Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.
Donata Vitagliano, Valentina De Falco, Anna Tamburrino, Sabrina Coluzzi, Giancarlo Troncone, Gennaro Chiappetta, Fortunato Ciardiello, Giampaolo Tortora, James A Fagin, Anderson J Ryan, Francesca Carlomagno, and Massimo Santoro
Hugo Prazeres, Joana P Couto, Fernando Rodrigues, João Vinagre, Joana Torres, Vitor Trovisco, Teresa C Martins, Manuel Sobrinho-Simões, and Paula Soares
Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS–RAF–ERK1/2, PIK3–AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10- and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5- and 3-fold versus 1.2- and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.
B Zierhut, K Mechtler, W Gartner, T Daneva, W Base, M Weissel, B Niederle, and L Wagner
In order to identify neuroendocrine tumour-specific protein expression, we generated monoclonal antibodies (mAbs) with a tumour-related reaction pattern using a human insulinoma as immunogen. One of the generated mAbs (mAb 1D4) exhibited striking immunoreactivity against various neuroendocrine tumours without staining pancreatic islets of Langerhans. Furthermore, mAb 1D4 immunostained a characteristic subtype of hypothalamic neurones. Using two-dimensional (2-D) gel electrophoresis, mAb 1D4 immunoblotting and mass spectrometry, heat shock protein 70 (Hsp70) isoforms were identified as the mAb 1D4-specific antigen. In hypothalamic tissue, the presence of two different Hsp70 isoforms (Hsp70-8 and Hsp70-1) was revealed by 2-D gel immunoblots and consecutive mass spectrometric peptide analysis. In contrast, insulinoma and other neuroendocrine tumours displayed solely Hsp70-8 expression. Moreover, the tumour-specific presence of an additional mAb 1D4 immunoreactive protein of 40 kDa was observed in eight out of eight tested neuroendocrine tumours. For this variant, exclusively, peptides derived from the C terminus excluding the 299 amino-terminal residues were detected. In cultured tumour-derived fibroblasts, expression of the truncated Hsp70-8 subtype was not present. In conclusion, we have demonstrated a neuroendocrine tumour-specific expression pattern of Hsp70 isoforms and identified an as yet unknown N-terminally truncated Hsp70-8 variant.
Lois M Mulligan
The focus of precision cancer medicine is the use of patient genetic signatures to predict disease occurrence and course and tailor approaches to individualized treatment to improve patient outcomes. The rearranged during transfection (RET) receptor tyrosine kinase represents a paradigm for the power of personalized cancer management to change cancer impact and improve quality of life. Oncogenic activation of RET occurs through several mechanisms including activating mutations and increased or aberrant expression. Activating RET mutations found in the inherited cancer syndrome multiple endocrine neoplasia 2 permit early diagnosis, predict disease course and guide disease management to optimize patient survival. Rearrangements of RET found in thyroid and lung tumors provide insights on potential disease aggressiveness and offer opportunities for RET-targeted therapy. Aberrant RET expression in a subset of cases is associated with tumor dissemination, resistance to therapies and/or poorer prognosis in multiple cancers. The potential of RET targeting through repurposing of small-molecule multikinase inhibitors, selective RET inhibitors or other novel approaches provides exciting opportunities to individualize therapies across multiple pathologies where RET oncogenicity contributes to cancer outcomes.
Toyoshi Endo and Tetsuro Kobayashi
A variant located on 14q13.3 nearest to thyroid transcription factor-1 (TTF1) predisposes individuals to thyroid cancer, but whether this variant is related to the RET/PTC rearrangement associated with human papillary thyroid carcinomas (PTCs) is unknown. The aims of this study were to investigate the effects of RET/PTC1 on the expression of thyroid-specific genes in thyrocytes and their relationship with malignant transformation of the thyrocytes. In the absence or presence of TSH, an extracellular signal-regulated kinase was phosphorylated in FRTL5 cells that stably expressed RET/PTC1, and these cells grew independently of TSH. FRTL (RET/PTC1) cells produced 566% more thyroglobulin mRNA and 474% more Na+/I− symporter mRNA than did the control FRTL (pcDNA) cells. FRTL (RET/PTC1) cells expressed 468% more Ttf1 mRNA than did FRTL (pcDNA) cells, but these two cell types did not differ significantly with respect to Pax8 or Ttf2 mRNA levels. When FRTL (RET/PTC1) cells and FRTL (pcDNA), cells were injected into each of nine nude mice, each mouse developed a single tumor at the site of FRTL (RET/PTC1) cell injection; in contrast, tumor formation never occurred at sites of FRTL (cDNA) cells injection. Tumors resulting from FRTL (RET/PTC1) cells retained 125I-uptake activity; moreover, the cells invaded into surrounding skeletal muscle. When overexpression of Ttf1 in FRTL (RET/PTC1) cells was silenced, the cells completely lost their tumorigenic potential. Exogenous TTF1 cDNA enhanced the tumorigenicity of BHP18-21v cells, human PTC cells that express RET/PTC1, in nude mice. These results indicated that concurrent overexpression of RET/PTC1 and TTF1 confers tumorigenicity to FRTL5 and BHP18-21v cells in nude mice.
