Pre lim ina ry C om m unication

Margherita Nannini*1, Annalisa Astolfi2, Paola Paterini2, Milena Urbini2, Donatella Santini3, Fausto Catena4, Valentina Indio5, Rita Casadio5, Antonio Daniele Pinna6, Guido Biasco1,2 & Maria A Pantaleo1,2

ro

of

1 Department of Hematology & Oncological Sciences ‘L&A Seràgnoli’, S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy 2 ’Giorgio Prodi’ Cancer Research Center, University of Bologna, Bologna, Italy 3 Pathology Unit, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 4 U.O. Chirurgia d’ Urgenza, AOU Parma, Italy 5 Biocomputing Group, Department of Biology, University of Bologna, Bologna, Italy 6 Transplant, General & Emergency Surgery Department, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy *Author for correspondence: Tel.: + 39 051 6364078 n Fax: + 39 051 6364037 n [email protected]

ut

ho

rP

KIT/PDGF receptor- a (PDGFRA) wild-type (WT) gastrointestinal stromal tumors (GIST) are characterized by an overexpression of IGF receptor 1 (IGF1R) at the mRNA and protein level. More recently, germline and somatic mutations in succinate dehydrogenase (SDH) subunits A, B and C have been identified in KIT/ PDGFRA WT sporadic GIST. Until now, the molecular basis of IGF1R overexpression in KIT/PDGFRA WT GIST has not been explained. In this brief report we investigate the status of the SDH complex at the genomic and protein level in relation to IGF1R expression at the mRNA and protein level in seven KIT/PDGFRA WT sporadic GIST patients. We found that IGF1R was upregulated in all patients harboring SDH mutation or displaying a SDH dysfunction, with respect to KIT/PDGFRA WT GIST without SDH mutations. The western blot analysis confirmed that all patients with an upregulation of IGF1R mRNA had detectable IGF1R protein expression. This report would suggest that IGF1R overexpression in KIT/PDGFRA WT GIST could be driven by the loss-of-function of the SDH mitochondrial complex.

A

Approximately 10–15% of gastrointestinal stromal tumors (GIST) occurring in adults do not harbor KIT or PDGF receptor-a (PDGFRA) mutations and are defined as KIT/PDGFRA wild-type (WT) [1] . It is well known that KIT/PDGFRA WT GIST differ from KIT/PDGFRA mutant GIST in clinical, pathological and biological features, thus they are considered a distinct subtype of GIST [2–7] . In particular, KIT/PDGFR A WT GIST, occurring both in adults and children, are characterized by an overexpression of IGF receptor 1 (IGF1R) at the mRNA and protein level without IGF1R genomic amplification, suggesting that other mechanisms may be involved [5–7] . Furthermore, germline and somatic mutations in succinate dehydrogenase (SDH) subunits A, B and C have recently been identified in KIT/PDGFR A WT sporadic GIST [8–10] . In addiction, irrespective of the presence of 10.2217/FON.12.170 © 2013 Future Medicine Ltd

SDH mutations, most KIT/PDGFR A WT GIST display a negative staining for SDHB that seems to reflect the loss-of-function of the mitochondrial complex [11–13] . Until now, the molecular basis of IGF1R overexpression in KIT/PDGFRA WT GIST with respect to KIT/PDGFRA mutant GIST has not yet been explained. It has been shown that SDH dysfunction may be linked to tumorigenesis by the induction of a pseudohypoxic pathway, involving hypoxia-inducible factor 2a (HIF2a), which exerts its proliferative effects by activating key receptor tyrosine kinases, including IGF1R [14–18] . Therefore, it would be reasonable to speculate that the overexpression of IGF1R occurs as a consequence of tumor pseudohypoxia/ hypoxia-inducible factor 2a activation induced by SDH dysfunction. We investigate the status of the SDH complex at the genomic and protein level in relation to IGF1R expression at the mRNA and protein level in seven KIT/PDGFRA WT sporadic GIST patients, with familiar or personal Future Oncol. (2013) 9(1), 1–6

Preliminary Communication

Future Oncology

Expression of IGF receptor 1 in KIT/ PDGF receptor-a wild-type gastrointestinal stromal tumors with succinate dehydrogenase complex dysfunction

