Receptor tyrosine kinase pathway analysis sheds light on similarities between clear-cell sarcoma and metastatic melanoma.

To highlight possible similarities and differences in receptor tyrosine kinase (RTK) and downstream signalling activation profiles between clear-cell sarcomas (CCS) and metastatic melanomas (MM), frozen, and paired-matched fixed samples of six CCS with EWSR1 rearrangement (EWSR1+), five CCS without EWSR1 rearrangement (EWSR1-), and seven MM were investigated by means of biochemical, immunohistochemical, FISH, molecular analyses, and immunofluorescence confocal microscopy. Fixed samples of a further 10 CCS and 14 MM were investigated by means of sequencing for BRAF, NRAS, and KRAS mutations and FISH analyses for the gain of chromosomes 22 and 8. RTK analysis of all CCS/MM samples showed activation of short-form (sf) recepteur d'origine nantais (RON) RTK and of PDGFRB, MET, and HER3. Analysis of downstream signaling revealed consistent phosphorylation patterns of PI3K/AKT, RSK, and the mTOR targets S6 and 4EBP1. Analysis of frozen and fixed material from 21 CCS and 21 MM showed the presence of the V600E BRAF mutation in 2/12 EWSR1+ and 3/9 EWSR1- CCS and 9/21 MM and demonstrated a significant (P < 0.001) correlation between the gain of chromosomes 22 and 8 and EWSR1- CCS. Our results show that BRAF mutation can also be present in CCS and support the proposed aberration of chromosomes 22 and 8 as a possibly useful nonrandom hallmark of EWSR1- CCS. Besides, they broaden the spectrum of the similarities of RTK pathway activation between CCS and MM, thus suggesting that new drugs found to be active in melanoma and RON inhibitors could have a role in CCS treatment. © 2011 Wiley Periodicals, Inc.

Receptor tyrosine kinase and downstream signalling analysis in diffuse malignant peritoneal mesothelioma.

Our aim was to assess the activation profile of EGFR, PDGFRB and PDGFRA receptor tyrosine kinases (RTK) and their downstream effectors in a series of cryopreserved diffuse malignant peritoneal mesothelioma (DMPM) surgical specimens to discover the targets for drug inhibition. We also made a complementary analysis of the cytotoxic effects of some kinase inhibitors on the proliferation of the human peritoneal mesothelioma STO cell line. We found the expression/phosphorylation of EGFR and PDGFRB in most of the tumours, and PDGFRA activation in half. The expression of the cognate ligands TGF-α, PDGFB and PDGFA in the absence of RTK mutation and amplification suggested the presence of an autocrine/paracrine loop. There was also evidence of EGFR and PDGFRB co-activation. RTK downstream signalling analysis demonstrated the activation/expression of ERK1/2, AKT and mTOR, together with S6 and 4EBP1, in almost all the DMPMs. No KRAS/BRAF mutations, PI3KCA mutations/amplifications or PTEN inactivation were observed. Real-time polymerase chain reaction revealed the decreased expression of TSC1 c-DNA in half of the tumours. In vitro cytotoxicity studies showed the STO cell line to be resistant to gefitinib and sensitive to sequential treatment with RAD001 and sorafenib; these findings were consistent with the presence of the KRAS mutation G12D in these cells although it was not detectable in the original tumour. Our results highlight the ligand-dependent activation and co-activation of EGFR and PDGFRB, as well as a connection between these activated RTKs and the downstream mTOR pathway, thus supporting the role of combined treatment with RTK and mTOR inhibitors in DMPM.

Chromosome band 17q21 in breast cancer: significant association between beclin 1 loss and HER2/NEU amplification.

Treatment success of breast cancer patients with trastuzumab alone or in combination depends not only on HER2/NEU amplification but also on PTEN and PI3K status and efficient cell death programs. In this pilot study, we found a significant association between loss of beclin 1 and HER2/NEU amplification (both on 17q21) in breast cancers. This finding was confirmed in two public copy number microarray datasets. Furthermore, there is a trend associating beclin 1 loss with TP53 mutations, PI3KCA gene gain, and PTEN mutations. Finally, the observation that beclin 1 gene loss predicted a response to trastuzumab alone or in combination with other drugs is worthy of further confirmation in larger cohorts. Our results suggest that, beclin 1 loss may contribute to genome instability and to a defective autophagy that may lead to tumoral cell death in presence of competent apoptosis or senescence pathways.

