SEARCH

SEARCH BY CITATION

Dear Sir,

Endocrine tumors include thyroid tumors, adreno cortical tumors, neuroendocrine tumors (NET) of the respiratory tract and gastrointestinal system, parathyroid tumors, pituitary tumors and paragangliomas.1 Although several genes involved in hereditary endocrine neoplasias have been identified, the mechanisms involved in tumorigenesis of sporadic NET remain largely unknown.2–5 This is due to several limitations, including the variability in anatomic accessibility and the paucity of both human functional cell lines and relevant animal models.6 Oncogene mutations commonly encountered in non-endocrine tumors are rarely found in these tumors. However, growth factor receptor expression and activation together with somatostatin receptors are frequently involved in the development of NET.4, 7 Thus, the identification of novel genetic mutations can be helpful for tumor diagnosis and classification, such as RET testing in MEN2, and may allow a better disease management.5

Moreover, conventional cytotoxic chemotherapies are often poorly effective in the treatment of metastatic NET patients.8 This observation supports the development of drug-targeted therapies, as exemplified by RET inhibitors in medullary and papillary thyroid carcinomas.9, 10 Indeed, attention is given to a range of genetic alterations in different signaling pathways, which constitute potential targets for drug-designed inhibitors.10

Among these alterations, activating mutations in the serine/threonine kinase BRAF gene have been described in a broad range of human cancers, including malignant melanomas, papillary thyroid carcinomas, ovarian and colon carcinomas.11 Activating mutations of epidermal growth factor receptor (EGFR) have been found predictive of the efficacy of new compounds in lung adenocarcinomas.12 The phosphatidylinositol 3′-kinase (PI3K) pathway plays a major role in oncogenesis and somatic mutations within its PI3K catalytic subunit, PIK3ca, are frequently detected in solid tumors,13 including as recently reported in poorly or undifferentiated thyroid carcinomas.14 Recently, dominant gain of function mutation V617F in the JAK2 gene pseudokinase domain (JH2) was identified in patients with myeloproliferative disorders.15

Therefore, we decided to screen well-characterized mutations of EGFR, BRAF, PI3KCA and JAK2 genes in a large series of thyroid carcinomas and NET samples (n = 160) obtained from the Center for Biological Resources at Institut Gustave-Roussy. They included NET from the pancreas (n = 11), small intestine (n = 8), medullary thyroid carcinomas (n = 25), paragangliomas (n = 6), parathyroid tumors (n = 4), pituitary adenomas (n = 25), metastasis from unknown NET primitive tumor (n = 2), differentiated thyroid tumors—hypofunctioning adenomas (n = 13), papillary carcinomas (n = 42), follicular carcinomas (n = 6)—, adreno cortical carcinomas (n = 18) and corresponding tissues without any sign of malignancy (n = 10) (Table I).

Table I. Histotypes and Genes Mutations in Neuroendocrine and Thyroid Tumors(%)
Tumor TypenBRAF exon 15JAK 2 exon 12PIK3CA exons 9 and 20EGFR exons 18, 19 and 21
Pancreas111/11 (9)0/110/110/11
Mesenteric10/10/10/10/1
Small intestine30/30/30/30/3
Ileum40/40/40/40/4
Metastasis with unknown primary site21/2 (50)0/20/20/2
Adreno cortical carcinomas180/180/180/180/18
Paragangliomas60/60/60/20/6
Parathyroid tumors40/40/40/40/4
Medullary thyroid carcinomas250/250/250/250/18
Follicular benign adenomas130/130/130/13nt
Follicular thyroid carcinomas60/60/60/6nt
Papillary thyroid carcinomas4222/42 (52)0/420/42nt
Pituitary adenomas250/250/250/250/25
Non tumoral tissues100/100/100/100/10

Genomic DNA was isolated from tissue samples using TriReagent® (Sigma-Aldrich, St. Louis, MO) after histological control by an expert pathologist. DNA samples were screened for the V600E BRAF exon 15 mutation, the V617F JAK2 exon 12 mutation; for PI3KCA gene mutations in exon 9 (codons 539, 542, 545 and 546) and exon 20 (codons 1008, 1025, 1043, 1047 and 1049),13, 16, 17 for EGFR exons 18 to 24 coding for tyrosine kinase domain18 and for RET exons 8, 10, 11, 13, 14, 15 and 16 mutations. Direct sequencing was performed, after PCR amplification of each exon, using the Big Dye Terminator sequencing kit (Applied Biosystems, Foster City, CA). The products were analyzed on an automated 3730 DNA Analyzer (Applied Biosystems). Sequence reading and alignment were performed with the SeqScape® software (Applied Biosystems).