J Cros, O Hentic, V Rebours, M Zappa, N Gille, N Theou-Anton, D Vernerey, F Maire, P Lévy, P Bedossa, V Paradis, P Hammel, P Ruszniewski, and A Couvelard
Temozolomide (TEM) showed encouraging results in well-differentiated pancreatic neuroendocrine tumors (WDPNETs). Low O 6-methylguanine-DNA methyltransferase (MGMT) expression and MGMT promoter methylation within tumors correlate with a better outcome under TEM-based chemotherapy in glioblastoma. We aimed to assess whether MGMT expression and MGMT promoter methylation could help predict the efficacy of TEM-based chemotherapy in patients with WDPNET. Consecutive patients with progressive WDPNET and/or liver involvement over 50% who received TEM between 2006 and 2012 were retrospectively studied. Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines. Nuclear expression of MGMT was assessed by immunochemistry (H-score, 0–300) and MGMT promoter methylation by pyrosequencing. Forty-three patients (21 men, 58years (27–84)) with grade 1 WDPNET (n=6) or 2 (n=36) were analyzed. Objective response, stable disease, and progression rates were seen in 17 patients (39.5%), 18 patients (41.9%), and 8 patients (18.6%), respectively. Low MGMT expression (≤50) was associated with radiological objective response (P=0.04) and better progression-free survival (PFS) (HR=0.35 (0.15–0.81), P=0.01). Disease control rate at 18months of treatment remained satisfying with an MGMT score up to 100 (74%) but dropped with a higher expression. High MGMT promoter methylation was associated with a low MGMT expression and longer PFS (HR=0.37 (0.29–1.08), P=0.05). Low MGMT score (≤50) appears to predict an objective tumor response, whereas an intermediate MGMT score (50–100) seems to be associated with prolonged stable disease.
A Berruti, A Mosca, M Tucci, C Terrone, M Torta, R Tarabuzzi, L Russo, C Cracco, E Bollito, R M Scarpa, A Angeli, and L Dogliotti
The presence of neuroendocrine (NE) differentiation in the context of predominantly exocrine prostate cancer may play a key role in androgen-independent tumor growth. The prognostic significance of plasma chromogranin A (CgA) was assessed in a series of consecutive prostate cancer patients with hormone-refractory disease.
One hundred and eight patients with newly diagnosed hormone-refractory prostate cancer entered the study. Plasma CgA levels and other biochemical parameters, such as serum prostate specific antigen, serum alkaline phosphatase, serum lactate dehydrogenase, serum albumin and hemoglobin concentration, were measured at baseline (i.e. when hormone refractoriness occurred) and their prognostic role was evaluated together with patient performance status, Gleason score (at diagnosis of prostate cancer) and the presence of visceral metastases. Furthermore, plasma CgA was prospectively evaluated in 50 patients undergoing chemotherapy.
At baseline, 45 patients (43.3%) showed elevated CgA values. Plasma CgA negatively correlated with survival, either in univariate analysis (P=0.008) or in multivariate analysis, after adjusting for previously mentioned prognostic parameters (P<0.05). In the patient subset undergoing chemotherapy, median CgA (range) values were 13.3 (3.0–141.0) U/l at baseline, 19.1 (3.0–486.0) U/l after 3 months, 20.8 (3.0–702.0) U/l after 6 months and 39.4 (3.0–414.0) U/l after 9 months (P<0.01). The corresponding supranormal rates were 17/50 (34%), 23/50 (46%), 26/50 (52%) and 34/50 (68%) respectively (P<0.005).
Elevated plasma CgA levels are frequently observed in prostate cancer patients with hormone-refractory disease and correlate with poor prognosis. NE differentiation in hormone-refractory patients is a time-dependent phenomenon and is not influenced by conventional antineoplastic treatments.
