Molecular profile and clinical features of patients with gliomas using a broad targeted next generation-sequencing panel.

Gliomas are the most common malignant primary brain tumors characterized by poor prognosis. The genotyping of tumors using next generation sequencing (NGS) platforms enables the identification of genetic alterations that constitute diagnostic, prognostic and predictive biomarkers. The present study investigated the molecular profile of 32 tumor samples from 32 patients with high-grade gliomas by implementing a broad 80-gene targeted NGS panel while reporting their clinicopathological characteristics and outcomes. Subsequently, 14 of 32 tumor specimens were also genotyped using a 55-gene NGS panel to validate the diagnostic accuracy and clinical utility of the extended panel. The median follow-up was 19.2 months. In total, 129 genetic alterations including 33 structural variants were identified in 38 distinct genes. Among 96 variants (single nucleotide variants and insertions and deletions), 38 were pathogenic and 58 variants of unknown clinical significance. TP53 was the most frequently mutated gene, followed by PTEN and IDH1 genes. Glioma patients with IDH1 mutant tumors were younger and had significantly longer overall survival compared to patients with wild-type IDH1 tumors. Similarly, tumors with TP53 mutations were more likely observed in younger patients with glioma. Subsequently, a comparison of mutational profiles of samples analyzed by both panels was also performed. Implementation of the comprehensive pan-cancer and the MOL panels resulted in the identification of 37 and 15 variants, respectively. Of those, 13 were common. Comprehensive pan-cancer panel identified 24 additional variants, 22 of which were located in regions that were not targeted by the MOL panel. By contrast, the MOL panel identified two additional variants. Overall, the present study demonstrated that using an extended tumor profile assay instead of a glioma-specific tumor profile panel identified additional genetic changes that may be taken into consideration as potential therapeutic targets for glioma diagnosis and molecular classification.

CHEK2 Pathogenic Variants in Greek Breast Cancer Patients: Evidence for Strong Associations with Estrogen Receptor Positivity, Overuse of Risk-Reducing Procedures and Population Founder Effects.

CHEK2 germline pathogenic variants predispose to breast cancer and possibly to other malignancies, with their spectrum and frequency being variable among populations. Τhe majority of CHEK2-associated breast tumors are hormone receptor positive; however, relevant clinical outcomes are not well defined. Herein, we illustrate the histopathological characteristics and clinical outcomes of 52 Greek breast cancer patients who are CHEK2 carriers. Genetic analysis was performed by Sanger/massively parallel sequencing, followed by MLPA. Subsequent haplotype analysis investigated possible founder effects. Blood relatives were offered cascade testing. CHEK2 variant spectrum was characterized by variability, while influenced by founder effects. The majority of carriers, i.e., 60.8%, were diagnosed with breast cancer before the age of 45. Notably, 91.5% of breast tumors were hormone receptor positive. Hormone therapy and mastectomy at diagnosis seem to have a positive trend on overall survival, after a median follow-up of 9.5 years. Remarkably, 41.9% of patients underwent risk-reducing surgery, one third of which involved salpingo-oophorectomy. Nearly half of families responded to cascade testing. Our data highlight the need for guideline-adherent choices, based on the evidence that CHEK2 carriers are at moderate risk for breast cancer and no risk for ovarian cancer, while underscore the possible role of chemoprevention with tamoxifen.

Is There an Independent Role of TERT and NF1 in High Grade Gliomas?