Unusual myogenic and melanocytic differentiation of soft tissue pPNETs: an immunohistochemical and molecular study of 3 cases.

All of the members of the peripheral primitive neuroectodermal tumor family (Ewing sarcomas, neuroectodermal tumors of bone, peripheral neuroepitheliomas, and Askin tumors) have similar morphologic and immunophenotypical features (ie, the proliferation of small and medium-sized round cells in a fibrous background showing strong and diffuse immunohistochemical positivity for CD99), and the common cytogenetic abnormality of a nonrandom translocation involving the EWS gene and one of several members of the erythroblastosis virus transforming sequence family of transcription factors. The combination of clinical information and morphologic/immunophenotypical characteristics is usually sufficient for a correct diagnosis, but there are rare cases in which an unusual predominant or multidirectional immunophenotypical differentiation makes diagnosis a challenge and requires the use of molecular cytogenetic or molecular techniques. We describe 3 such cases in which we employed fluorescence in-situ hybridization analysis to detect translocation involving the EWS gene and reverse transcription polymerase chain reaction followed by sequencing to detect the fusion transcript EWS-FLI1.

Analysis of receptor tyrosine kinases (RTKs) and downstream pathways in chordomas.

We have previously demonstrated that chordomas express activated platelet-derived growth factor receptor (PDGFRB) and that treatment with imatinib, which is capable of switching off the activation of various receptor tyrosine kinases (RTKs) including PDGFRB, benefits a number of patients. The aim of this study was to identify the possible presence of other activated RTKs and their downstream signaling effectors. Cryopreserved material from 22 naïve sporadic chordomas was investigated for the presence of activated RTKs and their cognate ligands and downstream signaling effectors by means of human phospho-RTK antibody arrays, Western blotting, and molecular analysis; immunohistochemistry and fluorescence in situ hybridization were used to analyze the corresponding formalin-fixed and paraffin-embedded samples. We detected activated PDGFRB, FLT3, and colony stimulating factor 1 receptor (CSF1R) of the PDGFR family and highly phosphorylated EGFR, HER2/neu, and (to a lesser extent) HER4 of the EGFR family. The detection of PDGFRB/PDGFB confirmed our previous data. The presence of activated EGFR was paralleled by the finding of high levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) and PDGFB co-expression and PDGFRB co-immunoprecipitation. Of the downstream effectors, the PI3K/AKT and RAS/MAPK pathways were both activated, thus leading to the phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 among the regulators involved in translational control. Taken together, our results (i) provide a rationale for tailored treatments targeting upstream activated receptors, including the PDGFR and EGFR families; (ii) support the idea that a combination of upstream antagonists and mTOR inhibitors enhances the control of tumor growth; and (iii) indicate that the 4E-BP1/eIF4E pathway is a major regulator of protein synthesis in chordoma.

Fluorescence in situ hybridization analysis and immunophenotyping of c-Kit/PDGFRA and Bcl-2 expression in gastrointestinal stromal tumors.

OBJECTIVE: To investigate c-Kit/PDGFRA genomic alterations, KIT-PDGFRA coexpression in gastrointestinal stromal tumors (GISTs) and the role of Bcl-2. STUDY DESIGN: We analyzed 70 primary tumors, 6 recurrences, 4 metastases and 1 recurrence plus metastasis, all molecularly characterized. Alterations in gene copy number were detected by fluorescence in situ hybridization (FISH) and expression of KIT, PDGFRA and Bcl-2 by immunohistochemistry. RESULTS: c-Kit/PDGFRA gene alterations affected 38% of all cases and 39% of primary tumors, with major changes accounting for 15% in both all the cases and primary tumors. Cytoplasmic KIT/PDGFRA coexpression was present in 96.5% of the c-Kit-mutated cases, 100% of the wt c-Kit/PDGFRA cases and 66.6% of the PDGFRA-mutated cases. Bcl-2 immunoreactivity was present in 70% of cases, with expression levels of +++ in 29%, ++ in 38% and + in 33%. CONCLUSION: FISH confirmed cytogenetic alterations in about 40% of primary GISTs at the onset. The high rate of c-Kit/PDGFRA coexpression suggests that both receptors are involved in oncogenicity and may affect imatinib efficacy. The assumption that Bcl-2 expression is supported by the KIT pathway and that its imatinib-mediated down-regulation contributes to autophagic cell death, although attractive, needs to be further confirmed.

