Luis Barrera-Herrera, Maria de la Merced Torres Carvajal, Rafael Andrade, Rocio. Lopez ...... Louise Andrade, Fernando Soares, Dirce Carraro, Isabela Cunha.
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ANNUAL MEETING ABSTRACTS
Gastrointestinal Pathology 565 Unusual Immunohistochemistry Staining Patterns Encountered in Cancers Screened for Lynch Syndrome Rocky Adams, Katherine Geiersbach, Sheryl Tripp, Wade Samowitz. University of Utah Health Sciences Center, ARUP Institute for Clinical & Experimental Pathology, Salt Lake City, UT; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT. Background: Mismatch repair deficiency is associated with Lynch syndrome in a subset of colorectal and endometrial cancers, and universal screening is now recommended for these tumors. Immunohistochemistry for four mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) is the most frequently used screening method. Although interpretation is usually straightforward for these stains, unusual patterns are encountered occasionally. Unusual staining patterns can lead to uncertainty about the possible underlying gene defects. Design: Retrospective review of 3,564 cases of cancer tested by immunohistochemistry indicated that 727 (20%) were abnormal. The usual abnormal pattern was defined as loss of both MLH1 and PMS2, loss of PMS2 alone, loss of both MSH2 and MSH6, or loss of MSH6 alone in the entire tumor tested. Of the abnormal cases, a significant fraction (93, almost 13%) showed unusual staining patterns. Unusual cases were stratified into categories based on the staining pattern and histopathologic features. Results: The most common categories of unusual staining are indicated in Table 1. The patterns indicate different underlying mechanisms, both intrinsic to the biology of the tumor (genetic heterogeneity or multiple gene defects) and extrinsic to the tumor (chemoradiation and tissue preservation artifacts). Colorectal Cancer (n = 57)
Endometrial Cancer (n = 31)
Other Cancers (n = 5)
Total
True heterogeneity (partial staining of the tumor; absent staining in whole regions of the tumor with retained internal control staining).
22
20
2
44
Non-standard loss of staining indicating a defect in more than one gene.
12*
3
1
16
Unusual staining in the setting of prior chemoradiation.
5
0
0
5
Artifacts of tissue preservation and staining, preventing interpretation of mismatch repair status in the tumor.
12
6
2
20
Unknown / other mechanisms.
7
2
0
9
Staining pattern
(*) 1 case showed two unusual staining patterns.
Conclusions: Multiple mechanisms can contribute to unusual staining patterns. These unusual patterns are encountered infrequently overall but represent a significant proportion of abnormal cases (13% in our series). Better understanding of the mechanisms causing unusual immunohistochemistry staining patterns will enable improved classification of tumors with mismatch repair deficiencies. 566 Prognostic Implication of Optimized Detection of CMV By Immunohistochemistry in CMV PCR-Positive Inflammatory Bowel Disease Patients Kajsa Affolter, Jessica Johnson, John Valentine, Kathryn Peterson, Xinjian Chen. University School of Medicine, Salt Lake City, UT. Background: The significance of superimposed CMV infection in patients with IBD has been an issue of debate. Independent studies have reported a significantly higher incidence of colon CMV infection in patients with steroid-refractory IBD as compared to those with non-refractory disease, suggesting a contributory role of this virus in the disease process. Despite the relative high incidence, CMV was identified only in a small proportion (20 – 40%) of the steroid resistant patients. The reported lack of colon CMV infection in the majority of these patients raises the question whether CMV infection is truly not associated with the patients or the negative result is due to the low sensitivity of the examination. Design: To address this question, we identified 26 IBD patients whose intestinal biopsies or resection specimens were CMV PCR positive, and performed CMV IHC on all the tissue blocks that were initially found to be CMV negative per H&E. Results: Re-examination of 153 tissue blocks derived from 37 separate colonoscopy and colectomy specimens from 26 IBD patients (representing 28 separate clinical CMV infections) identified additional CMV inclusions in patients whom were diagnosed CMV negative by H&E or IHC performed only on selected blocks in the initial examination. However, despite the enhanced sensitivity, IHC on all the blocks only detected CMV in about half the patients. While the reason for the negative results in the remaining patients remains to be completely understood, it appears at least partially due to the small number of biopsies taken. In the procedures where 6 or more segments of the intestine were sampled, the CMV positive rate was as high as 83%, whereas in the patients who had only 1 – 5 areas biopsied, the positive rate was only 29% (p < 0.01). The majority of IHCpos patients underwent colectomy or died, contrasting most of the IHCneg patients who did well with conservative management, suggesting IHC positivity is a better predictor of prognosis than PCR positivity alone. Conclusions: Our results demonstrate that while performing IHC on all blocks increases the sensitivity, adequate sampling is essential for optimal CMV detection.