Fourteen of the 25 medullary thyroid carcinomas were hereditary cases bearing germinal RET mutation at codons 611, 618, 634, 790 or 918. Furthermore, 3 of the 11 sporadic tumors presented a somatic RET mutation at codon 918.

No V600E BRAF mutation was detected in hypofunctioning follicular adenomas, follicular carcinomas and non-tumoral contralateral thyroid tissues. In contrast, 22 of 42 (52%) tissues from papillary thyroid carcinomas were mutated. The V600E BRAF mutation was also observed in two tissue samples from gastroenteropancreatic (GEP) tumors: the first corresponded to a 31-year-old male patient presenting a 45-mm well-differentiated pancreatic tumor, with lymph node invasion but without evidence of distant metastasis, and secreting chromogranin A and α-fetoprotein. The second mutated sample corresponded to a 47-year-old male patient presenting a poorly differentiated neuroendocrine lymph node metastasis in the neck from an unknown primary cancer. No mutation was detected in pituitary adenomas, in line with recent findings demonstrating that, in these tumors, molecular alterations mainly affect BRAF mRNA and protein expressions.19 Investigation of BRAF gene expression in samples of thyroid carcinoma (n = 10) and NET (n = 11) in comparison to that in their normal counterpart did not shown any changes in the expression level (data not shown). None of other NET samples presented any mutation within the analyzed BRAF sequences.

No mutation in the PI3KCA exons 9 and 20, EGFR exons 18, 19 and 21, and JAK2 exon 12 was observed in NET and thyroid tumors. Two papillary carcinomas showed synonymous polymorphisms in PI3KCA exon 20 at codon 1025, without any change at the amino acid sequence.

The V600E BRAF mutation constitutes a major somatic genetic event in papillary thyroid carcinomas and our results are in agreement with this observation.16 We also confirm that this mutation is rare in NET of the digestive tract and is absent in other thyroid tumors of follicular cell origin, in medullary thyroid carcinoma, parathyroid tumors, pituitary adenomas, paragangliomas and adreno cortical carcinomas.20–22

EGFR and phosphorylated-EGFR expressions appeared to be increased in GEP tumors and are related to poor survival.23 Mutations in the EGFR kinase domain are uncommon in primary and metastatic GEP tumors, in line with the modest efficacy of EGFR inhibitors in these tumors.24 Our results are in agreement with these observations in GEP tumors and also demonstrate that EGFR mutations are infrequent or absent in medullary thyroid carcinoma, in parathyroid tumors, in pituitary adenomas, in paragangliomas and in adreno cortical carcinomas. We did not include poorly or undifferentiated thyroid cancers in which EGFR overexpression has been reported.25

PI3KCA is one of the most commonly mutated oncogenes in human cancers,13 including anaplastic thyroid carcinomas, indicating that this pathway could be a major therapeutic target in thyroid cancers.26, 27 Our results show that PI3KCA mutations are uncommon in differentiated thyroid tumors and in NET. Critical changes in this pathway may also occur through the constitutive activation of stimulatory molecules (e.g., Ras) and/or the loss of function of the inhibitory PTEN protein. However, to date, no mutations in RAS and PTEN have been identified in NET.3, 28 The Jak/Stat pathway, involved in cytokine receptor signaling, is also involved in neoplastic phenotype. Particularly JAK2 pseudokinase domain mutation is responsible for myeloproliferative disorders.15 Interestingly, JAK2 also plays a critical role in the signaling of prolactin (PRL) hormone and the PRL signaling may be involved in the development of medullary thyroid carcinoma.29, 30 Furthermore, in this tumor, RET activates a Src/JAK/STAT3 pathway, independently of its ligand.31 Our results appear to exclude mutations in the JAK2 pseudokinase domain as responsible for the oncogenic development of NET, particularly of sporadic medullary thyroid carcinoma without RET mutations. This study provides one of the first evidence that JAK2, EGFR and PI3KCA hot spot mutations are uncommon in endocrine tumors and that the BRAF-V600E mutation is also uncommon in endocrine tumors other than thyroid papillary carcinomas. Further studies are warranted to find molecular abnormalities in these tumors that can be targeted by new therapeutic agents.