BACKGROUND: High grade glioma molecular profiling is of particular interest in neurooncology. The role of telomerase reverse transcriptase (TERT) varies dependent upon other molecular parameters. We explored the role of TERT in 101 high-grade gliomas. METHODS: A total of 101 patients (pts) with grade III-IV gliomas treated with standard of care and informative tumor genotypes were included in the present study. Of 55 genes targeted with the next-generation sequencing panel, mutations (muts) were found in 37; these were included in the analysis. TERT mut were tested with Sanger sequencing. MGMT promoter methylation status was determined by methylation specific PCR. RESULTS: 270 mut were detected in 92/101 tumors (91.1%). TERT was the most frequently mutated gene (74.3%). IDH1/2 mut were mutually exclusive with mut in the neurofibromin 1 (NF1) gene. Mutated TERT was associated with wild-type (wt) IDH1/2 (p = 0.025). The 12-month overall survival (OS) rate was 74.3% (median OS: 22 months). Pts with TERT and NF1 wt had a median OS of 40.8 months, while among pts with NF1 wt/TERT mutant, the median OS was 18.5 months. NF1 and TERT mut univariately conferred shorter OS (HR = 3.19; p = 0.004 and HR = 2.28; p = 0.002). Upon multivariate analysis, mutated TERT showed marginal unfavorable prognostic significance for OS (p = 0.049), while NF1 lost its unfavorable significance (p = 0.151). CONCLUSIONS: TERT is herein proven to confer poor prognosis in high grade gliomas, independent of IDH and MGMT. NF1 seems to also confer poor prognosis although our small numbers do not allow for firm conclusions.

Bridging Cancer Biology with the Clinic: Comprehending and Exploiting IDH Gene Mutations in Gliomas.

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are enzymes that play a major role in the Krebs cycle. Mutations in these enzymes are found in the majority of lower gliomas and secondary glioblastomas, but also in myeloid malignancies and other cancers. IDH1 and IDH2 mutations are restricted to specific arginine residues in the active site of the enzymes and are gain-of-function, i.e. they confer a neomorphic enzyme activity resulting in the accumulation of D-2-hydroxyglutarate (2-HG). 2-HG is an oncometabolite causing profound metabolic dysregulation which, among others, results in methylator phenotypes and in defects in homologous recombination repair. In this review, we summarize current knowledge regarding the function of normal and mutated IDH, explain the possible mechanisms through which these mutations might drive malignant transformation of progenitor cells in the central nervous system, and provide a comprehensive review of potential treatment strategies for IDH-mutated malignancies, focusing on gliomas.

The impact of paclitaxel and carboplatin chemotherapy on the autonomous nervous system of patients with ovarian cancer.

BACKGROUND: Paclitaxel-based regimens are frequently associated with the development of peripheral neuropathy. The autonomous nervous system (ANS) effects, however, of this chemotherapeutic agent remain unexplored. METHODS: We investigated a group of 31 female patients with ovarian cancer receiving treatment with paclitaxel and carboplatin, as well as a group of 16 healthy age- and gender-matched healthy volunteers. All study participants completed a questionnaire and were assessed neurophysiologically at three time points (baseline, 3-4 months and 6-8 months following the onset of chemotherapy). The evaluation of the ANS included assessment of the adrenergic cardiovascular function (orthostatic hypotension-OH), parasympathetic heart innervation (30/15 ratio) and sympathetic skin response (SSR). RESULTS: At the 3-4 months ANS assessment, 19.2 % of the patients had systolic OH and the same percentage had diastolic OH, but at the 6-8 months evaluation no patient had systolic OH and only 13.8 % had diastolic OH. The values of the 30/15 ratio were significantly reduced at both time points, whereas the SSR was not affected. CONCLUSIONS: Combined paclitaxel and carboplatin chemotherapy is associated with significant effects on the parasympathetic heart innervation and occasionally with effects on the adrenergic cardiovascular reaction. The SSR remained unaffected. Physicians should be alert to the possibility of these treatment-emergent side effects, so as to monitor ANS parameters and introduce treatment modifications accordingly. Our findings however, should be validated in larger cohorts.

Therapies in the pipeline for small-cell lung cancer.

INTRODUCTION OR BACKGROUND: Small-cell lung cancer (SCLC) represents ~15% of all cases of lung cancer and is characterized by a rapid tumour doubling time, early onset disease dissemination and high sensitivity to chemotherapy. SOURCES OF DATA: We searched MEDLINE and OVID databases for articles in English published from January 1980 to February 2015. AREAS OF AGREEMENT: Platinum-based chemotherapy, thoracic radiotherapy and prophylactic cranial irradiation are standard of care. Benefit from second-line chemotherapy is limited. AREAS OF CONTROVERSY: The role of platinum/irinotecan chemotherapy in the Western population and the role of maintenance therapies remain to be established. GROWING POINTS: Knowledge of the biology of SCLC has expanded exponentially and many potential therapeutic targets have been identified. AREAS TIMELY FOR DEVELOPING RESEARCH: The use of circulating tumour cells can help investigating molecular alterations occurring within tumour cells, understanding drug resistance mechanisms and evaluating new treatments.

