Clinical and molecular characteristics of non-small-cell lung cancer (NSCLC) harboring EGFR mutation: results of the nationwide French Cooperative Thoracic Intergroup (IFCT) program.

BACKGROUND: EGFR mutations cause inconsistent response to EGFR tyrosine-kinase inhibitors (TKI). To better understand these features, we reviewed all cases of EGFR-mutated non-small-cell lung cancer collected in the Biomarkers France database. PATIENTS AND METHODS: Of 17 664 patients, 1837 (11%) with EGFR-mutated non-small-cell lung cancer were retrospectively analyzed for clinical and molecular characteristics. Results were correlated with survival and treatment response for the 848 stage IV patients. RESULTS: EGFR exon 18, 19, 20 and 21 mutations were found in 102 (5.5%), 931 (51%), 102 (5.5%) and 702 (38%) patients, respectively. Over 50% of exon 18 and 20 mutated patients were smokers. The median follow-up was 51.7 months. EGFR mutation type was prognostic of overall survival (OS) versus wild-type {exon 19: hazard ratio (HR)=0.51 [95% confidence interval (CI): 0.41-0.64], P < 0.0001; exon 21: HR = 0.76 (95% CI: 0.61-0.95), P = 0.002; exon 20: HR = 1.56 (95% CI: 1.02-2.38), P = 0.004}. EGFR mutation type was prognostic of progression-free survival versus wild-type [exon 19: HR = 0.62 (95% CI: 0.49-0.78), P < 0.0001; exon 20: HR = 1.46 (95% CI: 0.96-2.21), P = 0.07]. First-line treatment choice did not influence OS in multivariate analysis. First-line TKI predicted improved progression-free survival versus chemotherapy [HR = 0.67 (95% CI: 0.53-0.85), P = 0.001]. OS was longer for del19 versus L858R, which was associated with better OS compared with other exon 21 mutations, including L861Q. TKI improved survival in patients with exon 18 mutations, while chemotherapy was more beneficial for exon 20-mutated patients. CONCLUSION: EGFR mutation type can inform the most appropriate treatment. Therapeutic schedule had no impact on OS in our study, although TKI should be prescribed in first-line considering the risk of missing the opportunity to use this treatment.

PTEN, ATM, IDH1 mutations and MAPK pathway activation as modulators of PFS and OS in patients treated by first line EGFR TKI, an ancillary study of the French Cooperative Thoracic Intergroup (IFCT) Biomarkers France project.

OBJECTIVES: Tumor mutation screening is standard of care for patients with stage IV NSCLC. Since a couple of years, widespread NGS approaches used in routine diagnostics to detect driver mutations such as EGFR, KRAS, BRAF or MET allows the identification of other alterations that could modulated the intensity or duration of response to targeted therapies. The prevalence of co-occurring alterations that could affect response or prognosis as not been largely analyzed in clinical settings and large cohorts of patients. Thanks to the IFCT program "Biomarkers France", a collection of samples and data at a nation-wide level was available to test the impact of co-mutations on first line EGFR TKI in patients with EGFR mutated cancers. MATERIALS AND METHODS: Targeted NGS was assessed on available (n = 208) samples using the Ion AmpliSeq™ Cancer Hotspot Panel v2 to screen for mutations in 50 different cancer genes. RESULTS: This study showed that PTEN inactivating mutations, ATM alterations, IDH1 mutations and complex EGFR mutations were predictors of short PFS in patients with a stage 4 lung adenocarcinoma receiving first line EGFR TKI and that PTEN, ATM, IDH1 and KRAS mutations as well as alterations in the MAPK pathway were related to shorter OS. CONCLUSION: These findings may lead to new treatment options in patients with unfavorable genotypes to optimize first line responses.

Identification of biological factors predictive of response to imatinib mesylate in aggressive fibromatosis.