As identification of CMV by histology/IHC rather than PCR has clinical implications, our data support the notion that optimized IHC identification of CMV may serve as a gold standard for the diagnosis of CMV colitis. 567 Retrospective Study of Clinicopathologic Features and Patient Outcome of Primary Adenosquamous Carcinoma of the Esophagus Atin Agarwal, Dongfeng Tan, Susan Abraham, Melissa Taggart, Huamin Wang, Jeannelyn Estrella, Miao Zhang, Asif Rashid, Dipen Maru. Baylor College of Medicine, Houston, TX; University of Texas MD Anderson Cancer Center, Houston, TX. Background: Primary adenosquamous carcinoma (ASC) of the esophagus is an infrequent tumor with limited information on its biology and clinical behavior. Design: In a retrospective search of the institutional database (2001-2014) of primary esophageal ASC, we identified 73 patients whose pathology was reviewed by a GI Pathologist. Forty-three of 73 patients with ASC diagnosed on pretreatment biopsies were included in the study. Pretreatment clinical staging was confirmed by endoscopic ultrasound and/or CT/PET CT scans. The presence of Barrett’s esophagus was assessed either on esophagogastroduodenoscopy or pathology specimens. Adenosquamous carcinoma was diagnosed based on presence of separate or admixed glandular and squamous component identified on H&E with or without mucicarmine or immunohistochemistry for keratin 5/6, p63, p40 and CDX-2. All patients with advanced locoregional disease underwent chemoradiation with or without surgery. All stage IV patients underwent chemotherapy with or without palliative localized radiation. Esophagectomy specimens after preoperative chemoradiation were reviewed for presence of Barrett’s esophagus, amount of residual tumor and pathologic stage. Results: The patient population included 36 men and 7 women with an age range of 4988 years. Tumor location was mid or distal esophagus in 25 (58%) and gastroesophageal junction in 18 (42%). Barrett’s esophagus was found in 13 (30%) patients. Two thirds (n=29) of patients presented with stage IV (n=28) or locally advanced unresectable disease (n=1). Six patients with locoregional disease demonstrated disease progression after preoperative chemoradiation. Five patients with locoregional disease had extensive residual tumor in surgical resection specimens. Three patients with locoregional disease demonstrated radiologic response to preoperative chemoradiation and were not operated due to high risk of operative morbidity. Only 3 of 41 (7%) patients survived more than 3 years. Conclusions: Primary esophageal adenosquamous carcinoma has higher stage at presentation and poor response to preoperative chemoradiation. 568 Prevalence and Clinical Significance of Microsatellite Instability in Colorectal Cancers With Retained DNA Mismatch Repair Proteins By Immunohistochemistry Agoston Agoston, Lynette Sholl, Neal Lindeman, Jason Hornick, Amitabh Srivastava. Brigham & Women’s Hospital, Boston, MA. Background: DNA mismatch repair (MMR) protein immunohistochemistry (IHC) is now used more often than microsatellite instability (MSI) testing by PCR to screen colorectal carcinomas (CRC) for Lynch Syndrome (LS). The rate of discordance reported in the literature between these two screening tests varies widely. The aim of our study was to determine the discordance rate between MMR IHC and MSI in a large series of CRC and to analyze its clinical significance. Design: MMR IHC to screen for LS was performed in 2116 CRC between 1991-2014. MSI testing was also performed in 947/2116 cases, most commonly due to young age (30% of tested loci showed instability. Results: Mean age of the study group was 54 years and M:F ratio was 1:1.1. MMR IHC was intact in 839/947 (89%) patients in whom both MMR IHC and MSI testing was performed. Only 6/839 cases (0.7%) with intact MMR IHC were MSI-H, and represented 6% of the 106 MSI-H cases. Germline testing in two of these 6 patients (due to young age, 35 and 46yrs, respectively, and strong family history of CRC) confirmed LS due to MLH1 point mutations in both cases. The results of genetic testing were not available in the remaining four patients but one had a family history suggestive of LS. Conversely, 8/841 (0.95%) patients with either microsatellite stable (MSS) tumors (n=738) or tumors with low level MSI (MSI-L) (n=103) showed abnormal results by MMR IHC . 