Overcoming resistance to first/second generation epidermal growth factor receptor tyrosine kinase inhibitors and ALK inhibitors in oncogene-addicted advanced non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) activating mutations and anaplastic lymphoma kinase (ALK) gene rearrangement in advanced non-small cell lung cancer (NSCLC) represent the two oncogenic events with an impact on current clinical practice. EGFR tyrosine kinase inhibitors (TKIs) and crizotinib are the standard of care for the treatment of EGFR mutant and ALK gene rearranged advanced NSCLC patients. Unfortunately, despite initial clinical benefit, acquired resistance to EGFR-TKIs or crizotinib usually develops after an average of 10-12 months of treatment. The aim of this review is to describe the mechanisms of resistance to first/second generation EGFR-TKIs and crizotinib. In particular, we focus on strategies to overcome resistance due to secondary EGFR T790M mutation and mutations of the ALK domain.

Management of NSCLC Disease Progression After First-Line EGFR Tyrosine Kinase Inhibitors: What Are the Issues and Potential Therapies?

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represent the standard of care for advanced non-small cell lung cancer (NSCLC) patients whose tumor harbors an activating EGFR mutation. The vast majority of patients will experience disease control with an EGFR-TKI but inevitably all patients will progress, often within a year of treatment. There is no current standard of care for this scenario but, in clinical practice, most of the patients will be offered platinum-based doublet chemotherapy. In some situations, continuation of the EGFR-TKI beyond radiological progression, with or without use of local treatments in case of oligo-progressive disease, represents a reasonable therapeutic option. The aim of this review is to describe the different treatment strategies that have been developed to tackle progression on EGFR-TKIs, including specific clinical scenarios and novel agents designed to tackle the common T790M resistance mutation.

EGFR gene gain and PTEN protein expression are favorable prognostic factors in patients with KRAS wild-type metastatic colorectal cancer treated with cetuximab.

INTRODUCTION: Cetuximab is a monoclonal epidermal growth factor receptor (EGFR)-targeting antibody, used in the treatment of colon cancer. KRAS mutation status is strongly predictive of cetuximab efficacy, but more predictive factors are needed for better patient selection. PTEN is a downstream inhibitor of the EGFR pathway and has been evaluated as a predictive factor of cetuximab efficacy in colorectal cancer. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tumor tissue samples were collected from 226 patients with advanced or metastatic colorectal cancer that had been treated with cetuximab. Clinical information was collected retrospectively from the patients' medical records. After central evaluation, 147 cases with adequate material were eligible for further evaluation. EGFR and PTEN status was evaluated with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Data were associated with cetuximab treatment outcome. Additional analysis was performed with previously published data on PIK3CA, BRAF and KRAS mutation status and EGFR ligand amphiregulin (AREG) and epiregulin intratumoral mRNA expression levels. PIK3CA mutation status and PTEN protein expression were also analyzed as a single complex parameter, to evaluate the predictive value of PI3K/PTEN axis dysfunction as one entity. RESULTS: Analysis showed a borderline association of overall response rate (ORR) and time to progression (TTP) with EGFR protein overexpression by IHC (p = 0.059 and p = 0.057, respectively) and a positive association of EGFR gain by FISH (found in only five cases) with longer TTP (p = 0.026). No association was found between ORR or TTP and PTEN IHC or FISH status. Comparative analysis with previously published data showed that PTEN protein expression is associated with longer TTP in patients with wild-type (WT) KRAS (p = 0.036) and especially in the ones with elevated AREG levels (p = 0.046), as well as in patients with both KRAS and BRAF WT (p = 0.019). Patients with both PIK3CA WT and PTEN protein expression had significantly longer TTP (p = 0.010) versus all others, in the absence of BRAF and KRAS mutations, a finding which persisted in the KRAS WT/AREG high subgroup (p = 0.046). CONCLUSIONS: In this cetuximab-treated colorectal cancer population, EGFR gain was associated with better outcome and PTEN protein expression with longer TTP in KRAS WT, KRAS WT/AREG high and KRAS/BRAF WT subpopulations. Cetuximab efficacy is greater with intact and activated EGFR signaling, without activating mutations of KRAS/BRAF and in the presence of preserved PTEN inhibitory activity upon the PI3K/AKT pathway. These results reflect a solid biological rationale and warrant further evaluation of the predictive role of PTEN in prospective studies.