Yours sincerely,

References

  1. Top of page
  • 1
    DeLellisRA,LlyodRV,HeitzPU,EngC, eds. Tumours of endocrine organs. World Health Organization Classification of Tumours. Lyon: Pathology & Genetics IARC Press, 2004. 320.
  • 2
    Leotlela PD,Jauch A,Holtgreve-Grez H,Thakker RV. Genetics of neuroendocrine and carcinoid tumours. Endocr Relat Cancer 2003; 10: 43750.
  • 3
    Zikusoka MN,Kidd M,Eick G,Latich I,Modlin IM. The molecular genetics of gastroenteropancreatic neuroendocrine tumors. Cancer 2005; 104: 2292309.
  • 4
    Oberg K. Neuroendocrine tumors of the gastrointestinal tract: recent advances in molecular genetics, diagnosis, and treatment. Curr Opin Oncol 2005; 17: 38691.
  • 5
    Weber F,Eng C. Update on the molecular diagnosis of endocrine tumors: toward-omics-based personalized healthcare? J Clin Endocr Metab 2008; 93: 1097104.
  • 6
    Syder AJ,Karam SM,Mills JC,Ippolito JE,Ansari HR,Farook V,Gordon JI. A transgenic mouse model of metastatic carcinoma involving transdifferentiation of a gastric epithelial lineage progenitor to a neuroendocrine phenotype. Proc Natl Acad Sci USA 2004; 101: 44716.
  • 7
    De Herder WW,Hofland LJ,van der Lely AJ,Lamberts SW. Somatostatin receptors in gastro-entero-pancreatic neuroendocrine tumours. Endocr Relat Cancer 2003; 10: 4518.
  • 8
    Baudin E. Gastroenteropancreatic endocrine tumors: clinical characterization before therapy. Nat Clin Pract Endocrinol Metab 2007; 3: 22839.
  • 9
    Yao JC,Hoff PM. Molecular targeted therapy for neuroendocrine tumors. Hematol Oncol Clin North Am 2007; 21: 57581.
  • 10
    De Groot JW,Links TP,Plukker JT,Lips CJ,Hofstra RM. RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors. Endocr Rev 2006; 27: 53560.
  • 11
    Garnett MJ,Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell 2004; 6: 31319.
  • 12
    Heymach JV,Nilsson M,Blumenschein G,Papadimitrakopoulou V,Herbst R. Epidermal growth factor receptor inhibitors in development for the treatment of nonsmall cell lung cancer. Clin Cancer Res 2006; 12: 4441s4445s.
  • 13
    Samuels Y,Wang Z,Bardelli A,Silliman N,Ptak J,Szabo S,Yan H,Gazdar A,Powell SM,Riggins GJ,Willson JK,Markowitz S, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004; 304: 554.
  • 14
    Satrapie L,El-Naggar AK,Cote GJ,Myers JN,Sherman SI. Phosphatidylinositol 3-kinase/akt and ras/raf-mitogen-activated protein kinase pathway mutations in anaplastic thyroid cancer. J Clin Endocrinol Metab 2008; 93: 27884.
  • 15
    James C,Ugo V,Casadevall N,Constantinescu SN,Vainchenker W. A JAK2 mutation in myeloproliferative disorders: pathogenesis and therapeutic and scientific prospects. Trends Mol Med 2005; 11: 54654.
  • 16
    Nikiforova MN,Kimura ET,Gandhi M,Biddinger PW,Knauf JA,Basolo F,Zhu Z,Giannini R,Salvatore G,Fusco A,Santoro M,Fagin JA et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 2003; 88: 5399404.
  • 17
    Motté N,Saulnier P,Le Couedic JP,Soria JC,Delaloge S,Boige V,Pautier P,Vainchenker W,Bidart JM,Villeval JL. Mutations in JAK2V617F homologous domain of JAK genes are uncommon in solid tumors. Int J Cancer 2007; 121: 211315.
  • 18
    Lacroix L,Pautier P,Duvillard P,Motté N,Saulnier P,Bidart JM,Soria JC. Response of ovarian carcinomas to gefitinib-carboplatin-paclitaxel combination is not associated with EGFR kinase domain somatic mutations. Int J Cancer 2006; 118: 10689.
