction reaction

oxidation

oxidation oxidation

R N H dihydro-β-carboline dihydro-B-carboline

R N H Beta-carboline beta-carboline

Fig. (4): Biosynthesis of beta-carboline alkaloids.

N

oxidation oxidation

N

Figure 4 Biosynthesis of beta-carboline alkaloids

The biosynthesis of ergot alkaloids starts with the alkylation of tryptophan via dimethylallyl pyrophosphate (DMAPP), in which the carbon atom in position 4 of the indole ring acts an important facilitator of the nucleophile (Figure 5). The consequential 4-dimetilallil-Ltryptophan is followed by N-methylation. Additional products that result from the biosynthesis are chanoclavine-I and agroclavine—the latter is hydroxylated to elymoclavine, that in turn oxidizes into paspalic acid OH

O

O O P O PO O O

COOH NH2

HN

COOH

DMAPP

H

N

H

HO

N

NH2

NN HH

N H H

L-tryptophan

which is converted to lysergic acid through the process of allyl rearrangement[16,21]. Biosynthesis of monoterpenoid indole alkaloids starts with the Mannich reaction of tryptamine and secologanin; this produces strictosidine, which is renewed to 4, 21-dehydrogeissoschizine (Figure 6). After that, the biosynthesis of alkaloids containing the composed monoterpenoid fraction (Corynanthe type) was shaped throughout cyclization with the configuration of

N N H H chanoclavine-1

4-dimetilallil-L-tryptophan

HOOC H

oxidation

N N H H agroclavine

N

HOOC H

N

H

oxidation

N N H H elymoclavine

N H H paspalic

N N H H D-(+)-lysergic acid

Figure 5 Ergot alkaloids biosynthesis. Mannich reaction Mannich reaction NH2 N N H H

OHC

NH

OGlc O

N N H H

secologanin N

N N H H

N H H

H3 COOC strictosidine

OAc OH COOCH3

N

N

O

CH3

vindoline Aspidospirma type

OGlc

N

N N H

OH H3 COOC dehydrogeissoschizine

N N H H

O H3 COOC

cathenamine

CH3

COOCH3

tabersonine Aspidospirma type

Figure 6 Biosynthesis of monoterpenoid indole alkaloids

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23

Indoles as anti-cancer agents

scores of important modules of therapeutic agents in medicinal chemistry such as anti-cancer[22], anti-oxidant[23], anti-rheumatoidal[24], anti-HIV[25,26], anti-microbial[27-29], anti-inflamatory[30] , analgesic [31] , antipyretic [32] , anti-convulsant[33,34], anthelmintic cardiovascular[35], selective COX-2(cyclooxygenase-2) inhibitory activities[36-39] (which is an enzyme accountable for inflammation and pain) and DNA binding ability[40]. Furthermore, countless essential indole derivatives are used in disease management, for example, the non-steroidal anti-inflammatory drug indomethacin (Indocin®), the beta blocker pindolol (Viskin®) for management of high blood pressure (hypertension), the psychedelic, dimethyltryptamine (DMT)[41] and BioResponse DIM® for estrogens for men and women (http://www.bioresponse.com/Home.asp) (Figure 7)[12,13].

cathenamine, and following reduction reaction into ajmalicine in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). With the biosynthesis of previous alkaloids, 4,21-dehydrogeissoschizine changed into preaquamycin (an alkaloid of subtype Strychnos, type Corynanthe) which ascends to other alkaloids of subtype Strychnos and of the types Iboga and Aspidosperma[21].

Indoles in medicinal chemistry Indole derivatives are imperative heterocycles in the drug-discovery studies. They are a very important category of compounds that play a key role in cell physiology and are probable intermediates for numerous biological reactions. Indole derivatives correspond to Me HN

OH

Me

Me

MeO

O

HO

N

Me

Me N

O

N H N H Pindolol

DMT

Cl

N H

N H DIM

Indomethacin

Figure 7 Marketed indole drugs

Cell death induction by indoles Anti-cancer agents have been usually evaluated for their ability to induce apoptosis. Indoles have been verified to inhibit proliferation, expansion and invasion of human cancer cells[1,2,42-44]. Many mechanisms of apoptosis stimulation of indole derivatives, I3C and DIM, were reported for, (a): down-regulation of anti-apoptotic gene products such as Bcl-2 (B-cell lymphoma 2) and Bcl-XL (B-cell leukemia-extra large), (b): down-regulation of the inhibitor of apoptosis proteins, e.g. CIAPs, X-chromosome linked inhibitor of apoptose protein (XIAP) and survival, (c): up-regulation of pro-apoptotic factors such as Bax gene, (d): liberation of mitochondrial cytochrome C in addition to stimulating of caspase-9 and caspase-3[45], and (e): inhibition of the NF-kB signaling pathway[46-51]. A vast number of diverse mechanisms of apoptosis induction by indoles have also been reported[52-56]. Figure 8 demonstrates the extrinsic and the intrinsic pathways of apoptosis (programmed cell death). The Extrinsic Route: In the extrinsic pathway, signal molecules identified as

24

ligands, which are released by the immune system’s natural killer cells possess the Fas ligand (FasL) on their exterior to connect to transmembrane death receptors on the target cell. After the binding of the death ligand to the death receptor the target cell triggers multiple receptors to aggregate together on the surface of the target cell. The aggregation of these receptors recruits an adaptor protein known as Fas-associated death domain protein (FADD) on the cytoplasmic side of the receptors. FADD, in turn, recruits Caspase-8. Caspase-8 will then be activated and will be now able to directly activate caspase-3 and caspase-7. The activation of caspase-3 will initiate the degradation of the cells[57]. The Intrinsic Route: The intrinsic pathway is triggered by cellular strain, particularly mitochondrial stress caused by factors such as DNA damage from chemotherapy or UV exposure. Upon delivery of the stress signal, the pro-apoptotic proteins in the cytoplasm (Bcl-2-like protein 4 (BAX) and BAX-like Bcl-2 homology domain 3 protein (BID)) bind to the outer membrane of the mitochondria to signal the release of the internal content.