BACKGROUND: Imatinib induces responses and disease stabilisations in non-resectable patients with aggressive fibromatosis (AF). The precise target of imatinib in AF and predictive factors for response to treatment are unknown. METHODS: We investigated factors potentially predictive of response to imatinib in a series of 40 patients with progressive AF included in a phase II trial of imatinib: we tested the presence of KIT exon 10 variant (M541L), the expression of imatinib-sensitive kinases and cell cycle proteins by immunohistochemistry (IHC), and other clinical and biological factors. RESULTS: Of 10 patients for whom DNA could be extracted, 3 had a KIT exon 10 variant (30%), with no correlation with response or progression-free survival (PFS). The expression of other imatinib targets (PDGFRA/B, macrophage colony-stimulating factor receptor (M-CSFR)) and of downstream components of the cell cycle, cell proliferation and proliferation pathway (cyclin D1, ERK, MEK 1-2) did not correlate with PFS. Pre-treatment lymphopenia (<1500/microl) and tumour size >120 mm correlated with shorter PFS in univariate and multivariate analyses. CONCLUSION: Our findings show that a baseline biological characteristic of the patient is the major parameter influencing response to imatinib in aggressive fibromatosis. Tumour characteristics, including the presence of a KIT exon 10 M541L variant, may influence tumour control but this needs to be confirmed and better explained.

A prospective epidemiological study of new incident GISTs during two consecutive years in Rhône Alpes region: incidence and molecular distribution of GIST in a European region.

BACKGROUND: Preliminary data indicate that the molecular epidemiology of localised gastrointestinal stromal tumour (GIST) may be different from that of advanced GIST. We sought to investigate the molecular epidemiology of sarcomas, including GIST, in the Rhone-Alpes region in France. PATIENTS AND METHODS: A prospective and exhaustive study in the Rhone-Alpes Region in France to assess the precise incidence of primary sarcomas with systematic centralised pathological review and molecular analysis was conducted for 2 consecutive years. RESULTS: Among 760 patients with a confirmed diagnosis of sarcoma, 131 (17%) had a GIST. The majority of patients had gastric primaries (61%). Mutational analysis could be performed in 106 tumour samples (74%), and 71 (67%) had exon 11 mutations. PDGFRA mutations were found in 16% of cases, which is twice as high as previously reported for advanced GIST. CONCLUSION: Data indicate that PDGFRA mutations in localised GIST may be twice as high as what was previously reported in patients with advanced disease. This finding may have important consequences for patients offered adjuvant imatinib, although most of these tumours are in the low-risk group.

Expression of beta-catenin in gastroenteropancreatic endocrine tumours: a study of 229 cases.

AIMS: To clarify the role of beta-catenin in digestive endocrine carcinogenesis, a large and representative series of gastroenteropancreatic endocrine tumours was analysed in order to determine the incidence and pattern of beta-catenin changes and to analyse the clinical and histological characteristics of the tumours presenting immunohistochemically detectable changes in beta-catenin expression. METHODS: 229 cases of gastroenteropancreatic endocrine tumours (stomach, 11; duodenum and ampulla, 29; jejunum and ileum, 51; appendix, 13; colon and rectum, 17; and pancreas, 108) were studied by immunohistochemistry to assess the pattern of distribution of beta-catenin (membranous, cytoplasmic or nuclear). DNA was analysed to detect mutations in exon 3 of the CTNNB1 gene. RESULTS: The distribution of immunoreactive beta-catenin protein was membranous in 164 cases, cytoplasmic in 58 cases and nuclear in seven cases. No mutation was detected in exon 3 of the CTNNB1 gene in any case. The seven cases with nuclear accumulation of beta-catenin were large tumours (mean size 44 (standard deviation (SD) 18.5) mm) with metastases, including liver metastases in five cases, high Ki-67 index (mean 34% (SD 16.5%)) and cyclin D1 overexpression; p53 accumulation was detected in six cases. Five patients died of disease; the mean (SD) survival was 13.6 (4.8) months. CONCLUSIONS: Immunohistochemically detectable nuclear accumulation of beta-catenin is infrequent in gastroenteropancreatic endocrine tumours and is usually not associated with mutations in CNNTB1 exon 3. Changes in beta-catenin expression are late events in digestive endocrine carcinogenesis, associated with tumour progression and dissemination.

Decreased E-cadherin immunoreactivity correlates with poor survival in patients with bladder tumors.