Treatment relevant target immunophenotyping of 139 salivary gland carcinomas (SGCs).

Salivary gland carcinomas (SGCs) are rare tumors encompassing a wide spectrum of histologic/biologic entities. Standard non-surgical treatments are ineffective in case of advanced disease. Our aim was to analyze SGCs deregulation gene profiles that could become target for innovative treatment options. Samples from 139 patients with primary, recurrent and/or metastatic SGCs were investigated by immunohistochemistry for protein encoded by tyrosine kinases receptors (TKRs) i.e. c-kit, HER2, EGFR and hormonal receptors, i.e. androgen (AR), estrogen (ER) and progesterone receptors (PgR). In 26 cases, the HER2 immunohistochemical analysis was complemented by fluorescence in-situ hybridization analysis. EGFR was the most expressed TKRs (71%) and it was found across all histotypes. c-Kit expression was mainly restricted to adenoid cystic carcinoma (78%) while HER2 expression, mostly sustained by gene amplification, correlated with salivary duct carcinoma (SDC) in 44% of cases and adenocarcinoma, not otherwise specified (AD, NOS) in 21% of cases. With respect to histogenetic classification, TKRs expression occurred more often in tumors derived from intercalated duct rather than excretory ones with the only exception of HER2. AR was found in 13% of samples, restricted to SDC and AD, NOS and it was co-expressed with HER2 in more than half of the SDC cases. ER and PgR positivity was never detected. This TK-hormonal receptors analysis identify a histotype-specific profiles that could be exploited for better selecting patients for innovative treatment within prospective studies.

Polymorphisms of Metabolizing Enzymes and Susceptibility to Ethmoid Intestinal-type Adenocarcinoma in Professionally Exposed Patients.

Intestinal-type adenocarcinoma (ITAC) of ethmoid is a rare tumor associated with occupational exposure to wood and leather dusts. Polymorphisms in xenobiotic metabolizing enzymes play an important role in gene-environment interactions and may contribute to a high degree of variance in individual susceptibility to cancer risk. The aim of this study was to investigate by polymerase chain reaction the role of polymorphisms at CYP1A1 and GSTM1 genes in 30 ethmoid ITAC patients and 79 healthy donors. The distribution of Thr/Asn genotype at CYP1A1 codon 461 was significantly overrepresented among the patients (23.3%; P = .0422), whereas the Ile/Val genotype at CYP1A1 codon 462 was not significantly different between cases and controls (P = .76). The GSTM1 null genotype was not significantly different between cases and control (P = 1), but we observed that the combined codon 461 Thr/Asn and GSTM1 null genotype was overrepresented in the patient group (P = .0019). The results reveal that patients with CYP1A1 codon 461 polymorphism may be at high genetic risk of ITAC and that the risk increases in the presence of combined polymorphism of CYP1A1 and GSTM1 null genotype. This strongly suggests that CYP1A1 codon 461 and GSTM1 null genotype may be useful in selecting exposed individuals at risk for ethmoid ITAC.

Response to sunitinib malate in advanced alveolar soft part sarcoma.