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El Sayed MT, et al.

The interaction between the pro-apoptotic (BAX and BID) and the antiapoptotic proteins (Bcl-2) on the surface of the mitochondria is thought to be important in the formation of the PT pores in the mitochondria, and hence, the release of cytochrome c and the intramembrane content from the mitochondria. Following the release, cytochrome c forms a multi protein complex known as apoptosome which consists of cytochrome c, Apaf-1, procaspase-9 and ATP. Following its formation, the complex will activate caspase-9. The activated caspase-9 will then turn the procaspase-3 and procaspase-7 into active cas-

pase-3 and active caspase-7. These activated proteins initiate cell degradation or cell death. Besides the release of cytochrome c from the intramembrane space, the intramembrane also releases Smac/Diablo proteins to inhibit the inhibitor of apoptosis (IAP). IAP is a protein family which consists of 8-human derivatives. Their function is to stop apoptotic cell death by binding to caspase-3, caspase-7 and caspase-9 and inhibit them, the schematic representation of these pathways are shown in Figure 8[12,13,58].

Figure 8 Intrinsic and extrinsic pathways leading to apoptosis. [Available from https://innspubnet files.wordpress.com/2015/04/ mitochondrial-pathway.jpg]

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25

Indoles as anti-cancer agents

Indoles for inhibition of invasion and metastasis Cancer cells are able to travel through the lymphatic and blood vessels, pass through the bloodstream, and then occupy and produce in healthy tissues elsewhere. This ability to spread to other tissues and organs leads to malignancy and makes cancer a potentially life threatening disease. Tumor angiogenesis is the propagation of a complex of blood vessels that penetrate into cancerous growths, supply blood and oxygen and eliminate waste products[59]. In reality, tumor angiogenesis starts with cancerous tumor cells releasing molecules that post signals to the neighboring host tissues. This signaling activates definite genes in the host tissue that, in turn, build proteins to support growth of new blood vessels. Indole derivatives, I3C and DIMs have been reported to restrain the invasion of cancer cells[57-60] and the expansion of new blood vessels (angiogenesis)[59,60].

Indole compounds for chemosensitization Chemosensitization is the progression by which compounds for example, indole compounds, I3C and DIM adapt the cellular signaling pathways leading to apoptosis and thus conquer the chemo- plus immune-resistance of well-known chemotherapeutic drugs[1,61]. I3C has been reported to sensitize multidrug resistant tumors to chemotherapeutic drugs without any related toxicity[1,62-64]. Mechanisms of anti-cancer and chemosensitizing effects of indole compounds were summarized in Figure 9. Indole compounds, such as I3C and its dimmer DIM, induce apoptosis through inhibition of several prosurvival pathways. Emerging evidence also documents the ability of indoles to reverse the process of EMT via regulation of key miRNAs. An efficient induction of apoptosis and

reversal of EMT not only ensures increased sensitivity to conventional drugs (chemosensitization) but also results in significantly reduced invasion and metastasis[1,62-64].

Reported indoles as anti-cancer active agents Nowadays, clinical association of human reproductive organ cancers requests new chemotherapeutics. In recent times, a lot of hard works have been done to organize antiproliferative signaling pathway of indole-3-carbinol and its foremost indole metabolite 3,3′-diindolylmethane (DIM)[65-71]. While DIM significantly reduces the occurrence of impulsive and carcinogen induced mammary tumor establishment (Figure 10)[72-74]. It also exhibits unpleasant promoting action in convinced investigation procedure[75,76]. As a result, the decision was to look for novel effective chemotherapeutics amongst 3,3′-diindolylmethane derivatives. Moreover, the X-ray studies of 5,5′-dimethoxy-3,3′-methanediyl-bis-indole[77] revealed its ‘butterfly’ conformation, which is analogous to the one proposed earlier for inhibitors of HIV-1 reverse transcriptase, sharing the mode of action of nevirapine[78]. Other diindolylmethane derivatives and their corresponding tetrahydroindolocarbazoles have been synthesized and screened for anti-cancer activity in which two compounds indicated were significantly more sensitive for several cancer cell lines corresponding to their GI50 values. The highest antipoliferative activity recorded for the carbazole derivatives in a nanomolar scale towards the three certain cancers cell lines: non-small lung cell NCIeH460 with GI = 616 nmol/L, ovarian cancer cell line OVCAR-4 with GI = 562 nmol/L and breast cancer cell line 50 nmol/L scale MCF7 with GI = 930 nmol/L (Figure 10)[16].

X

X H

N H H

H

N N H H N N H H

X== F, X X=CN X = CN

Figure 9 Summary of mechanisms of anti-cancer and chemosensitizing effects of indole compounds. [Original source from http://www.mdpi.com/cancers/cancers-03-02955/article_deploy /html/images/cancers-03-02955f 1-1024.png]

26

N N H H

Carbazole Carbazole derivative derivative

Figure 10 3,3′-diindolylmethane derivatives and tetrahydroindolocarbazoles

Dorota et al.[79,80] in 2005 synthesized the disubstituted diindolylmethanes flouro and cyano derivatives which decrease the expansion of MCF7 (breast), NCI–H460

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El Sayed MT, et al.