E-cadherin, an intercellular adhesion molecule, has been shown to behave like an invasion suppressor gene in vitro. This may explain the inverse relation between expression of E-cadherin and tumor grade that was found in certain cancers. We therefore examined E-cadherin expression in bladder cancer samples from patients with known clinical follow-up. Forty-nine snap-frozen specimens (24 superficial and 25 invasive tumors) and 4 samples of normal urothelium were retrospectively analyzed with anti-E-cadherin monoclonal antibodies. In normal urothelium E-cadherin is expressed homogeneously with a typical membranous staining at cell-cell borders. Decreased expression is found in 5 of 24 superficial tumors and in 19 of 25 invasive cancers. Completely negative tumors are infrequent (4 cases). Most of the time a heterogeneous staining, which may correspond to an unstable E-cadherin expression during tumor development, is seen. Decreased E-cadherin expression correlates with both increased grade and stage (chi 2 = 9.5, P < 0.01, and chi 2 = 14.9, P < 0.005, respectively). More importantly, abnormal E-cadherin expression correlates with shorter survival (log rank test: chi 2 = 16.5, P < 0.001). In keeping with its in vitro invasion suppressor function, decreased E-cadherin expression correlates with the clinical aggressiveness of bladder tumors. This is the first report of E-cadherin as a marker with prognostic value. This parameter must now be tested in a large prospective study to assess its precise clinical relevance.

Complex cadherin expression in renal cell carcinoma.

E-cadherin is an intercellular adhesion protein expressed by most epithelia. Decreased expression of E-cadherin correlates with tumor aggressiveness in most carcinomas. In renal cell carcinoma (RCC), however, this correlation is not well established and the prevalence of negative tumors is higher than in other carcinomas. Our immunofluorescence study of alpha-catenin expression in 20 RCC cell lines revealed a typical honeycomb staining pattern in all of the lines, whereas only six expressed E-cadherin. This suggested that other cadherins are expressed in RCC lines. Indeed, immunoprecipitation with an anti-alpha-catenin antibody resulted in coprecipitation of proteins of Mr 125,000-135,000. Using Western blot, these proteins react with a pan-cadherin antibody. To identify these cadherin related proteins, RT-PCR using degenerated primers and sequence comparisons was carried out. We then assessed the expression of the identified cadherins. N-cadherin mRNA was present in all cell lines; and cadherin 6 mRNA was seen in 16 lines. Cadherin 11 (mRNA) and E-cadherin (protein) were expressed in five and six lines, respectively. A cadherin 4 transcript was observed in only one line, whereas no P-cadherin protein could be detected. Expression of the four main cadherins was also found in normal kidney (two samples tested) and RCC specimens (four samples). Thus, RCC and normal kidney express a complex set of cadherins.

Prognostic value of cadherin-associated molecules (alpha-, beta-, and gamma-catenins and p120cas) in bladder tumors.

Loss of E-cadherin-mediated adhesion is an important step in the progression of many carcinomas. In model systems, it has been shown that cadherin function requires not only proper E-cadherin expression but also its linkage to the cytoskeleton through catenins. Hence, defects in catenins may cause defective E-cadherin function, and catenins as well as E-cadherin might constitute prognostic indicators. Here, we extend our previous study on E-cadherin in bladder cancer (Cancer Res., 53: 3241-3245, 1993). We have evaluated the expression of E-cadherin-associated cytoplasmic molecules (alpha-, beta-, and gamma-catenins and p120cas) to clarify whether or not the pattern of their expression could provide additional prognostic information beyond that from E-cadherin alone. Forty-eight frozen bladder tumor specimens and 9 samples of normal urothelium were studied by immunohistochemistry. A discrepancy between the E-cadherin and catenin expression pattern was seen in 20.8% of cases. Abnormal expression of each molecule is significantly correlated with tumor grade (P < 0.01) and stage (P < 0.01). Reduced expression of all of the molecules correlates with poor survival (P < 0.01 for each variable). Proportional hazard regression analysis showed that beta-catenin, E-cadherin, and alpha-catenin have strong predictive value, whereas plakoglobin and p120cas have a somewhat lower predictive value. Within patients with invasive tumors, those with a normal staining for either E-cadherin, alpha-catenin, or beta-catenin show a trend toward better survival. However, the difference in survival is significant only for E-cadherin (P < 0.05). Thus, beta-catenin, E-cadherin, and alpha-catenin have similar prognostic values. Therefore, from a practical point of view, the expression of any of these proteins can be of prognostic value for patients with bladder cancer.