Keywords gastrointestinal stromal tumors n GIST n KIT n PDGFRA n SDHA n SDHB n SDHC n SDHD n succinate dehydrogenase n wild-type

n

part of

ISSN 1479-6694

1

Nannini, Astolfi, Paterini et al.

history negative for additional tumors, especially GIST and paragangliomas. Patients & methods Patients & tumor samples

Patient’s main characteristics belonging to each subgroup are listed in Table 1. The analyses were conducted on fresh tissue specimens collected during surgery and were obtained from GIST primary lesions in six patients and from metastases in one patient. The specimens were immediately frozen in liquid nitrogen and stored until RNA and DNA extraction. Mutational analysis

ut

ho

rP

ro

SDHA gene exons (1–15), SDHB gene exons (1–8), SDHC exons (1–6) and SDHD gene exons (1–4) were sequenced on fresh frozen tumor specimens of KIT/PDGFRA WT GIST patients using the Sanger Sequencing method. DNA was extracted by the QIAmp DNA Mini kit (Qiagen, Milan, Italy) in accordance with manufacturer’s directions. Each exon was amplified with PCR amplification using specific primer pairs designed with Primer Express 3.0 Software (Applied Biosystems, Milan, Italy) to amplify exons, but not SDHA pseudogenes located on chromosomes 3 and 5. Then, PCR products were purified with the Qiaquick PCR purification kit (Qiagen) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems). Sanger sequencing was performed on an ABI 3730 Genetic Analyzer (Applied Biosystems).

1 h with gentle agitation at 4°C. Lysates were centrifuged at 13,000 × g for 15 min at 4°C and supernatants were stored at -80°C. Protein concentrations were determined with the BCA protein assay (Pierce Rockford, IL, USA). A total of 30 µg of protein was resolved on a 12% SDSPAGE gel and transferred onto polyvinylidene difluoride membranes. Nonspecific binding sites were blocked by incubation in blocking buffer (PBS containing 0.1% Tween-20 with 5% w/v BSA) for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary antibodies specific for the proteins of interest and for a-Actin as the housekeeping protein. Membranes were then washed and incubated with peroxidase conjugate secondary antibodies diluted in blocking buffer for 1 h at room temperature. Antigens were revealed using Enhanced Chemiluminescence Reaction (ECL Advance, Amersham Pharmacia Biotech, Les Ulis, France). The following antibodies were used: mouse monoclonal SDHA antibody (Abcam, Cambridge, UK); mouse monoclonal SDHB antibody (Abcam); and rabbit polyclonal IGF1 receptor-b antibody (Cell Signaling, MA, USA); rabbit polyclonal b-Actin antibody (Santa Cruz Biotechnology, CA, USA).

of


A

Protein extraction & western blot analysis

Fresh frozen tissues were disrupted in RIPA buffer (Sigma Aldrich, MO, USA), supplemented with protease inhibitors (1 mM PMSF, 10 µg/ ml Aprotinin, 10 µg/ml Leupeptin and 1 mM Orthovanadate Sodium salt) and lysed for

Quantitative PCR

Total RNA was extracted by RNeasy spin column method (Qiagen) following manufacturer’s instructions. Altogether, 1 µg total RNA was reverse transcribed to single-stranded cDNA using the Transcriptor first strand cDNA synthesis kit (Roche Diagnostics, Monza, Italy) with oligo-dT primers (2.5 µM). Gene-specific primers amplifying IGF1R and GAPDH were designed with Primer Express 3.0 Software (Applied Biosystems) and quantitative PCR was performed using FastStart Sybr Green (Roche Diagnostics) on the LightCycler 480 apparatus

Table 1. Patient’s characteristics. Patient Age Tumor (years)/ site sex

Risk Metastases at diagnosis stratification Subunit

1 2

26/F 29/M

Stomach Stomach

High High

3

38/F

Stomach

High

4

18/F

Stomach

Very low

5 6 7

63/F 58/M 50/M

High Ileum Duodenum Very low Intermediate Stomach

Lymph nodes; liver and lung A Lymph nodes; liver A A Lymph nodes; liver A A A Yes (sites not available) No No No

SDH-mutational status Exon

SNV

SAV

9 2 13 13 5 8

1151C>G 91C>T 1765C>T 1766G>A 511C>T 1046_1047delTG

S384* R31* R589W R589Q R171C L349R fs*11

Wild-type for SDHA, SDHB, SDHC and SDHD Wild-type for SDHA, SDHB, SDHC and SDHD Wild-type for SDHA, SDHB, SDHC and SDHD

SAV: Single amino acid substitution; SDH: Succinate dehydrogenase; SNV: Single nucleotide variation.