(lung) and SF-268(NCS) cells, considerably 5,5′difluoro-3,3′-methanediyl-bis-indole and 5,5′-dicyano-3, 3′-methanediyl-bis-indole were tested against the MCF7 (breast), NCI-H460 (lung) and SF-268 (CNS) tumor cell lines. The results are reported as the proportion of growth of the tested cells to untested control cells (Figure 10). F-derivative at concentration 1.10–4.00 mol/L reduces the growth of MCF7, NCI-H460, and SF-268 cell lines to 1%, 0% and 2%, whereas the CN derivative at concentration 5.10–5.00 mol/L to 4%, 1% and 9%, respectively. Both compounds are extremely cytotoxic in vitro towards those tumor lines. Their cytotoxicity indicates that they could be motivating as prospective antitumoral chemotherapeutics[79,80]. Indoles (I3C and DIM or its derivatives) have been revealed to induce apoptosis in breast [81-87] , squamous cell carcinoma[88], cholangiocarcinoma[89], colon[90-93], Me2N

N

NMe2

N

R2

N N

O

I

Indole-PMM

Me

Z

R1

Me N

H N

HN

C2H3

MeO

O

cervical[94], ovarian[95], pancreatic[96,97] and prostate[98-101] cancer cells. Many other indole derivatives that were reported as active anti-cancer agents as follow: the potential prodrug (1,2-dimethyl-3-(N-(4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)-N-trideuteronmethylaminome thyl)-5-methoxyindole-4,7-dione), pentamethylmelamine (PMM) in which the labeled pentamethylmelamine is attached to an indole-4,7-dione moiety has attracted much interest as an anti-tumor agent for over 35 years (Figure 11). It entered clinics in the 1970s for the treatment of ovarian carcinoma but difficulties were encountered, as it was insoluble in water and thus is difficult to formulate. However, it has recently been recognised as a second-line treatment for ovarian carcinoma [12-19,102-104]. Schoentjes et al.[105] reported a patent of indole derivatives with general formula (I) in 2011 and reported its use for the treatment of cancers (Figure 11).

N H

Figure 11 Structure of prodrug indole-PMM derivative and tryptamine derivative

Numerous aroylamide indole derivatives have been synthesized and preliminarily screened for their in vitro anti-cancer activity in A431 and H460 cell lines (Figure 12). All the compounds examined exhibited strange effectiveness in a tumor cell cytotoxicity evaluation. The findings indicated that the indole derivatives would be talented candidates for the improvement of new anticancer agents. 3-aroylindole is a probable anti-cancer drug candidate designed and projected from in vitro human microsome studies with better pharmacokinetics and enhanced influence in the tumor xenograft represenH3CO R1

OCH3

H3 CO N

N R2

NH2

O

O R3

Aroylamide indoles

H3CO

NN H H

Aroylindole

Figure 12 Molecular structure of aroyl- and aroylamide-indoles

doi: 10.18282/amor.v1.i1.12

tation [106,107]. Dragmacidin is an inaccessible bis-indole alkaloid (Figure 13) isolated from a deep water marine sponge[108,109] collected from the southern Australian coast[110]. Dragmacidin was set up to hold two indole groups fixed by a piperazine ring system. Dragmacidin exhibited in vitro cytotoxicity with IC50 values of 15 µg/mL against P-388 cell lines and 1–10 µg/mL against A-549 (human lung), HCT-8 (human colon) and MDAMB (human mammary) cancer cell lines. In 1995, Murray et al.[110] reported the isolation of dragmacidin D (Figure 13). Dragmacidin D was found to be active against human lung tumour cell lines and inhibited in vitro growth of the P-388 murine and A-549 with IC50 values of 1.4 and 4.5 μg/mL, respectively[108,109]. Four new bis-indole alkaloids nortopsentins A–D (Figure 13) were extracted from the Caribbean deep sea sponge Spongosorites ruetzleri[110]. These derivatives of nortopsentins A–C exhibited anti-cancer activity against P-388 cells with IC50 values of 7.6, 7.8 and 1.7 respectively, and for Trimethylnortopeentin B derivative is 0.9 µg/mL.