Decreased E-cadherin expression is associated with poor prognosis in patients with prostate cancer.

Decreased levels of the cell-cell adhesion molecule E-cadherin are associated with loss of differentiation in a number of human carcinomas. However, the value of E-cadherin as a prognostic marker in these cancers is largely undetermined. A previous study of E-cadherin levels in prostate cancer revealed that almost 50% of tumors examined had reduced or absent levels of this protein (Umbas et al., Cancer Res., 52: 5104-5109, 1992). To determine the potential prognostic significance of this finding, prostate cancer specimens from 89 patients were evaluated immunohistochemically for E-cadherin expression, and the results were related to histopathological grade, tumor stage, presence of metastases, and survival. As previously observed, a significant inverse correlation was found between E-cadherin expression and tumor grade. Importantly, we also found significant correlations between E-cadherin expression and tumor stage and overall survival. Sixty-three percent of the tumors that extended beyond the prostate capsule (T3-4) versus 33% of the tumors confined to the prostate (T1-2) had aberrant expression (chi 2 = 8.1, P < 0.005). Seventy-six percent of the primary tumors from patients that presented with metastases showed aberrant staining compared to 32% from patients without metastases (chi 2 = 14.9; P < 0.001). The life table analysis showed a significantly higher survival rate for patients with normal staining compared to patients with aberrant expression (chi 2 = 20.4, P < 0.001 by log rank test). Moreover, abnormal expression of E-cadherin correlated significantly with progression after radical prostatectomy (P < 0.005). These results suggest that E-cadherin expression can serve as a prognostic indicator for the biological potential of prostate cancer.

Expression of cyclin D1 and EMS1 in bladder tumours; relationship with chromosome 11q13 amplification.

11q13 amplifications have been found in several cancers, including bladder tumours. However, the biological significance of this genetic alteration is not yet fully understood. To get more insight into the role of 11q13 amplification in bladder tumour development, we have studied the level of amplification and expression of 4 (protoonco)genes lying within the amplicon; cyclin D1, FGF3, FGF4 and EMS1 DNA amplification was found in 5/46 tumours. There was no correlation between amplification and clinico-pathological data. No expression of FGF3 and FGF4 was detected whereas both cyclin D1 and EMS1 were expressed at higher level in tumours with amplifications. Thus cyclin D1 and EMS1, but not FGF3 and FGF4, are likely to play a pathogenic role in the 11q13 amplification in bladder cancer. However, amplification is not the unique way of activation of these genes. Indeed, in situ hybridisation and Northern blot analysis have shown that most bladder tumours have a fair to high expression of cyclin D1 and EMS1 in contrast to normal urothelium with a moderate expression. Interestingly, a trend towards higher expression occurs in superficial versus invasive tumours (8.8 +/- 2.0 versus 1.9 +/- 0.4; P approximately equal to 13% for cyclin D1 and 4.5 +/- 1.4 versus 2.0 +/- 0.4; P approximately equal to 8% for EMS1). Moreover, the 9 tumours with low expression are all highly malignant, leading to the hypothesis that the tumours developing through a cyclin D1/EMS1 independent pathway are more aggressive.

Astrocytomas and choroid plexus tumors in two families with identical p53 germline mutations.

Germline p53 mutations carry an increased risk of development of breast cancer, soft tissue and osteosarcomas, brain tumors, leukemia and adrenocortical carcinomas. Cerebral neoplasms are usually of astrocytic lineage and occur in 40% of affected families. This report presents clinical, neuropathological and molecular genetic data from 2 families in France with an identical p53 germline mutation in codon 248 (CGG->TGG; Arg->Trp) and a clustering of CNS tumors. The youngest patient in each family developed a malignant choroid plexus tumor while several young adults of both kindred succumbed to low-grade astrocytoma, anaplastic astrocytoma or glioblastoma. The only non-neural neoplasm was an adrenocortical carcinoma in a boy aged 4 years who developed an anaplastic choroid plexus papilloma 2 years later. Of 2 previously reported inherited choroid plexus tumors, 1 occurred in a family which also carried a germline mutation in codon 248. It remains to be shown whether this unusual pattern of CNS tumors is due to an organ-specific effect of this particular p53 mutation or whether it reflects the genetic background of the affected families.