2

Future Oncol. (2013) 9(1)

future science group


IGF1R overexpression in KIT/PDGFRA WT GIST with SDH dysfunction

Discussion

of

ro

10

1

0.1

0.01 Pt 1

A

ut

ho

It is well known that KIT/PDGFRA WT GIST occurring both in adults and children display an overexpression of IGF1R at the mRNA and protein level [5–7] . This molecular peculiarity is also common in the precursor of interstitial cells of Cajal (ICCs), identified by Lorincz et al., and characterized by an IGF1R+, KITlow, CD44 +, CD34 + and Insr+ phenotype [19] . It is likely that the differentiation of precursor ICCs into mature ICCs is driven by IGF1, therefore the IGF1R pathway may play a key role in GIST pathogenesis and development, especially in the KIT/PDGFRA WT subtype [4] . However, the molecular basis of IGF1R overexpression in KIT/PDGFRA WT GIST has not been explained yet, since no gene amplification of IGF1R has been found by SNP array analysis [5–7] . Germline and somatic mutations in SDH subunits A, B and C have recently been identified in KIT/PDGFRA WT sporadic GIST [8–10] . Moreover, irrespective of the presence of SDH mutations, most KIT/PDGFR A WT GIST display a negative staining for SDHB that seems to reflect the loss-of-function of the mitochondrial complex [11–13] . In the present study a positive correlation between the SDH mutational status/dysfunction

100

rP

Results

At the mRNA level (Figure 1A) it was found that the expression of IGF1R was significantly higher in four out of seven KIT/PDGFRA WT GIST patients, and the quantitative analysis of protein level by western blot confirmed that all patients with a high expression of IGF1R mRNA had detectable IGF1R protein expression (Figure 1B) . All of the four patients displaying increased IGF1R expression showed inactivating nonsense or missense SDHA mutations in the coding sequence (Table 1) that led to a complete loss of SDHA and SDHB protein expression (western blot analysis, Figure 2 ). This result shows that only KIT/PDGFR A WT GIST with SDH complex inactivation are characterized by IGF1R overexpression with respect to either KIT/PDGFRA mutant or KIT/PDGFRA WT GIST without SDH mutations, and confirms that an association exists between IGF1R expression and SDH-dysfunction (p = 0.0286, Fisher’s exact test).

and IGF1R expression in seven KIT/PDGFRA WT GIST patients was found. In particular it was found that all patients with SDH complex dysfunction had an upregulation of IGF1R at the mRNA level with respect to SDH WT, KIT/ PDGFRA WT patients, and an overexpression at the protein level. Of note, among those patients, two have shown an overexpression of IGF1R at immunohistochemical (IHC) analysis, as previously described [7] . Unfortunately, it was not possible to perform the IHC analysis of all seven patients due to the lack of the formalinfixed paraffin-embedded blocks. The lack of this finding does not affect the final result of the study, although this analysis should be performed on a larger sample in the future. Despite the small sample size analyzed, by the fact that GIST is a rare disease means this short report may provide an explanation for the

IGF1R mRNA expression

(Roche Diagnostics). The DDCt method was used to quantify gene product levels relative to the GAPDH housekeeping gene.