27

Indoles as anti-cancer agents R2

R3 R1

NH

N

Br

R5

N R1 HN

R2

R1 HN

HN

N H H

HN

Topsentin R1=H, R2=OH Bromotopsentin R1 =Br, R2 =OH Deoxytopsentin R1 =R2=H

HO

N

NH

NH R1=R2 = Br R1=Br, R2= H R1=H, R2=Br R1=R2 = H

HO

OH

R2

Nortopsentins A, Nortopsentins B, Nortopsentins C, Nortopsentins D,

NH NH2

Dragmacidin D O

N

HN

HN

N

Dragmacidin

H H N

OH

N

R2 HN

O

H N

O

O

OH

NH

N Hyrtinadine A

N N H H

N N H H

Hyrtiosins B

Figure 13 Marine natural bisindole alkaloids as anti-cancer agents

Topsentin showed self-conscious proliferation of cultivated human and murine tumor cells. It exhibited in vitro anti-cancer activity against P-388 with IC50 value of 3 μg/mL, human tumor cell (HCT-8, A-549, T47D) with IC50 value of 20 μg/mL and in vivo activity against P-388 (test/control (TC) 137%, 150 mg/kg) and B16 melanoma (T/C 144%, 37.5 mg/kg). Bromotopsentin showed antiproliferative activity against human bronocopuemonary cancer cells (NSCLC-N6) with an IC50 = 6.3 μg/mL. Deoxytopsentin showed antiproliferative activity against human bronocopulmanary cancer cells (NSCLCN6) with an IC50 value of 6.3 μg/mL. It also displayed moderate activity against breast cancer and hepatoma (HepG2) with an IC50 of 10.7 and 3.3 μg/mL, respectively[111-113]. Recently, Kobayashi et al. reported a new cytotoxic bisindole alkaloid hyrtinadine A (Figure 13) from an Okinawan marine sponge Hyrtios sp[114-116]. Hyrtinadine reported to exhibit in vitro cytotoxicity against murine leukemia L-1210 and human epidermis carcinoma KB cells. Schupp et al. isolated a couple of new indolocarbazole alkaloids, staurosporines (Figure 14) from the marine ascidians Eudistoma toealensis and its predator[116,117]. Schupp et al. reported the prospects of these staurosporine derivatives as inhibitors of cell explosion and macromolecule synthesis[117]. Staurosporine D was found to be the main vigorous staurosporine candidate as a MONO-MAC-6 (human monocytic cell lines) inhibitor and inhibit the RNA and DNA synthesis. The IC50 values of staurosporine A, D and E for inhibiting MONOMAC-6 cells were 24.4, 13.3 and 33.9 mg/mL, respectively while those of staurosporine B and C were >100

28

µg/mL. The percentage of inhibition of RNA and DNA synthesis of compounds staurosporine A and D were 93 and >98, 98 and >98, respectively. Analysis of structure activity relationship verified that hydroxylation of staurosporine at position 3 of the indolocarbazole moiety causes an increase in antiproliferative activity. The position of the–OH group is critical to determine the antiproliferative property of a range of staurosporine analogues. A novel carbazole alkaloid, coproverdine (Figure 14) was isolated from a nameless ascidians, Anchorina sp. collected from the North Island of New Zealand[118]. Coproverdine was screened against a diversity of murine and human tumor cell lines such as P-388, A-549, HT-29, MEL-28 and DU-145 exhibiting IC50 values of 1.6, 0.3, 0.3, 0.3 and 0.3 µmol/L, respectively. The hyrtioerectine alkaloid A (Figure 15) was inaccessible from a red coloured marine sponge Hyrtios erectus[119,120]. Hyrtioerectine A was tested for its cytotoxic activity towards HeLa cells and showed sensible cytotoxicity with IC50 assessment of 10 µg/mL. Foderaro et al. published the isolation of a new tetrahydro-β-carboline alkaloid (Figure 15) bengacarboline from the Fijian ascidians Didemnum sp[121]. Bengacarboline was found to be cytotoxic against a 26 human tumor cell line panel in vitro with a mean IC50 value around 0.9 µg/mL and also inhibited the catalytic activity of topoisomerase II at 32 µmol/L. In 1994, Bifulco et al. reported the isolation of two tris-indole alkaloids, gelliusines A and B (Figure 15) from a deep water new caledonian sponge Gellius or Orina sp[122]. Gelliusin A and B were found to be diastereomeric alternatives prepared by the coupling of three indole units in which two 6-bromo tryptamine units

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El Sayed MT, et al.

are linked through their aliphatic chains to the C-2 and C-6 position of a middle serotonin moiety. The coupling of the indole unit appears to be non-stereoselective giving two enantiomeric pairs, having dissimilar comparaH N

O

tive configuration at C-8 and C-8 named (±) gelliusines A and B. Gelliusines A and B showed anti-cancer activity with an IC50 value of between 10 and 20 μg/mL against KB, P-388, P-388/dox, HT-29 and NSCLCN-6 cell lines. O

H N

O

R1

CHO N H H H H

N

OH

N

Me R3

O

HH R2

N

OH O

O

Coproverdine

O

Staurosporine A: R1=H, R2 =CH3, R3=OCH3, R4 =H Staurosporine B: R1=OH, R2 =CH3, R3=OH, R4 =H Staurosporine C: R1=H, R2 =H, R3=OCH3, R4 =OH Staurosporine D: R1=OH, R2 =CH3, R3=OCH3, R4=H Staurosporine E: R 1=H, R2 =CH3, R3=OH, R4 =H

N

N

H

Me

Staurosporine G

Figure 14 Chemical structures of marine natural products, staurosporines and coproverdine H2N

HO HO

O

O

H N

N

NH2 OH

HO

NH

N H

N H NH

N H Hyrtioerectine A

NH2

HN N H Br

H2N (±) Gelliusines

N H

Br

Bengacarboline

Figure 15 Molecular structures of Hyrtioerectine A, Bengacarboline and (±) Gelliusines

Dendridine A (Figure 16), a distinctive C2-symmetrical 4,4′-bis(7-hydroxy) indole alkaloid was isolated from an Okinawan marine sponge Hyrtios sp[123]. It exhibited reasonable cytotoxicity towards murine leukemia L-1210 cells with IC50 value of 32.5 µg/mL. Chetomin was acknowledged as a natural product anti-tumor candidate which prevents the configuration of the HIF-1,