Kimberly Kamp, Ronald A M Damhuis, Richard A Feelders, and Wouter W de Herder
An increased association between neuroendocrine tumors of the gastrointestinal tract and pancreas (GEP-NET) and other second primary malignancies has been suggested. We determined whether there is indeed an increased risk for second primary malignancies in GEP-NET patients compared with an age- and sex-matched control group of patients with identical malignancies. The series comprised 243 men and 216 women, diagnosed with a GEP-NET between 2000 and 2009 in a tertiary referral center. The timeline, before-at-after diagnosis, and the type of other malignancies were studied using person-year methodology. Poisson distributions were used for testing statistical significance. All data were cross-checked with the Dutch National Cancer Registry. Out of 459 patients with GEP-NET, 67 (13.7%) had a second primary cancer diagnosis: 25 previous cancers (5.4%), 13 synchronous cancers (2.8%), and 29 metachronous cancers (6.3%). The most common types of second primary cancer were breast cancer (n=10), colorectal cancer (n=8), melanoma (n=6), and prostate cancer (n=5). The number of patients with a cancer history was lower than expected, although not significant (n=25 vs n=34.5). The diagnosis of synchronous cancers, mainly colorectal tumors, was higher than expected (n=13 vs n=6.1, P<0.05). Metachronous tumors occurred as frequent as expected (n=29 vs n=25.2, NS). In conclusion, our results are in contrast to previous studies and demonstrate that only the occurrence of synchronous second primary malignancies, mainly colorectal cancers, is increased in GEP-NET patients compared with the general population.
P D Leotlela, A Jauch, H Holtgreve-Grez, and R V Thakker
Neuroendocrine tumours (NETs) originate in tissues that contain cells derived from the embryonic neural crest, neuroectoderm and endoderm. Thus, NETs occur at many sites in the body, although the majority occur within the gastro-entero-pancreatic axis and can be subdivided into those of foregut, midgut and hindgut origin. Amongst these, only those of midgut origin are generally argentaffin positive and secrete serotonin, and hence only these should be referred to as carcinoid tumours. NETs may occur as part of complex familial endocrine cancer syndromes, such as multiple endocrine neoplasia type 1 (MEN1), although the majority occur as non-familial (i.e. sporadic) isolated tumours. Molecular genetic studies have revealed that the development of NETs may involve different genes, each of which may be associated with several different abnormalities that include point mutations, gene deletions, DNA methylation, chromosomal losses and chromosomal gains. Indeed, the foregut, midgut and hindgut NETs develop via different molecular pathways. For example, foregut NETs have frequent deletions and mutations of the MEN1 gene, whereas midgut NETs have losses of chromosome 18, 11q and 16q and hindgut NETs express transforming growth factor-alpha and the epidermal growth factor receptor. Furthermore, in lung NETs, a loss of chromosome 3p is the most frequent change and p53 mutations and chromosomal loss of 5q21 are associated with more aggressive tumours and poor survival. In addition, methylation frequencies of retinoic acid receptor-beta, E-cadherin and RAS-associated domain family genes increase with the severity of lung NETs. Thus the development and progression of NETs is associated with specific genetic abnormalities that indicate the likely involvement of different molecular pathways.
Z Hamze, C Vercherat, A Bernigaud-Lacheretz, W Bazzi, R Bonnavion, J Lu, A Calender, C Pouponnot, P Bertolino, C Roche, R Stein, J Y Scoazec, C X Zhang, and M Cordier-Bussat
The protein MENIN is the product of the multiple endocrine neoplasia type I (MEN1) gene. Altered MENIN expression is one of the few events that are clearly associated with foregut neuroendocrine tumours (NETs), classical oncogenes or tumour suppressors being not involved. One of the current challenges is to understand how alteration of MENIN expression contributes to the development of these tumours. We hypothesised that MENIN might regulate factors maintaining endocrine-differentiated functions. We chose the insulinoma model, a paradigmatic example of well-differentiated pancreatic NETs, to study whether MENIN interferes with the expression of v-MAF musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA), a master glucose-dependent transcription factor in differentiated β-cells. Immunohistochemical analysis of a series of human insulinomas revealed a correlated decrease in both MENIN and MAFA. Decreased MAFA expression resulting from targeted Men1 ablation was also consistently observed in mouse insulinomas. In vitro analyses using insulinoma cell lines showed that MENIN regulated MAFA protein and mRNA levels, and bound to Mafa promoter sequences. MENIN knockdown concomitantly decreased mRNA expression of both Mafa and β-cell differentiation markers (Ins1/2, Gck, Slc2a2 and Pdx1) and, in parallel, increased the proliferation rate of tumours as measured by bromodeoxyuridine incorporation. Interestingly, MAFA knockdown alone also increased proliferation rate but did not affect the expression of candidate proliferation genes regulated by MENIN. Finally, MENIN variants with missense mutations detected in patients with MEN1 lost the WT MENIN properties to regulate MAFA. Together, our findings unveil a previously unsuspected MENIN/MAFA connection regarding control of the β-cell differentiation/proliferation balance, which could contribute to tumorigenesis.