Pt 2

Pt 1

Pt 3

Pt 2

Pt 3

Pt 4

Pt 4

Pt 5

Pt 5 Pt 6

Pt 6

Pt 7

Pt 7

IGF1Rβ β Actin

Figure 1. IGF receptor 1 expression in KIT/PDGF receptor-a wild-type gastrointestinal stromal tumor patients. (A) Quantitative RT-PCR analysis of gene expression. IGF1R expression was normalized to the GAPDH housekeeping gene. (B) Western blot evaluation of IGF1R in KIT/PDGFRA wild-type gastrointestinal stromal tumor patients compared with a-Actin. IGF1R: IGF receptor 1; Pt: Patient.

www.futuremedicine.com

3

Pt 1

Pt 2

Pt 3


Pt 4

Pt 5

Pt 6

Pt 7

SDHA

SDHB

Actin

Figure 2. Evaluation of succinate dehydrogenase A and B expression in KIT/ PDGF receptor-a wild-type gastrointestinal stromal tumor patients by western blot. Pts 1–4 show a remarkable inhibition of SDHA and a complete loss of SDHB protein. SDH: Succinate dehydrogenase; Pt: Patient.

A

ut

ho

rP

ro

increased IGF1R expression already observed in KIT/PDGFRA WT GIST. It could be supposed that it may be driven by the loss-of-function of the SDH complex that leads to an increase in succinate levels and a subsequent stabilization of HIF into cytosol, mimicking the status of pseudohypoxia [14] . This results in the nuclear transcription of several genes involved in tumorigenesis, including IGF1R, EGF receptor and c-myc [15–18] . Even though only SDHA mutations were found in this small sample, it is believed it is the dysfunction of the SDH complex and not the type of mutation found that correlates with IGF1R overexpression, as Chou and colleagues have recently shown [19] . Furthermore, the current findings suggest that the altered metabolic phenotype in tumors, known as the Warburg effect, may also be genetically determined in KIT/PDGFRA WT GIST, which could be considered a metabolic disorder as inherited paraganglioma/pheochormocytomas and renal cancer [21,22] . This introduces the possibility of studying all of the main genes related to the hypoxic pathway, such as the Von Hippen Lindau gene product (pVHL), and considering them as novel targets for KIT/PDGFRA WT GIST treatment, in particular for the subset of patients defined as ‘type 2’ or ‘pediatric-type GIST’ [10–12,23,24] . In fact, although data are scarce, these patients represent a very peculiar subtype of GIST who besides having in common several pathological and clinical features, such as the epithelioid pattern, the multifocal presentation, the female prevalence, the gastric primary tumor localization, and the indolent course of disease despite the presence of lymph nodes and liver metastases up-front, it seems that they showed a more prolonged response to

sunitinib in comparison with imatinib. These further data suggest that the impairment of the hypoxic pathway, driven by SDH mutations or by other genes involved, may play a key role in tumor biology. [10,25] . Finally, consistent with data recently published by Chou et al., increased IGF1R expression may not be a feature of KIT/PDGFRA WT GIST or pediatric GIST with respect to mutant GIST per se, but rather a feature of the specific subgroup of WT GIST defined as SDH deficient, suggesting that IGF1R inhibition may therefore represent a rational therapeutic target for this small subgroup of GIST patients, which account for between 5.0 and 7.5% of all GIST in adults [20] . Of note, IGF1R expression has not yet been evaluated in KIT/PDGFRA WT GIST associated to Carney-Stratakis Syndrome and Carney Syndrome, which are very similar both in clinical presentation and pathological features to SDH-deficient sporadic GIST [26–29] . In particular, GIST also associated to CarneyStratakis Syndrome and Carney Syndrome belong to a SDH-deficient subgroup, as they are typically characterized by a loss of expression of SDHB at IHC [29] . Therefore, the evaluation of IGF1R status and its correlation with SDH dysfunction could be extended to this subgroup of GIST.

of


4


Conclusion & future perspective

This brief report would suggest that IGF1R overexpression in KIT/PDGFRA WT GIST with respect to KIT/PDGFRA mutant GIST could be driven by the loss-of-function of the SDH mitochondrial complex. This preliminary data should be confirmed in large series and evaluated in in vitro models in order to understand what leads to this status of pseudohypoxia that seems to be necessary to maintain this functional relationship. Furthermore, large series are required for evaluating the potential clinical impact of these molecular findings and improving the clinical management of KIT/ PDGFRA WT GIST. Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. future science group