3/8 discordant cases were MSI-L and two of these three were LS patients (both MSH6 mutations) . The third MSI-L patient had loss of MLH1/PMS2 on IHC but no genetic testing was available and family history was not suggestive of LS. Of the 5/8 discordant MSS cases (3 with MSH2/MSH6 loss, 2 with only MSH6 loss), 3 were negative for LS by genetic testing, and 2 did not have genetic testing and did not have a family history suggestive of LS. Conclusions: Discordance between intact MMR IHC and MSI testing is rare (0.7%) in CRC, and there was no clear trend favoring either method. Performing MSI testing in patients with intact MMR IHC when they present at a young age (25% admixed normal colonic mucosa and serrated polyps with dysplasia were excluded. DNA methylation array was performed using the Illumina Infinium Human Methylation 450k beadchip (Illumina, San Diego, CA) which simultaneously profiles >485,000 CpG sites covering all CpG islands. Polyps were also analyzed for BRAF and KRAS mutation. We used principal component analysis to assess most prevalent sources of variability across the entire array of methylation values. The association between the top principal components and clinical and demographic variables was then examined using linear regression. Results: The methylation array analysis included 35 HPs (21 left; 14 right) and 42 SSA/Ps (20 left; 22 right). The majority of samples were positive for BRAF V600E mutation (80% HPs; 88% SSA/Ps tested). The principal component analysis showed that DNA methylation was most strongly related to smoking status, gender and polyp size. A locus specific analysis using methylation status as the dependent variable and morphology (SSA/P vs HP) as the predictor, with adjustment for age, gender, polyp size, and anatomic site, showed very few loci with low P values and none reached the Bonferroni threshold for a statistically significant difference. Hierarchical clustering analysis of the 50 loci with the lowest p values failed to show separate clustering by morphologic type. Conclusions: HPs and SSA/Ps do not cluster separately in methylation array analysis supporting the hypothesis that they arise through similar epigenetic pathways. Our findings provide a pathogenetic basis for developing clinical surveillance guidelines based on the number and size of serrated polyps rather than histological distinction between HP and SSA/P.
ANNUAL MEETING ABSTRACTS 575 Histopathologic Features of IBD in Patients With PSC Do Not Predict Need for Orthotopic Liver Transplantation John Aranake-Chrisinger, Ilke Nalbantoglu. Washington University, St. Louis, MO. Background: Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease of the biliary tree associated with inflammatory bowel disease (IBD) in 70-80% of patients (pts). Ulcerative colitis (UC) is most often associated with PSC, though a minority of pts have Crohn disease (CD). There is evidence that gut-liver crosstalk may contribute to the pathogenesis of PSC-IBD. It has been reported that more severe PSC is associated with lower IBD disease severity in PSC-IBD pts. These reports suggest that histopathologic findings in colonic and ileal specimens may be useful in predicting the risk of orthotopic liver transplant (OLT) in PSC-IBD pts. Design: The aim of this study was to determine if histopathologic features of IBD in PSC-IBD pts correlate with the need for OLT. Colonic biopsies (bx) and resection specimens from PSC-IBD pts between 1999 and 2013 were identified. Histologic and clinical features including disease severity and distribution, age, IBD duration at the time of bx, medical treatment, colectomy, and need for OLT were obtained. CD20, CD3 and IgG4 immunostains were performed, enumerated and averaged over 10 high power fields (400x). Results: 26 PSC-UC pts and 6 PSC-CD pts were identified. IBD duration, average age, and gender were not significantly different between PSC-UC and PSC-CD pts (p=0.3635, p=0.764, and p=0.4056, respectively). Follow-up duration was 4.8 years in PSC-CD