[Heterogeneity of nucleolar distribution in prostatic cancer. Comparison with the cytological grade and DNA content]. / Hétérogénéité de distribution des nucléoles dans le cancer de la prostate. Comparaison avec le grade cytologique et le contenu en ADN.

Thirty seven prostatic carcinomas at a D2 metastatic stage were studied after transrectal fine-needle aspiration in a prospective multicentric study. The number and the distribution of nucleoli were compared, in each cytological grade, to the flow cytometric nuclear DNA content. In 9 grade I, 13 grade II and 15 grade III the proportion of bimodal DNA profiles was 11%, 30.7% and 66.7%. The total number of nucleoli per 100 nuclei was respectively 114.11 (SD = 8.12), 158.31 (SD = 47.97) and 194.40 (SD = 58.42). A statistically significant difference (t = 2.78; P = 0.009) was found between the total number of nucleoli of unimodal (22 cases) and bimodal DNA profiles (15 cases). However, no significant difference in nucleolar parameters was found between unimodal (14 cases) and bimodal DNA profiles (14 cases) in cytological grades II and III. These results suggest that the nucleolar parameters do not necessarily parallel the increase in the nuclear DNA content, and that the two factors are independent of one another.

[Molecular biology studies of bladder cancer]. / Molekularbiologische Untersuchungen beim Blasenkarzinom.

The mechanisms that control the biological behaviour of urothelial cancer are complex, and many regulative interactions are involved. So far, few aspects of these control mechanisms have been recognized and characterized. A precondition for better understanding is knowledge the interaction of this factors. Some markers (e.g., chromosomal aberrations, EGFR expression) are correlated with progression of tumour. Whether they are the cause or the result of the aggressive behaviour of growth remains unknown. Only a few markers, especially in flow cytometry, will have any benefit in clinical routine. Whether it is possible to find a marker with prognostic value remains uncertain.

Classical cadherins in urological cancers.

Decreased E-cadherin expression assessed by immunohistochemistry correlates with poor survival of bladder and prostate cancer patients. The clinical usefulness of this parameter should therefore be evaluated in a large scale prospective study. In proximal kidney tubule and in its derived tumours cadherin-6 seems to take over E-cadherin function. Impaired E-cadherin function leads to increased invasive capacity of the cells. It has been shown that defective function can result from several mechanisms: mutation of the gene, alteration of transcription, post translational modifications or changes in the interaction of E-cadherin with cytoskeleton anchoring proteins: the catenins. A major mechanism leading to decreased E-cadherin expression in tumours lies in decreased transcription of the gene. Hence, a better understanding of the regulation of E-cadherin transcription might open avenues for therapy by restoring a normal expression.

Frequencies of KIT and PDGFRA mutations in the MolecGIST prospective population-based study differ from those of advanced GISTs.

Gastrointestinal stromal tumors (GISTs) are the most common human sarcoma. Most of the data available on GISTs derive from retrospective studies of patients referred to oncology centers. The MolecGIST study sought to determine and correlate clinicopathological and molecular characteristics of GISTs. Tumor samples and clinical records were prospectively obtained and reviewed for patients diagnosed in France during a 24-month period. Five hundred and ninety-six patients were included, of whom 10% had synchronous metastases. GISTs originated from the stomach, small bowel or other site in 56.4, 30.2 and 13.4% of cases, respectively. The main prognostic markers, tumor localization, size and mitotic index were not independent variables (P < 0.0001). Mutational status was determined in 492 (83%) patients, and 138 different mutations were identified. KIT and PDGFRA mutations were detected in 348 (71%) and 74 (15%) patients, respectively, contrasting with 82.8 and 2.1% in patients with advanced GIST (MetaGIST) (P < 0.0001). Further comparison of localized GISTs in the MolecGIST cohort with advanced GISTs from previous clinical trials showed that the mutations of PDGFRA exon18 (D842V and others) as well as KIT exon11 substitutions (W557R and V559D) were more likely to be seen in patients with localized GISTs (odds ratio 7.9, 3.1, 2.7 and 2.5, respectively), while KIT exon 9 502_503dup and KIT exon 11 557_559del were more frequent in metastatic GISTs (odds ratio of 0.3 and 0.5, respectively). These data suggest that KIT and PDGFRA mutations and standardized mitotic count deserve to be investigated to evaluate the relapse risk of GISTs.