  • 19
    Ewing I,Pedder-Smith S,Franchi G,Ruscica M,Emery M,Vax V,Garcia E,Czirják S,Hanzély Z,Kola B,Korbonits M,Grossman AB. A mutation and expression analysis of the oncogene BRAF in pituitary adenomas. Clin Endocrinol (Oxf) 2007; 66: 34852.
  • 20
    Perren A,Schmid S,Locher T,Saremaslani P,Bonvin C,Heitz PU,Komminoth P. BRAF and endocrine tumors: mutations are frequent in papillary thyroid carcinomas, rare in endocrine tumors of the gastrointestinal tract and not detected in other endocrine tumors. Endocr Relat Cancer 2004; 11: 85560.
  • 21
    Tannapfel A,Vomschloss S,Karhoff D,Markwarth A,Hengge UR,Wittekind C,Arnold R,Horsch D. BRAF gene mutations are rare events in gastroenteropancreatic neuroendocrine tumors. Am J Clin Pathol 2005; 123: 25660.
  • 22
    Wang GG,Yao JC,Worah S,White JA,Luna R,Wu TT,Hamilton SR,Rashid A. Comparison of genetic alterations in neuroendocrine tumors: frequent loss of chromosome 18 in ileal carcinoid tumors. Mod Pathol 2005; 18: 107987.
  • 23
    Papouchado B,Erickson LA,Rohlinger AL,Hobday TJ,Erlichman C,Ames MM,Lloyd RV. Epidermal growth factor receptor and activated epidermal growth factor receptor expression in gastrointestinal carcinoids and pancreatic endocrine carcinomas. Mod Pathol 2005; 18: 132935.
  • 24
    Gilbert JA,Lloyd RV,Ames MM. Lack of mutations in EGFR in gastroenteropancreatic neuroendocrine tumors. N Engl J Med 2005; 353: 20910.
  • 25
    Schiff BA,McMurphy AB,Jasser SA,Younes MN,Doan D,Yigitbasi OG,Kim S,Zhou G,Mandal M,Bekele BN,Holsinger FC,Sherman SI et al. Epidermal growth factor receptor (EGFR) is overexpressed in anaplastic thyroid cancer, and the EGFR inhibitor gefitinib inhibits the growth of anaplastic thyroid cancer. Clin Cancer Res 2004; 10: 8594602.
  • 26
    Garcia-Rostan G,Costa AM,Pereira-Castro I,Salvatore G,Hernandez R,Hermsem MJ,Herrero A,Fusco A,Cameselle-Teijeiro J,Santoro M. Mutation of the PIK3CA gene in anaplastic thyroid cancer. Cancer Res 2005; 65: 10199207.
  • 27
    Hou P,Liu D,Shan Y,Hu S,Studeman K,Condouris S,Wang Y,Trink A,El-Naggar AK,Tallini G,Vasko V,Xing M. Genetic alterations and their relationship in the phosphatidylinositol 3-kinase/Akt pathway in thyroid cancer. Clin Cancer Res 2007; 13: 116170.
  • 28
    Van Nederveen FH,Perren A,Dannenberg H,Petri BJ,Dinjens WN,Komminoth P,de Krijger RR. PTEN gene loss, but not mutation, in benign and malignant phaeochromocytomas. J Pathol 2006; 209: 27480.
  • 29
    Kedzia C,Lacroix L,Ameur N,Ragot T,Kelly PA,Caillou B,Binart N. Medullary thyroid carcinoma arises in the absence of prolactin signaling. Cancer Res 2005; 65: 8497503.
  • 30
    Costa P,Catarino AL,Silva F,Sobrinho LG,Bugalho MJ. Expression of prolactin receptor and prolactin in normal and malignant thyroid: a tissue microarray study. Endocr Pathol 2006; 17: 37786.
  • 31
    Plaza Menacho I,Koster R,van der Sloot AM,Quax WJ,Osinga J,van der Sluis T,Hollema H,Burzynski GM,Gimm O,Buys CH,Eggen BJ,Hofstra RM. RET-familial medullary thyroid carcinoma mutants Y791F and S891A activate a Src/JAK/STAT3 pathway, independent of glial cell line-derived neurotrophic factor. Cancer Res 2005; 65: 172937.

Nabahet Ameur, Ludovic Lacroix, Nelly Motte, Eric Baudin, Bernard Caillou, Michel Ducreux, Dominique Elias, Philippe Chanson, Martin Schlumberger, Jean Michel Bidart.