PURPOSE: Alveolar soft part sarcoma (ASPS) is a rare, chemoresistant soft tissue sarcoma. ASPS harbors the t(17-X) (p11.2;q25) translocation, resulting in the ASPACR1-TFE3 fusion protein, causing MET autophosphorylation and activation of downstream signaling. The tumor vascular pattern prompted us to use sunitinib malate (SM), a tyrosine kinase inhibitor with antiangiogenic properties. EXPERIMENTAL DESIGN: Since July 2007, five patients with progressive metastatic ASPS have been treated with continuous SM 37.5 mg/d on a named basis. Four patients are evaluable for response. In four cases, cryopreserved material was available. Upstream and downstream targets of receptor tyrosine kinase (RTK) pathways, as well as mechanisms of activation, were investigated by biochemical profiles, including human phospho-receptor RTK antibody arrays and immunoprecipitation/Western blotting, molecular analyses, immunohistochemistry, and fluorescence in situ hybridization analyses. RESULTS: After 3 months, two patients had RECIST (response evaluation criteria in solid tumor) partial response, as well as positron emission tomography response and subjective improvement. One had a RECIST stable disease. One progressed and stopped treatment. One patient is still responding after 12 months. The upstream analysis showed activation of all the platelet-derived growth factor receptor (PDGFR) family members, as well as epidermal growth factor receptor, MET families, and RET. Vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) were activated only in one case. The downstream target analysis showed strong activation of phosphatidylinositol 3-kinase/AKT, extracellular signal-regulated kinase 1/2, and mTOR and its targets (S6K and S6). The absence of any upstream mTOR effector deregulation and the presence of RTK cognate ligands support an autocrine-paracrine activation loop mechanism. CONCLUSION: SM may have antitumor activity in ASPS, possibly through a mechanism involving PDGFR and RET. The role of MET, epidermal growth factor receptor, and mTOR, as well as PDGFR inhibition, needs to be further explored.

PDGFRA, PDGFRB, EGFR, and downstream signaling activation in malignant peripheral nerve sheath tumor.

We investigated the activation of platelet-derived growth factor (PDGF) receptor A (PDGFRA), PDGF receptor B (PDGFRB), epidermal growth factor receptor (EGFR), and their downstream pathways in malignant peripheral nerve sheath tumors (MPNSTs). PDGFRA, PDGFRB, and EGFR were immunohistochemically, biochemically, cytogenetically, and mutationally analyzed along with the detection of their cognate ligands in 16 neurofibromatosis type 1 (NF1)-related and 11 sporadic MPNSTs. The activation of the downstream receptor pathways was also studied by means of v-akt murine thymoma viral oncogene homolog (AKT), extracellular signal-regulated kinase (ERK), and mammalian target of rapamycin (mTOR) Western blotting experiments, as well as rat sarcoma viral oncogene homolog (RAS), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), phosphoinositide-3-kinase, catalytic, alpha polypeptide (PI3KCA), and phosphatase and tensin homolog deleted on chromosome ten (PTEN) mutational analysis and fluorescence in situ hybridization. PDGFRA, PDGFRB, and EGFR were expressed/activated, with higher levels of EGFR expression/phosphorylation paralleling increasing EGFR gene copy numbers in the NF1-related cases (71%). Autocrine loop activation of these receptors along with their coactivation were suggested by the expression of the cognate ligands in the absence of mutations and the presence of receptor tyrosine kinase (RTK) heterodimers, respectively. Both MPNST groups showed AKT, ERK, and mTOR expression/phosphorylation. No BRAF, PI3KCA, or PTEN mutations were found in either group of MPNSTs, but 18% of the sporadic MPNSTs showed RAS mutations. PTEN monosomy segregated with the NF1-related cases (50%, p = 0.018), but PTEN protein was expressed in all but two cases. In conclusion, PDGFRA, PDGFRB, and EGFR seem to be promising molecular targets for tailored treatments in MPNST. In particular, the ligand- and heterodimerization-dependent RTK activation/expression coupled with a downstream signaling phosphorylation, mediated by the upstream receptors or RAS activation, may provide a rationale to apply combined RTK and mTOR inhibitor treatments both to sporadic and NF1-related cases.

Prolonged remission of disseminated atypical adenomatous hyperplasia under gefitinib.

Atypical adenomatous hyperplasia (AAH) is a putative precursor of bronchioloalveolar carcinoma (BAC) and adenocarcinoma of the lung, developing from terminal respiratory unit cells. AAH and BAC lesions typically present as ground-glass opacities at spiral chest computed tomography. Epidermal growth factor receptor polysomy/mutations, conferring higher sensitivity to Gefitinib, are frequent in BAC but less common in AAH. We describe an interesting case of disseminated AAH showing a sustained remission under Gefitinib therapy.

Molecular characterization of synovial sarcoma in children and adolescents: evidence of akt activation.