[Incidence rate, epidemiology of sarcoma and molecular biology. Preliminary results from EMS study in the Rhône-Alpes region]. / Incidence, épidémiologie des sarcomes et biologie moléculaire. Résultats préliminaires de l'étude EMS en Rhône-Alpes.

Sarcomas comprise a heterogeneous group of mesenchymal neoplasms. They can be grouped into 3 general categories, soft tissue sarcoma, visceral and primary bone sarcoma, which have different staging and treatment approaches. Soft tissue sarcomas are typically classified on the basis of genetic alterations and light-microscopic examination of hematoxylin-eosin-stained tissue, in which recognizable morphological characteristics of normal tissues are identified. Sarcomas are further characterized by histologic grade. The 3 most important prognostic variables are grade, size, and location of the primary tumor. This review includes a discussion of both soft tissue sarcomas (unclassified sarcoma, liposarcoma, leiomyosarcoma, synovial sarcoma, dermatofibrosarcoma protuberans, angiosarcoma, Kaposi sarcoma, gastrointestinal stromal tumor, rhabdomyosarcoma, ...) and primary bone sarcomas (osteosarcoma, Ewing sarcoma and chondrosarcoma). The approach to a patient with a sarcoma begins with a biopsy that obtains adequate tissue for diagnosis without interfering with subsequent optimal definitive surgery. Subsequent treatment depends on the specific type of sarcoma. Due to the absence of clear knowledge for incidence rate, we conducted in 2005 and 2006 an exhaustive analysis of all diagnosed cases in the Rhône-Alpes region. Because sarcomas are relatively uncommon yet comprise a wide variety of different entities, second opinion was systematically performed for all included cases.

Defective E-cadherin function in urological cancers: clinical implications and molecular mechanisms.

Decreased E-cadherin expression assessed by immunohistochemistry correlates with poor survival of bladder and prostate cancer patients. The clinical usefulness of this parameter should therefore be evaluated in a large-scale prospective study. E-cadherin is an epithelial cell-cell adhesion molecule and impaired function presumably leads to increased invasive capacity of the cells. It has been shown that defective function can result from several mechanisms: mutation of the gene, alteration of transcription, posttranslational modification or changes in the interaction of E-cadherin with cytoskeleton anchoring proteins--the catenins. A major mechanism leading to decreased E-cadherin expression in tumors lies in decreased transcription of the gene. Hence, a better understanding of the regulation of E-cadherin transcription might open avenues for therapy by restoring normal expression.

[Analysis of tumor DNA by flow cytometry on prostate cyto-aspiration product. Value of routine cytologic evaluation]. / Analyse de l'ADN tumoral par cytométrie en flux sur produit de cyto-aspiration prostatique. Intérêt du contrôle cytologique de routine.

In this prospective study, flow cytometry DNA profile of 169 stage D2 prostatic carcinomas were compared with conventional cytologic data. Two transrectal fine-needle aspiration biopsies were performed in each patient. Aspirates were immediately fixed in 2% polyethylene glycol 1500* alcoholic solution (Merck). Suspensions were mixed and studied by a conventional cytologic technique (Papanicolaou) and by flow cytometry (propidium iodide stain on isolated nuclei). A highly significant correlation was found between the DNA profile and the cytologic grade (p less than 0.001). The DNA profile was bimodal in 14% (3/21) of aspirates containing benign or atypical cells, 18% (3/17) of grade I aspirates, 30% (13/43) of grade II aspirates and 71% (24/34) of grade III aspirates. Routine cytologic evaluation of cell suspensions evaluated by flow cytometry is important in clinical practice since both falsely unimodal and falsely bimodal profiles occur. Leukocytes or benign epithelial cells can interfere with the tumor cell population. In fine-needle aspirates, the broad range of non-malignant contaminating cells limits the value of immunocytochemistry and emphasizes the usefulness of routine conventional cytologic evaluation.