pts and 8.2 years in PSC-UC pts (p=0.1317). Confirming previous studies, a high proportion of PSC-UC pts had ileal involvement (31%), right colon only involvement (29%), and pancolitis (67%). Rectal involvement (67%) was also less frequently seen compared to pts with UC without IBD. In PSC-UC pts, OLT was not associated with colectomy, medical treatment, disease severity or distribution, mucosal CD3, CD20, or IgG4 counts. But, PSC-UC pts that required OLT had more severe inflammation of the left colon compared to PSC-UC pts who did not require OLT (p=0.0568). And, no PSC-CD pts required OLT, compared to 10 of 26 pts (38.5%) with PSC-UC (p=0.06). Conclusions: None of the histopathologic variables examined showed a significant association with OLT, suggesting histopathologic features of IBD may not be independent predictors of the need for OLT in PSC-IBD pts. Still, the lack of OLT in PSC-CD pts and the increase in disease activity in the left colon of PSC-UC pts requiring OLT are interesting findings, although not statistically significant. Larger studies with longer follow-up are necessary to further characterize the link between OLT and IBD type as well as histologic features. 576 The 12-Gene Colon Cancer Assay: Experience With 12,776 Stage 2 Patients Helen Bailey, Anson Tharayanil, Ruixiao Lu, Joseph Anderson, Frederick Baehner, Amy Sing. Genomic Health, Inc., Redwood City, CA. Background: Genomic assays have the potential to provide information beyond the traditional methods used for assessing risk of recurrence. The 12-gene Colon Cancer Assay (Oncotype DX) is clinically validated and predicts recurrence risk after surgical resection in patients (pts) with stage 2 colon cancer (CC).1-3 We report the Genomic Health Clinical Laboratory experience with stage 2 CC since the product became commercially available. The assay incorporates the expression of 12 genes (7 cancer related [3 cell-cycle genes, 3 stromal genes, and the early response gene, GADD45B] and 5 reference genes) and gives a Recurrence Score result (a numeric score between 0 and 100) that is a quantitative estimate of the risk of recurrence based on individual tumor biology in MMR-proficient tumors. Design: 12,776 samples from stage 2 CC pts submitted 4/2010 to 8/2014 passed pathology review and RT-PCR quality measures. Descriptive statistics for the clinical characteristics of pts, Recurrence Score results and distributions were calculated. Low, intermediate, and high risk groups are defined as: SM), with muscularization of SM. Fibrosis is most prominent in SM, followed by SS, but only minimal in MU and negligible in MP. Neural hypertrophy is common in MP and SM. Adipocyte proliferation is also present in SM and MP but less prominent. Overall, the weight of histopathologic elements is in the order of chronic inflammation > muscle hyperplasia > active inflammation > fibrosis > neural hypertrophy. The volume expansion is most significant in muscular layer (MP > SM > SS > MU). Conclusions: In Crohn’s ‘fibrostenosis’, the most prominent histologic change secondary to inflammation is smooth muscle hyperplasia. Fibrosis is less significant. The predominant volume-expanding compartment is also muscular layer. The ‘inflammation-smooth muscle hyperplasia axis’ may be more important than fibrosis
in the development of Crohn’s stricture and in the consideration of novel therapeutic target. Our scoring scheme would be a useful tool for studying the prevalence of different histopathologic components. 603 Improving HER2 Assessment in Gastroesophageal Junction (GEJ) and Gastric Adenocarcinoma (GADC): Lessons From Concurrent Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) – Study of 117 Specimens Zongming Chen, Hong Yin, Jinhong Li, Shaobo Zhu, Fan Lin. Geisinger Medical Center, Danville, PA. Background: Since the ToGA trial demonstrated a survival benefit with trastuzumab therapy in GEJ or GADC patients with HER2 overexpression, assessment of HER2 status in these tumors has been routinely performed. Over 100 cases were tested, and useful lessons were learned to further improve test accuracy. Design: A total of 117 invasive GEJ and GADC specimens were tested for HER2 status by both IHC (4B5, Ventena) and FISH (Dako). Results were interpreted according to the modified criteria by Hofmann et al (Histopathology. 