Synovial sarcoma (SS) is the most frequent nonrhabdomyosarcomatous soft tissue sarcoma encountered in adolescents and young adults, and despite advances in the treatment of local disease, metastases remain the main cause of death. The aim of this study was to characterize a single-center series of pediatric SS molecularly to seek any biomarkers or pathways that might make suitable targets for new agents. Seventeen cases of pediatric SS showing the SYT-SSX fusion transcript were screened immunohistochemically, biochemically, molecularly, and cytogenetically (depending on the available material) to investigate any expression/activation of epidermal growth factor receptor, platelet-derived growth factor receptor alpha (PDGFRalpha), PDGFRbeta, Akt, and deregulated Wnt pathway. The most relevant outcome was the finding of activated epidermal growth factor receptor, PDGFRalpha, and PDGFRbeta, which activated Akt in both the monophasic and biphasic histologic subtypes. Consistently, Akt activation was completely abolished in an SS cell line assay when stimulated by PDGF-AA and treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Our results also showed the nuclear localization of beta-catenin and cyclin D1 gene products in monophasic SS and the movement of beta-catenin into the cytoplasm in the glandular component of the biphasic subtype. Although they need to be confirmed in larger series, these preliminary data suggest that therapeutic strategies including specific inhibitors of the phosphatidylinositol 3-kinase/Akt pathway might be exploited in SS.

Cyclooxygenase-2 and platelet-derived growth factor receptors as potential targets in treating aggressive fibromatosis.

PURPOSE: To explore the molecular bases of potential new pharmacologic targets in aggressive fibromatosis (desmoid tumor). EXPERIMENTAL DESIGN: Tumor specimens from 14 patients surgically treated for aggressive fibromatosis (6 familial adenomatous polyposis and 8 sporadic cases), analyzed for adenomatous polyposis coli (APC) and CTNNB1 (beta-catenin) mutations, were further investigated for beta-catenin, cyclooxygenase-2 (COX-2), platelet-derived growth factor (PDGF) receptor alpha (PDGFRA)/PDGF receptor beta (PDGFRB), their cognate ligands (PDGFA and PDGFB), and KIT using a comprehensive immunohistochemical, biochemical, molecular, and cytogenetic approach. RESULTS: No CTNNB1 (beta-catenin) mutations were found in the familial adenomatous polyposis patients, but previously reported activating mutations were found in six of the eight sporadic patients. All of the cases carrying an altered WNT pathway showed nuclear and cytoplasmic immunoreactivity for beta-catenin, whereas beta-catenin expression was restricted to the cytoplasm in the sporadic patients lacking CTNNB1 mutations. COX-2 protein and mRNA overexpression was detected in all 14 cases, together with the expression and phosphorylation of PDGFRA and PDGFRB, which in turn paralleled the presence of their cognate ligands. No PDGFRB mutations were found. The results are consistent with PDGFRA and PDGFRB activation sustained by an autocrine/paracrine loop. CONCLUSIONS: Aggressive fibromatosis is characterized by WNT/oncogene pathway alterations triggering COX-2-mediated constitutive coactivation of PDGFRA and PDGFRB, and may therefore benefit from combined nonsteroidal anti-inflammatory drug + tyrosine kinase inhibitor treatment.

p53 codon 72 polymorphisms in human papillomavirus-negative and human papillomavirus-positive squamous cell carcinomas of the oropharynx.

BACKGROUND: Tobacco smoking, alcohol abuse, and high-risk human papillomavirus (HPV) are risk factors in the etiology of oropharyngeal squamous cell carcinomas (SCCs). The TP53 polymorphism, in which an arginine (R) is changed to proline (P) at codon 72, is functionally significant and could therefore be a predisposing genetic defect. METHODS: The aim of the study was to investigate the role of codon 72 polymorphism by means of double gradient-denaturing gel electrophoresis in 77 oropharyngeal SCC patients including 33 TP53 mutated and 16 HPV-16-positive cases. The controls consisted of 141 consecutive healthy blood donors. RESULTS: The cases and controls showed significantly different genotype distribution (P = .0005): the frequencies of the RR, RP, and PP genotypes among the cases were, respectively, 81.8%, 10.4%, and 7.8%, as opposed to 59.6%, 33.3%, and 7.1% among the controls, in agreement with the Hardy-Weinberg equilibrium (P = .35). The PP genotype was significantly overrepresented among females (22.2% vs 3.4%; P = .0243) and in HPV-16-positive cases (25.0% vs 3.3%; P = .0152). No segregation was found between either of the codon 72 genotypes and age or TP53 mutations. CONCLUSIONS: The significantly lower frequency of the RP genotype in the patients as a whole suggests that it has a protective effect on oropharyngeal SCCs. Moreover, the PP genotype may be a risk factor for the development of oropharyngeal SCC by females and the development of HPV-16-related SCC, although the findings need to be validated in a larger number of tumors.