IGF1R overexpression in KIT/PDGFRA WT GIST with SDH dysfunction


Executive summary Background KIT/PDGF receptor-a (PDGFRA) wild-type (WT) gastrointestinal stromal tumors (GIST) differ from KIT/PDGFRA mutant GIST in clinical, pathological and biological features, thus they are considered a distinct subtype of GIST. KIT/PDGFRA WT GIST occurring both in adults and children are characterized by an overexpression of IGF receptor 1 (IGF1R) at the mRNA and protein level with respect to KIT/PDGFRA mutant GIST, without IGF1R genomic amplification, suggesting that other mechanisms may be involved. Germline and somatic mutations in succinate dehydrogenase (SDH) subunits A, B and C have recently been identified in KIT/PDGFRA WT sporadic GIST. Irrespective of the presence of SDH mutations, most KIT/PDGFRA WT GIST display a negative staining for SDHB that seems to reflect the loss-of-function of the mitochondrial complex.

Results All patients with SDH complex dysfunction had an upregulation of IGF1R at the mRNA level and an overexpression at the protein level with respect to KIT/PDGFRA WT, SDH WT GIST.

of

Conclusions Increased IGF1R expression in KIT/PDGFRA WT GIST compared with KIT/PDGFRA-mutant GIST may be driven by the loss-of-function of the SDH complex. IGF1R overexpression may be not a feature of KIT/PDGFRA WT GIST or pediatric GIST per se with respect to mutant GIST, but rather a feature of the specific subgroup of WT GIST defined as SDH-deficient GIST.

ro

Papers of special note have been highlighted as: nn of considerable interest

3.

4.

5.

6.

7.

nn

Janeway KA, Kim SY, Lodish M et al. Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations. Proc. Natl Acad. Sci. USA 108, 314–318 (2011). This is the first finding of succinate dehydrogenase (SDH) B and SDHC mutations in sporadic KIT/PDGF receptor-a wild-type GIST.

ho

2.

This paper describes in detail the biology of gastrointestinal stromal tumors (GIST).

8.

Astolfi A, Nannini M, Pantaleo MA et al. A molecular portrait of gastrointestinal stromal tumors: an integrative analysis of gene expression profiling and high-resolution genomic copy number. Lab. Invest. 90, 1285–1294 (2010).

9.

ut

nn

Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J. Clin. Oncol. 15(22), 3813–3825 (2004).

Belinsky MG, Skorobogatko YV, Rink L et al. High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors. Genes Chromosomes Cancer 48, 886–896 (2009).

A

1.

rP

expression in wild-type GISTs: a potential novel therapeutic target. Int. J. Cancer 125, 2991–2994 (2009).

References

Pantaleo MA, Astolfi A, Nannini M et al. Differential expression of neural markers in KIT and PDGFRA wild-type gastrointestinal stromal tumours. Histopathology 59, 1071–1080 (2011). Janeway KA, Zhu MJ, Barretina J, PerezAtayde A, Demetri GD, Fletcher JA. Strong expression of IGF1R in pediatric gastrointestinal stromal tumors without IGF1R genomic amplification. Int. J. Cancer 127, 2718–2722 (2010). Tarn C, Rink L, Merkel E et al. Insulin-like growth factor 1 receptor is a potential therapeutic target for gastrointestinal stromal tumors. Proc. Natl Acad. Sci. USA 105, 8387–8392 (2008). Pantaleo MA, Astolfi A, Di Battista M et al. Insulin-like growth factor 1 receptor


nn

Pantaleo MA, Astolfi A, Indio V et al. SDHA loss-of-function mutations in KIT-PDGFRA wild-type gastrointestinal stromal tumors identified by massively parallel sequencing. J. Natl Cancer Inst. 103, 983–987 (2011). This is the first finding of SDHA mutations in sporadic KIT/PDGF receptor-a wildtype GIST.

10. Pantaleo MA, Nannini M, Astolfi A,

Biasco G, GIST Study Group Bologna. A distinct pediatric-type gastrointestinal stromal tumor in adults: potential role of succinate dehydrogenase subunit A mutations. Am. J. Surg. Pathol. 35, 1750–1752 (2011). 11. Miettinen M, Wang ZF, Sarlomo-Rikala M,

Osuch C, Rutkowski P, Lasota J. Succinate dehydrogenase-deficient GISTs: a clinicopathologic, immunohistochemical, and molecular genetic study of 66 gastric GISTs with predilection to young age. Am. J. Surg. Pathol. 35, 1712–1721 (2011). 12. Gill AJ, Chou A, Vilain R et al.