2008;52(7):797-805). Data from the two methods were compared (Table). Potential causes for discordance were analyzed in detail. Results: HER2-positive rates were 21% and 23% by IHC and FISH, respectively. Four of 69 IHC-negative cases were FISH positive. Upon retrospective review, consensus opinion reclassified 2 cases from 1+ to 2+. Thus, a false-negative rate was 2% (2/117). Three of 24 IHC 3+ cases were FISH negative, but a true false-positive error was a case of a signet ring cell carcinoma demonstrating reproducible cytoplasmic positivity mimicking a membranous staining pattern. The other 2 included a case with an IHCpositive focus not present on the FISH slide and a case with polysomy 17; both were regarded as true-positive events. Thus, the false-positive rate was 0.8% (1/117). The results are summarized in the Table. IHC Results Negative (0 and 1+)
FISH Amplified
Not amplified
Concordance Rate (%)
4 (1+ in 3; 0 in 1)
65
Equivocal (2+)
2
22
65/69 (94) N/A
Positive (3+)
21
3
21/24 (88)
Conclusions: Based on the current criteria, a false-negative result is the more frequent error in HER2 assessment. Performing FISH in cases with a borderline 2+ IHC result may improve result accuracy. Although false-positive results are rare, signet ring cell carcinoma with aberrant HER2 expression in cytoplasm may represent a potential pitfall. 604 “Indefinite for Dysplasia” in Barrett”s Esophagus: Active Inflammation and Abnormal Flow Cytometric DNA Content Are Significant Predictors of Early Detection of Dysplasia or Adenocarcinoma Won-Tak Choi, Mary Emond, Peter Rabinovitch, Melissa Upton, Maria Westerhoff. University of Washington Medical Center, Seattle, WA; University of Washington, Seattle, WA. Background: Dysplasia arising from Barrett’s esophagus (BE) precedes esophageal adenocarcinoma (EAC). However, some cases are difficult to diagnose as dysplastic, especially in the setting of inflammation, ulceration, or technical issues; these may be designated “indefinite for dysplasia (IND).” At present, histologic identification of dysplasia is the best marker of an increased risk of malignancy, although flow cytometric analysis of DNA content has been shown to be valuable for detecting patients at high risk for developing EAC. Few reports in the literature have specifically evaluated the natural history or outcome of IND. In fact, neither the American College of Gastroenterology nor American Gastroenterological Association specifies guidelines for the management of IND. Design: We analyzed a series of 96 IND patients initially diagnosed between 2005 and 2013 to determine the outcome of IND and to identify factors (including histologic features and flow cytometric data) to predict subsequent detection of dysplasia or EAC. Results: Our data show that 25% of IND cases were found to have low-grade dysplasia (LGD), high-grade dysplasia (HGD), or EAC within 1 year, with 37% and 47% detected within 2 and 3 years, respectively. The 1-, 2- and 3-year detection rates of HGD or EAC were 10%, 13%, and 20%, respectively. Active (neutrophilic) inflammation in the area of IND and abnormal DNA flow cytometric results were significant risk factors for detection of dysplasia or EAC (hazard ratio (HR) = 3.4, p = 0.0005; and HR = 5.7, p = 0.003, respectively). When active inflammation and DNA flow cytometric results were considered together, the resulting HR for the combined markers was 18.8 (p < 0.0001). The sensitivity and specificity of the combined markers for predicting detection within 3 years was 100% (95% confidence interval (CI) = [91%, 100%]) and 60% (95% CI = [31%, 83%]), respectively, resulting in 100% negative predictive value (95% CI = [61%, 100%]) and 89% positive predictive value (95% CI = [76%, 96%]). Conclusions: IND in BE is a diagnosis that may cause difficulty in planning further patient management. Our results show that in the context of IND, histology with the support of DNA flow cytometry can identify a subset of patients who may merit more frequent endoscopic surveillance for early detection of dysplasia or EAC.