N H

Dendridine A

N H Chetomin

Figure 16 Chemical structure of Dendridine A and Chetomin

doi: 10.18282/amor.v1.i1.12

P300 complex (Figure 16). Universal management of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth[124]. It has been established by Lee et al. that 1,1,3-tri (3-indolyl)cyclohexane (Figure 17) inhibits cancer cell expansion in lung cancer cells of xenograft models[125]. Consequently, it is a potential anti-cancer product derived from its strong tumor growth inhibition and positive pharmacologic properties. It also increases the manufacture of reactive oxygen species (ROS) and triggers DNA damage[126-128]. Cyclohepta [b] indole and benzo[6,7] cyclohepta [1,2-b] indole (Figure 17) were subsequently screened for cytotoxic activity against human nasopharyngeal carcinoma (HOME-1) and gastric adenocarcinoma (NUGC-3) cell lines, where the product showed imperative cytotoxicity at a concentration of 4 µg[126]. In 2014, it has been proved that an indole and its derivatives NC001-8 could be novel therapeutic agents for spinocerebellar ataxias (SCA17). The development of indole-

29

Indoles as anti-cancer agents O

HN

NH N H

N N H

N H

cyclohepta[b]indole NH benzo[6,7]cyclohepta[1,2-b]indole 3,3',3''-(cyclohexane-1,1,3-triyl)tris(1H-indole)

NC001-8

Figure 17 Chemical structure of some cycloalkano indoles have anti-cancer activity

the cytotoxic effects of ionizing radiation. Clinical applications of combining Au(I)-indoles with ionizing energy are discussed with developing a new strategy to achieve chemosensitization of cancer cells[129].

based compounds offers a promising strategy for the treatment of polyglutamine (polyQ) diseases via activation of haperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models[127]. Sandra et al.[129], reported the relevance of goldcontaining indoles as anti-cancer agents. It displayed cytostatic effects against leukemia and adherent cancer cell lines. However, two gold-bearing indoles showed unique behavior by increasing the cytotoxic property of clinically relevant levels of ionizing emission (Figure 18). Quantification of the amount of DNA demonstrates that each gold-indole enhances apoptosis by restraining DNA fixation. Both Au(I)-indoles were screened for inhibitory property towards a mixture of cellular targets counting thioredoxin reductase, an identified target of numerous gold compounds and a variety of ATP-dependent kinases. Both compounds showed inhibitory property against numerous kinases connected with the beginning of cancer and its progression. The inhibition of these kinases provides a probable mechanism for the capability of these Au(I)-indoles to potentiate

P

Au P

N O

Au N

O

O

O

Figure 18 Gold-containing indoles compounds

A progression series of novel 5-(2-Carboxyethenyl) indole derivatives were designed and synthesized (Figure 19). Two of the seven recently prepared compounds were screened for their anti-cancer activities towards K562 and HT-29 cell lines resulting in 5-(2-Carboxyethenyl)indole derivatives being verified for major anti-cancer activity against HT-29 cell, their effectiveness was around 4.67, 8.24 and 6.73 μmol/L[130]. R4

H3 COOC

R1 N

R1= H, CH3 , CF3

R1

5-(2-Carboxyethenyl) indole derivatives

CH3

N N H

R1 CH3

R1= H, F 2,3-Dimethylindole

R2 R3

N H

R1= H, F, CH3, OCH3 R2 = H, F, R3 = H,F R4 = H, CH3, Ph, PhCN Tetrahydrocarbazole Tetrahydrocarbazole

Figure 19 Simple indoles recently evaluated for anti-cancer activity

Kumar et al.[131] have synthesized 2,3-dimethylindoles and tetrahydrocarbazoles using Phenylhydrazine hydrochlorides and different cyclic and acyclic ketones in presence of antimony phosphate as catalyst (Figure 19). The products were tested for anti-cancer activity against five different cell lines such as kidney adenocarcinoma (ACHN), pancreas carcinoma (Panc1), lung car-

30

cinoma (GIII) (Calu1), non-small cell lung carcinoma (H460), colon cancer cell (HCT116) and normal breast epithelium (MCF10A) cell lines. The results showed that 2,3-dimethylindole (R1 = H, F) exhibited promising activity against both lung carcinoma and pancreas carcinoma cell line with IC50 value 2.7, 3.1, 2.8 and 3.2 nmol/L. Tetrahydrocarbazole (R1 = OCH3, R2 = F, R3 =

doi: 10.18282/amor.v1.i1.12

El Sayed MT, et al.

F, R4=PhCN) showed high activity against lung carcinoma cell line only, with IC50 2.5 nmol/L.

Conclusion The current review covered three important topics about indoles: Firstly, indoles as natural products and its biosynthesis. Secondly, the mechanisms of induction of cell death for numerous cancers cell lines by indoles. Thirdly, recent studies with indoles and associated compounds that are investigated for anti-cancer screening and that are directly forwarded for drug approval.

Conflict of interest The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

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CASE REPORT

Primary diffuse large B-cell lymphoma of the endometrium Yuqing Zou, Hongmin Zhao*, Jianhua Gao, Hong Zhu, Yong Li, Jianhua Zheng Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China

Abstract: This article was designed to analyze the prognosis and to explore the clinical characteristics and treatment of a case of primary diffuse large B-cell lymphoma (DLBCL) of the endometrium, through a detailed report. Methods: This report was done by analyzing a case through its clinical features, pathological examination and immunophenotyping. We believe that this would allow us to forecast the prognosis of the case. Case Description: The patient was admitted because of irregular postmenopausal vaginal bleeding for more than a month. She underwent a series of surgical procedures that included abdominal hysterectomy, bilateral adnexectomy, pelvic lymph node dissection and enterolysis owing to the assessed pathological results after uterine curettage. The postoperative pathological results showed that it was a DLBCL. Conclusion: Primary DLBCL of the endometrium is rarely reported. Its diagnosis and differential diagnosis mainly depends on pathological examination and immunophenotyping. Radical surgery combined with chemotherapy may be an ideal mode of treatment. The prognosis of the disease has a varied range but radical surgery combined with chemotherapy may improve her quality of life and prolong her survival period. Keywords: endometrial cancer; diffuse large B-cell lymphoma; non-Hodgkin lymphoma (NHL) Citation: Zou YQ, Zhao HM, Gao JH, et al. Primary diffuse large B-cell lymphoma of endometrium. Adv Mod Oncol Res 2015; 1(1): 36–40; http://dx.doi.org/10.18282/amor.v1.i1.33. *Correspondence to: Hongmin Zhao, Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China, [email protected]. Received: 3rd September 2015; Accepted: 18th September 2015; Published Online: 6th October 2015