Immunohistochemistry for SDHB divides gastrointestinal stromal tumors (GISTs) into 2 distinct types. Am. J. Surg. Pathol. 34, 636–644 (2010).

www.futuremedicine.com

13. Gill AJ, Chou A, Vilain RE, Clifton-Bligh RJ.

‘Pediatric-type’ gastrointestinal stromal tumors are SDHB negative (‘type 2’) GISTs. Am. J. Surg. Pathol. 35, 1245–1247 (2011). 14. Harris AL. Hypoxia – a key regulatory factor

in tumour growth. Nat. Rev. Cancer 2(1), 38–47 (2002). 15. Selak MA, Armour SM, MacKenzie ED et al.

Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-a prolyl hydroxylase. Cancer Cell 7, 77–85 (2005). 16. Franovic A, Holterman CE, Payette J, Lee S.

Human cancers converge at the HIF-2a oncogenic axis. Proc. Natl Acad. Sci. USA 106, 21306–21311 (2009). 17. Franovic A, Gunaratnam L, Smith K,

Robert I, Patten D, Lee S. Translational upregulation of the EGFR by tumor hypoxia provides a nonmutational explanation for its overexpression in human cancer. Proc. Natl Acad. Sci. USA 104, 13092–13097 (2007). 18. Gordan JD, Bertout JA, Hu CJ, Diehl JA,

Simon MC. HIF-2a promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity. Cancer Cell 11, 335–347 (2007). 19. Lorincz A, Redelman D, Horváth VJ,

Bardsley MR, Chen H, Ordög T. Progenitors of interstitial cells of cajal in the postnatal murine stomach. Gastroenterology 134, 1083–1093 (2008). 20. Chou A, Chen J, Clarkson A et al. Succinate

dehydrogenase-deficient GISTs are characterized by IGF1R overexpression. Mod. Pathol. 25(9), 1307–1313 (2012). 21. Favier J, Brière JJ, Strompf L et al. Hereditary

paraganglioma/pheochromocytoma and inherited succinate dehydrogenase deficiency.

5

Preliminary Communication Horm. Res. 63, 171–179 (2005).


25. Carney JA, Sheps SG, Go VL et al. The triad

22. Favier J, Brière JJ, Burnichon N et al. The

Warburg effect is genetically determined in inherited pheochromocytomas. PLoS One 4(9), e7094 (2009). 23. Linehan WM, Srinivasan R, Schmidt LS. The

genetic basis of kidney cancer: a metabolic disease. Nat. Rev. Urol. 7, 277–285 (2010). 24. Rege TA, Wagner AJ, Corless CL,

26. Carney JA, Stratakis CA. Familial

paraganglioma and gastric stromal sarcoma: a new syndrome distinct from the Carney triad. Am. J. Med. Genet. 108, 132–139 (2002). 27. Stratakis CA, Carney JA. The triad of

parangangliomas, gastric stromal tumors and pulmonary chondromas (Carney triad), and the dyad of parangangliomas and gastric striomal sarcomas (Carney-Stratakis

syndrome): a molecular genetics and clinical implications. J. Intern. Med. 266, 43–52 (2009). 28. Zhang L, Smyrk TC, Young WF Jr et al.

Gastric stromal tumors in Carney triad are different clinically, pathologically, and behaviorally from sporadic gastric gastrointestinal stromal tumors: findings in 104 cases. Am. J. Surg. Pathol. 34, 53–64 (2010). 29. Gaal J, Stratakis CA, Carney JA et al. SDHB

immunohistochemistry: a useful tool in the diagnosis of carney-stratakis and carney triad gastrointestinal stromal tumors. Mod. Pathol. 24, 147–151 (2011).

A

ut

ho

rP

ro

of

Heinrich MC, Hornick JL. ‘Pediatric-type’ gastrointestinal stromal tumors in adults: distinctive histology predicts genotype and clinical behavior. Am. J. Surg. Pathol. 35, 495–504 (2011).

of gastric leiomyosarcoma, functioning extraadrenal paraganglioma and pulmonary chondroma. N. Engl. J. Med. 296, 1517–1518 (1977).

6