Ileal inflammation may trigger the development of GP2-specific pancreatic autoantibodies in patients with Crohn's disease.

Why zymogen glycoprotein 2 (GP2), the Crohn's disease (CD)-specific pancreatic autoantigen, is the major target of humoral autoimmunity in inflammatory bowel diseases (IBD) is uknown. Recent evidence demonstrates that GP2 is also present on the apical surface of microfold (M) intestinal cells. As the colon lacks GP2-rich M cells, we assumed that patients with colonic CD are seronegative for anti-GP2. Anti-GP2 antibodies were tested in 225 CDs, including 45 patients with colonic location (L2), 45 with terminal ileum (L1) and 135 with ileocolonic involvement; 225 patients with ulcerative colitis (UC) were also tested. Anti-GP2 reactivity was detected in 59 (26.2%) CDs and 15 (6.7%) UCs (P < 0.001). Only 5 CDs with L2 had anti-GP2 antibodies, compared to 54/180 (30.0%, P = 0.0128) of the CDs with L1 and L3. Anti-GP2 antibody positive CD patients had higher ASCA titres compared to seronegative cases. Amongst the 128 CD patients with previous surgical intervention, 45 (35.0%) were anti-GP2 antibody positive compared to 14/97 (14.0%) without surgical (P < 0.001). Our data support the assumption that ileal inflammation is required for the development of anti-GP2 antibodies in CD, and suggest that the intestine rather than the pancreatic juice is the antigenic source required for the initiation of anti-GP2 antibodies.

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis.

BACKGROUND: Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody--the serological marker of type 2 autoimmune hepatitis (AIH-2)--is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1)--either in association with anti-LKM1, or in isolation--and anti-soluble liver antigen antibodies (anti-SLA) have been considered as useful and specific diagnostic markers for AIH. However, their specificity for AIH has been questioned by some recent studies, which have shown the detection of anti-LC1 and anti-SLA by immunoprecipitation assays in HCV patients irrespective of their anti-LKM1 status. The aim of the present study was to test the anti-LC1 and anti-SLA presence by specific enzyme linked immunosorbent assays (ELISAs), in a large group of Greek HCV-infected patients with or without anti-LKM1 reactivity as firstly, immunoprecipitation assays are limited to few specialized laboratories worldwide and cannot be used routinely and secondly, to assess whether application of such tests has any relevance in the context of patients with viral hepatitis since antibody detection based on such ELISAs has not been described in detail in large groups of HCV patients. METHODS: One hundred and thirty eight consecutive HCV patients (120 anti-LKM1 negative and 18 anti-LKM1 positive) were investigated for the presence of anti-LC1 and anti-SLA by commercial ELISAs. A similar number (120) of chronic hepatitis B virus (HBV) infected patients seronegative for anti-LKM1 was also tested as pathological controls. RESULTS: Six out of 18 (33%) anti-LKM(pos)/HCV(pos) patients tested positive for anti-LC1 compared to 1/120 (0.83%) anti-LKM(neg)/HCV(pos) patients and 0/120 (0%) of the anti-LKM1(neg)/HBV(pos) patients (p < 0.001 for both comparisons). Anti-SLA antibodies were not present in any of the HCV (with or without anti-LKM1) or HBV-infected patients. CONCLUSION: We showed that anti-LC1 and anti-SLA autoantibodies are not detected by conventional assays in a large group of anti-LKM1 negative patients with chronic hepatitis B and C infections. Based on these results we cannot find any justification for the application of anti-LC1 and anti-SLA tests in the routine laboratory testing of viral hepatitis-related autoantibody serology with the only potential exception being the anti-LC1 screening in anti-LKM1(pos)/HCV(pos) patients.