D

iffuse large B-cell lymphoma (DLBCL) is a high-grade invasive lymphoma. DLBCL accounts of about 30% of all non-Hodgkin lymphoma (NHL) cases and is the most common subtype of NHL. NHL rarely presents itself in a female genital tract. Only 0.2%–1.1% of all extranodal cases occur in a female genital tract. Its main distribution being in the ovary and uterine neck[1]. Fox et al. defined the primary uterine malignant lymphoma as: 1) one whose site is examined to be in the ovary, the first time; 2) it is the only lymphoma that can be found through a general checkup; 3) its incidence shows no evidence of leukemia in the peripheral blood test; 4) if a secondary lymphoma occurs where the hysterectomy was performed, it should be a few months between the date of the initial discovery of the primary lymphoma and the appearance

of the secondary one[2].

Clinical data–general data Patient: Female, 72 years old. Chief complaint: Bleeding irregularly for more than a month, 20 years post-menopause. Personal history: Regular menstruation started from 15 years old; 5–6/30 days; age at menopause was 52 years old; not addicted to tobacco or alcohol. Past history: Suffering from diabetes for more than 20 years and treated with insulin; blood glucose has been controlled effectively (fasting blood glucose 6.5 mmol/L; after breakfast 8.0 mmol/L; before lunch 7.1 mmol/L; after lunch 11.6 mmol/L; before supper 6.8 mmol/L; after supper 7.2 mmol/L; before sleep 5.9 mmol/L); suffering from coronary disease and rheumatoid for more than 20 years. Pregnancy-labor history: Pregnant four

Copyright © 2015 Zou YQ, et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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times and gave birth four times. Family history: Parents deceased; sons and daughters are healthy; no family history of tumor. History of present illness: Irregular vaginal bleeding for a month before admission, small amount; bright red; no fever or stomach ache; examined with gynecological ultrasonography, indicated endometrial organic changes; curettage was performed; histopathology further revealed lymphoma; normal sleep, diet, bowel movement and urination without obvious weight changes during admission.

Materials and methods Physical examination No difference in general condition; free of spots; clear consciousness; stable life signs; heart and lungs are normal; flat abdomen; soft abdomen due to palpations; no tenderness or rebound pain. Gynecological examination: senile change of vulva; vagina patency and mild atrophy observed; anterior wall of vagina has one degree of expansion; fornix does not reach haphalgesia or nodules; humble vaginal mucosa; vagina is atrophic at cervical neck; flat fornix; cervical neck has one degree of erosion; bleeds when touched; uterus is anteversion and soft; uterus size is similar to that of one at 12 weeks of pregnancy; not active; no tenderness; bilateral accessory is normal.

Auxiliary examination Gynecological ultrasonography: Uterine empyema (endometrial neoplasm is to be excluded; exquisite dot and band echoes covering about 7 × 5 cm); small cysts in the left ovarian (3 × 3 cm) according to clinical features. Pathologic findings of curettage: (Intrauterine substances) DLBCL, immune phenotype of germinal center. Immunohistochemistry: CA125(−), CK(−), EMA(−), Vimentin (portion of +), CD3(−), CD20(+), Ki-67(+, 80%), MPO(−), ER(−), P53(+), CD79a(+), CEA(−), CK20(−), TTF-1(−), CK19(−), CK7(−), CD34(−), CD56(−), CD43(+), CD10(+), α-inhibin(−), CD99(+), TdT(−), CD117(−), PR(−), CD5(−), CD7(−), MUM-1(+), Bcl-6(+), Bcl-2(+), desmin(−), SMA(−).

file presents gray and yellow like fish meat, pliable but strong, about 8 × 7 × 3 cm. Chemoinjections were administered into the internal iliac artery during operation (30 mg/side).

Postoperative chemotherapy According to the result of the postoperative pathologic histology, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone were taken as R-CHOP chemotherapy step I one month after operation, the secondary reaction was so serious after chemotherapy that the patient was still in recovery two months after leaving the hospital.

Results PET/CT result of the whole body (Figure 1) 1.

The space occupying the lesion of the endometrium is accompanied by higher glycometabolism. It conforms to the image feature of malignant lesions. Thus, the primary lesion is taken into consideration. 2. The right basal ganglia shows softening focus, lacunar infarction. 3. The left sphenoid sinus is inflamed. 4. Higher glycometabolism was observed in the upper lobe of the left lung and the inferior lobe of the right lung. Optimum node is likely to be considered. 5. Higher glycometabolism was observed in the mediastinum and both hilus pulmonis lymphaden, thus inflammatory lymphonodus is likely to be considered. 6. Higher glycometabolism was observed in the chest, upside of the esophagus, inflammatory lesion or physiologic ingestion is taken into consideration. 7. Chronic cholecystitis. 8. Accessory spleen splenulus. 9. Calcification in both kidneys. 10. Cyst in the left accessory area. 11. Rarefaction of bone. 12. Degenerative change in several centrums.