154A 605 Outcome of “Indefinite for Dysplasia” in Inflammatory Bowel Disease: Correlation With Flow Cytometry and Other Risk Factors of Colorectal Cancer Won-Tak Choi, Peter Rabinovitch, Dongliang Wang, Maria Westerhoff. University of Washington Medical Center, Seattle, WA; SUNY Upstate Medical University, Syracuse, NY. Background: Dysplasia that develops in the background of inflammatory bowel disease (IBD) precedes colorectal cancer (CRC). The category of “indefinite for dysplasia (IND)” is used often in equivocal cases, but its clinical significance remains unclear. Additional biomarkers in the setting of IND are needed to better define risk of progression to high-grade dysplasia (HGD) or CRC, as well as to differentiate between non-dysplastic and truly dysplastic epithelium. Flow cytometric analysis of DNA content (aneuploidy) has shown some promise in stratifying patients into low or high risk for CRC, but there are few reports that specifically evaluate its potential in the setting of IND. Design: We analyzed a series of 84 IBD cases with IND diagnosed between 2003 and 2013 to determine the outcome of IND (mean follow-up of 28 months). Among the 84 IND cases, 53 patients had concurrent flow cytometric analysis of tissue taken from the same endoscopic location. Electronic medical records and our pathology information system were further reviewed to correlate outcomes with the following factors: type of lesion identified (flat vs. polypoid), primary sclerosing cholangitis (PSC), active inflammation, and flow cytometric data. Results: Thirteen percent of IND cases yielded low-grade dysplasia (LGD) after a mean follow-up of 28 months, whereas only 2% of IND cases developed advanced neoplasia (HGD or CRC). There was no statistical difference between ulcerative colitis and Crohn’s disease groups (p = 0.10 from log-rank test). The risk of LGD, HGD, or CRC was not significantly associated with the type of lesion (p = 0.94), PSC (p = 0.94), or active inflammation (p = 0.41) in our cohort of IND patients. However, the finding of aneuploidy at baseline IND was predictive of LGD and HGD (p = 0.037). Patients with IND and aneuploidy had 1-year, 2-year, and 3-year progression rates to neoplasia of 12%, 17%, and 30%, respectively. In contrast, IND patients with normal baseline flow cytometric results had 1-year, 2-year, and 3-year progression rates to neoplasia of 0%, 5%, and 5%, respectively. Conclusions: Flow cytometry may be a useful adjunct in determining patient surveillance and management in the setting of IBD with IND. IND patients with abnormal flow cytometry may warrant more intensive follow-up, but conversely, IND in the setting of normal flow cytometry may require less frequent surveillance colonoscopy. 606 MCM4 and MCM7, Significantly Correlated With Ki67 Expression and Prograssion in Esophageal Adenocarcinoma, and Precancerous Lesions Bonnie Choy, Amy Lalonde, Sofia Taboada, Tongtong Wu, Zhongren Zhou. University of Chicago, Chicago, IL; University of Rochester Medical Center, Rochester, NY. Background: Minichromosome maintenance (MCM) proteins play important roles in DNA replication. The deregulation of these proteins has been shown to contribute to tumorigenesis and cell proliferation. The present study investigated the correlation between MCM4, MCM7, and a conventional proliferative marker, Ki67, expression levels in esophageal carcinoma and precancerous lesions to determine the predictive value of these markers for the progression of esophageal diseases. Design: Esophageal tissue microarrays consisting of 82 squamous epithelium (SE), 60 columnar cell metaplasia (CM), 33 Barrett’s esophagus (BE), 38 low-grade dysplasia (LGD), 14 high-grade dysplasia (HGD), 108 esophageal adenocarcinoma (EAC), and 24 esophageal squamous cell carcinoma (ESCC) were immunohistochemically stained. Nuclear staining of each marker was considered positive. The percentage (0-100%) positively stained cells were recorded. The association of markers with clinicopathological charateristics was analyzed. A p-value of