c-Kit/PDGFRA gene status alterations possibly related to primary imatinib resistance in gastrointestinal stromal tumors.

PURPOSE: To correlate morphologic changes with molecular, biochemical, and cytogenetic profiles in gastrointestinal stromal tumor (GIST) patients before and after imatinib treatment. EXPERIMENTAL DESIGN: We investigated 132 tumor samples obtained from 35 patients with advanced disease who underwent resective surgery after imatinib treatment according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group protocol. On the basis of imaging findings, 27 patients were responders and 8 progressors, and retaining this radiological subdivision, we analyzed posttreatment morphologic changes correlating them with molecular, biochemical, and cytogenetic analyses. RESULTS: On the basis of morphology (residual viable cellularity/proliferation markers), three subgroups were identified showing high, moderate, or low response. All of the progressing cases clustered in the low-response subgroup, whereas the responding cases were distributed in all three subgroups. The correlation between morphology and the molecular findings showed that secondary mutations segregated with the low-response subgroup, whereas c-Kit primary resistance mutations were randomly distributed in the three subgroups. Fluorescence in situ hybridization analysis of c-Kit/PDGFRA genes showed that all of the progressing cases were disomic. Referring to morphology, among the responding cases, a disomic pattern was mainly restricted to the high responders, whereas the moderate and low responders were aneusomic. Comparison of post-imatinib genomic profiles with the 23 available primary tumors showed that 17 cases carried the same cytogenetic pattern. Overall, 12 of the 27 primary tumors presented a gain/loss of c-Kit/PDGFRA gene copy number. CONCLUSIONS: Our findings show that c-Kit/PDGFRA genomic alterations were present at disease onset in 1/3 of the examined cases. They therefore represent an early event possibly related to primary imatinib resistance in GISTs.

Molecular and cytogenetic subgroups of oropharyngeal squamous cell carcinoma.

PURPOSE: The aim of this study was to acquire further insights into the pathogenetic pathways of head and neck squamous cell carcinomas (HNSCC) that may be useful for identifying new biomarkers instrumental in developing more specific treatment approaches. EXPERIMENTAL DESIGN: Cell cycle regulators and epidermal growth factor receptor (EGFR) and BRAF genes were analyzed in a series of 90 oropharyngeal SCCs of a cohort of surgically treated patients from a single institution, and the results were matched with the presence of high-risk human papillomavirus (HR-HPV) DNA and the TP53 status. RESULTS: At least four distinct groups of tumors were identified sharing a common histology but displaying different molecular/cytogenetic patterns: (a) 19% were HPV-positive SCCs whose lack of alterations of the investigated genes could explain their particular natural history, which requires less aggressive treatment; (b) 37% were HPV-negative SCCs carrying TP53 mutations, which may be more effectively treated by drugs acting through p53-independent apoptosis; (c) 34% were HPV-negative SCCs carrying wild-type TP53 and loss of 9p21 (p16INK4a and p15INK4b) and/or cyclin D1 overexpression that justify treatment with DNA-damaging drugs followed by cell cycle inhibitors; and (d) 10% were HPV-negative lacking tumor suppressor genes and cell cycle alterations. The second, third, and fourth groups also showed an increased copy number of EGFR and chromosome 7 (43%) that might justify the additional or alternative use of EGFR inhibitors. CONCLUSIONS: Our findings suggest that assessing HPV, TP53, 9p21, and EGFR status may be crucial to finding more tailored and beneficial treatments for oropharyngeal SCCs.