Surgical method and findings

Histopathologic examination after operation—general findings

The patient underwent a series of surgical procedures that included abdominal hysterectomy, bilateral adnexectomy, pelvic lymph node dissection and enterolysis. Uterus was observed to be enlarged in the shape of a sphere during operation, the size is close to one in 12 weeks of pregnancy. 12 × 9 × 6 cm, uterus pro-

The uterus and both accessories: the whole uterus V: 12 × 9 × 5 cm, gray and yellow goiter in intracavity V: 8 × 7 × 2 cm, takes up the whole uterine cavity. Cervical length is 2 cm, ectocervix d: 6.5 cm, 2 × 2 × 1 cm solid tumor in endostoma. Length of left fallopian tube is 6.5 cm, d: 0.4 cm, ovarian bursa V: 3.5 × 3 × 2.5 cm, length

doi: 10.18282/amor.v1.i1.33

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Primary diffuse large B-cell lymphoma of the endometrium

of right fallopian tube is 5 cm, d: 0.3 cm, ovary V: 3 × 1.5 × 0.8 cm, by the way, lymph gland is included. Microscopic examination (Figure 1 and Figure 2): abnormally shaped, large cell diffuse infiltration and karyokinesis can be seen obviously.

Histopathology results Uterus: Diffuse large B-cell lymphoma, germinal center immunophenotyping, leiomyoma, cervix uteri chronic inflammation: (left ovary) simple cyst, no change in right accessory and left fallopian tube, (left and right groin lymph gland, both obturator lymph gland, right ilium overall lymph gland, both internal ilium lymph gland, both external ilium lymph gland) 0/1, 0/2, 0/4, 0/2, 0/1, 0/1, 0/1, 0/1 and 0/2 are to be found without any change. (Left ilium overall lymph gland) soft tissue is seen without any change. Immunohistochemistry: CD3 (−), CD20 (+), CD79a (+), PAX-5(+), Ki-67 (>50%, +), CD10 (+), BCL-6(+), MUM-1 (+), p53 (individual cell+), CD5 (−), BCL-2 (+).

Discussion

Figure 1 PET/CT results of the patient

Figure 2 Pathological section after surgery, large cell diffuse infiltration is visible

Figure 3 Similar to Figure 2, diffuse infiltration is seen among tumor cells in the same size, obvious atypia

38

Incidences of primary endometrial non-Hodgkin lymphoma are rare. There is little specificity of clinical manifestation. Therefore, it is difficult to clearly diagnose in the early phases, the diagnosis, however, mainly depends on the histopathologic examination and the immunohistochemistry phenotype. Diffuse large B-cell lymphoma always metastasizes in the early phase. Patients undergoing irregular treatment or no treatment at all, may survive for less than a year[3]. At present, there is no clear etiology for this kind of disease. There are, however, some factors that are taken into consideration these days: 1. There is no lymphoid tissue in genital organs, thus the lymphocytes in blood flow maybe the basis of the occurrence of endometrial non-Hodgkin lymphoma. 2. Chronic inflammation stimulates B-cell clone of NF-KB access mediated. 3. Endometrial non-Hodgkin lymphoma always manifests itself post-menopause, thus the morbidity may be related to female hormonal readiness. 4. Virus infection. 5. Immunodeficiency. 6. Occupational factors: long-term exposure to chemi[4] cal coloring agents, dyestuff, etc. Primary endometrial non-Hodgkin lymphomas are rare. During diagnosis, it should be distinguished from myoma of uterus canceration, endometrial proliferation, inflammatory pseudotumors, primitive neuroectodermal tumors, endometrium mesenchymal tumors, and other small cellular tumors[5]. Endometrial primary non-Hodgkin lymphoma has no specific characteristics in terms of clinical symptoms. It usually occurs among post-menopausal women, which

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Zou YQ, et al.

may be related to excessive endometrial proliferation that is caused by hormone level changes after menopause. The most common symptom is irregular post-menopausal vaginal bleeding or drainage. Endometrial primary non-Hodgkin lymphoma is diagnosed using information from the pathological and histological diagnosis of preoperative diagnostic curettage, but since this disease is rare, it sometimes fails to receive enough attention from doctors in the department of obstetrics and gynecology. A majority of patients are diagnosed according to the result of intraoperative and postoperative pathologic histology. Using the pathological histology report from this case and related literature reports, we can say that most primary non-Hodgkin lymphomas of the uterus belong to type B lymphocyte lymphomas, mostly diffuse large B-cell lymphoma tumors. Heterotypic large B-cell diffuse infiltration can be seen through a microscope, but only confined to the shallow endometrium. It can erode the endometrial glands and go far into the deep muscle layer. It grows diffusely in muscle tissue, characterized by pleomorphism. The types of form of the tumor can be divided into polyps, erosion and nodular types, generally in the shape of a fishes together with a small amount of hemorrhage and necrosis[6,7]. A lot of research and data show that p53, Ki-67, part of the CD family and expressions of other genes have changed in a variety of tumor which include endometrial carcinomas and lymphomas. Therefore, research of the related gene expression changes has important significance to predict diseases in the future. Tumor suppressor gene p53, encoding p53 protein, which are called “genome guardian”, were discovered by Linzer[8] in 1979, p53 can be divided into wild type and mutant type. Under normal circumstances, p53 usually exists in wild-type, maintaining a low level state within cells and is nonfunctional. When DNA suffers damage or other stimuli, p53 can be activated, and participates in regulating the cell cycle and promoting apoptosis. The p53 mutations lose normal biological function of tumor suppressing genes and p53 protein lose vitality for tumor suppressing. The p53 is a tumor suppressor gene and excessive expression of p53 means that the survival rate is low, studies show that excessive expression of p53 exists in most type II endometrial carcinomas. In this case, immunohistochemical prompts ER (−), PR (−) and p53 (+) are in conformity with type II endometrial cancer. Immunohistochemical, female and progesterone negative receptors suggest that hormone therapy does not promote good effects, high expression of p53 suggests easy relapse and low survival rate. Ki-67, namely the nuclei associated antigen, is associated with cell proliferation and reflects cell proliferation

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activity, studies have shown that Ki-67 is closely related with the development, transfer and prognosis of a variety of malignant tumors[9], the positive expression rate of Ki-67 can be used to determine the strength of the tumor. Ki-67 has a short half-life, it can reflect the activity of cell proliferation more accurately. High Ki-67 expression rate indicates a stronger degree of tumor malignancy. Ki-67 is in lower expression in normal endometrium tissues. Relevant literature data shows[10]: in normal endometrium tissues, endometrial hyperplasia tissues and endometrial polyps, the positive expression rate of Ki-67 showed a trend of increasing (12.00%, 37.33%, 17.33% and 82.00% respectively), these differences are statistically significant (p 50%), both Ki-67 showed high expressions, which means tumor cells proliferate actively. There is a high degree of tumor malignancy, they grow invasively, progress fast, and hence easily fall into relapse. The primary DLBCL of the endometrium was treated by radical surgery combined with postoperative R-CHOP chemotherapy, which enabled a lower relapse rate than treatment by means of radical surgery combined with radiotherapy[6]. The primary diffuse large B-cell lymphoma of the endometrium is a highly malignant tumor with poor prognosis; however, the quality of life of patients with lymphoma can be improved by using rituximab combined with chemotherapy. According to Jones, et al.[12], the complete remission rates of RCHOP21 and CHOP-21 are 76% and 63% respectively (p = 0.0005), and the 2-year survival rates are 70% and 57% respectively (p = 0.007); it can be concluded that the effect of the R-CHOP method is superior to that of the CHOP method, which is confirmed by five and 10 years follow-ups[13]. The prognosis is improved by radical surgery combined with R-CHOP chemotherapy and some patients’ survival period can be prolonged by this method, although some patients may still suffer a poor prognosis. The selected case underwent radical surgery and one course of R-CHOP chemotherapy which caused serious side effects, so she failed to undergo chemotherapy. According to the aforementioned information, the selected case suffered from a highly malignant tumor and the progression was fast. There was also a slight possibility of tumor recurrence. We conclude that if she could

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Primary diffuse large B-cell lymphoma of the endometrium

successfully undergo chemotherapy, the survival period post-surgery may be prolonged but further follow-up would be necessary.

5.

Conclusion

6.

To conclude, the primary DLBCL of the endometrium is rare and its diagnosis and differential diagnosis mainly depends on pathological examination and immunophenotyping. The prognosis is poor, but radical surgery combined with chemotherapy may improve the life quality of patients and prolong their survival period.

7.

8.

Conflicts of interest

9.

The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

10.

References 1.

2. 3.

4.

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Kosari F, Daneshbod Y, Parwaresch R, et al. Lymphomas of the female genital tract: a study of 186 cases and review of the literature. Am J Surg Pathol 2005; 29(11): 1512–1520. Fox H, More JR. Primary malignant lymphoma of the uterus. J Clin Pathol 1965; 18(6): 723–728. Fisher RI, Miller TP, O’Connor OA. Diffuse aggressive lymphoma. Hematology Am Soc Hematol Educ Program 2004; 2004(1): 221–236. Zou H, Fu J. Advances of primary lymphoma in the

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doi: 10.18282/amor.v1.i1.1

ORIGINAL RESEARCH ARTICLE

Role of immunoexpression of cyclin D1, D3, retinoblastoma (Rb) mutant and clinical risk factors on complete moles as risk factors of persistent moles Yudi M. Hidayat1*, Sofie R. Krisnadi1, Supriadi Gandamihardja1, Mieke H. Satari2, Bethy S. Hernowo3, Leri Septiani1, Ahmad Faried4 1

Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Padjadjaran (FK UNPAD)–Dr. Hasan Sadikin Hospital (RSHS), Bandung, Indonesia 2 Faculty of Dentistry, Universitas Padjadjaran, Bandung, Indonesia 3 Department of Pathology Anatomy, FK UNPAD–RSHS, Bandung, Indonesia 4 Oncology Working Group, FK UNPAD–RSHS, Bandung, Indonesia

Abstract: Introduction: Changes in complete hydatidiform mole (CHM) that become persistent are difficult to handle because the malignant pathogenesis of CHM is still unclear. The growth of abnormal cells in CHM is thought to be caused by cell cycle abnormalities. Some components that play a role in this phase include cyclin D and retinoblastoma (Rb). The aim of our study was to determine the role of clinical risk factors, as well as cyclin D1, cyclin D3 and Rb-protein, in the occurrence of persistent moles. Materials and Method: This study involves 68 CHM cases at Dr. Hasan Sadikin Hospital from 2007–2011. The protein expression of cyclin D1, cyclin D3, and Rb were determined by immunohistochemistry. The results were analyzed by comparing the two groups of CHM that became persistent to those that returned to normal, as determined by a Mochizuki regression curve assessment. Results: 20 cases (29%) of CHM became persistent and that 48 cases (71%) returned to normal. Significant clinical variables were age (p