Microsatellite Instability Is Insufficiently Used as a Biomarker for Lynch Syndrome Testing in Clinical Practice.

PURPOSE: The pan-cancer presence of microsatellite instability (MSI)-positive tumors demonstrates its clinical utility as an agnostic biomarker for identifying immunotherapy-eligible patients. Additionally, MSI is a hallmark of Lynch syndrome (LS), the most prevalent cancer susceptibility syndrome among patients with colorectal and endometrial cancer. Therefore, MSI-high results should inform germline genetic testing for cancer-predisposing genes. However, in clinical practice, such analysis is frequently disregarded. METHODS: A next-generation sequencing (NGS)-based technique was used for MSI analysis in 4,553 patients with various tumor types. Upon request, somatic BRAF gene analysis was conducted. In addition, hereditary testing of cancer-associated genes was performed in MSI-high cases using a capture-based NGS protocol. MLH1 promoter methylation analysis was conducted retrospectively in patients with colorectal and endometrial cancer to further investigate the origin of MSI at the tumor level. RESULTS: The MSI positivity rate for the entire cohort was 5.27%. Endometrial, gastric, colorectal, urinary tract, and prostate cancers showed the highest proportion of MSI-high cases (15.69%, 8.54%, 7.40%, 4.55%, and 3.19%, respectively). A minority of 45 patients (22.73%) among the MSI-high cases underwent germline testing to determine whether the mismatch repair pathway deficiency was inherited. 24.44% of those who performed the genetic test carried a pathogenic variant in an LS-associated gene. Three MSI-high individuals had non-LS gene alterations, including BRCA1, BRCA2, and CDKN2A pathogenic variants, indicating the presence of non-LS-associated gene alterations among MSI-high patients. CONCLUSION: Although MSI analysis is routinely performed in clinical practice, as many as 77% of MSI-high patients do not undergo LS genetic testing, despite international guidelines strongly recommending it. BRAF and MLH1 methylation analysis could shed light on the somatic origin of MSI in 42.50% of the MSI-high patients; however, MLH1 analysis is barely ever requested in clinical practice.

RediScore: Prospective validation of a pipeline for homologous recombination deficiency analysis.

Tumors harboring homologous recombination deficiency (HRD) are considered optimal candidates for poly(ADP-ribose) polymerase 1 (PARP) inhibitor treatment. Such deficiency can be detected by analyzing breast cancer type (BRCA)1/2 gene mutations, as well as mutations in other genes of the homologous recombination pathway. The algorithmic measurement of the HRD effect by identifying genomic instability (GI) has been used as biomarker. As compared with the direct measurement of somatic gene alterations, this approach increases the number of patients who could benefit from PARP inhibitor treatment. In the present study, the performance of the Oncoscan CNV assay, accompanied by appropriate bioinformatic algorithms, was evaluated for its performance in GI calculation and was compared with that of a validated next-generation sequencing (NGS) test (myChoice HRD test). In addition, the clinical utility of the GI score (GIS) and BRCA1/2 tumor analysis were investigated in a cohort of 444 patients with ovarian cancer. For that reason, single nucleotide polymorphism (SNP) arrays and appropriate bioinformatics algorithms were used to calculate GIS in 29 patients with ovarian cancer with known GIS status using a validated NGS test. Furthermore, BRCA1/2 analysis results were compared between the aforementioned assay and the amplicon-based Oncomine™ BRCA Research Assay. BRCA1/2 analysis was performed in 444 patients with ovarian cancer, while GIS was calculated in 175 BRCA1/2-negative cases. The bioinformatics algorithm developed for GIS calculation in combination with NGS BRCA1/2 analysis (RediScore), and the OncoscanR pipeline exhibited a high overall agreement with the validated test (93.1%). In addition, the Oncomine NGS assay had a 100% agreement with the validated test. The BRCA1/2 mutation frequency was 26.5% in the examined patients with ovarian cancer. GIS was positive in 40% of the BRCA1/2-negative cases. The RediScore bioinformatics algorithm developed for GIS calculation in combination with NGS BRCA1/2 analysis is a viable and effective approach for HRD calculation in patients with ovarian cancer, offering a positive prediction for PARP inhibitor responsiveness in 55% of the patients.

Revisiting the Implications of Positive Germline Testing Results Using Multi-gene Panels in Breast Cancer Patients.

BACKGROUND/AIM: The use of multi-gene panels for germline testing in breast cancer enables the estimation of cancer risk and guides risk-reducing management options. The aim of this study was to present data that demonstrate the different levels of actionability for multi-gene panels used in genetic testing of breast cancer patients and their family members. MATERIALS AND METHODS: We performed an analysis in our clinical database to identify breast cancer patients undergoing genetic testing. We reviewed positive results in respect of risk estimation and management, cascade family testing, secondary findings and information for treatment decision-making. RESULTS: A total of 415 positive test reports were identified with 57.1%, 18.1%, 10.8% and 13.5% of individuals having pathogenic/likely pathogenic variants in high, moderate, low and with insufficient evidence for breast cancer risk genes, respectively. Six point seven percent of individuals were double heterozygotes. CONCLUSION: Germline findings in 92% of individuals are linked to evidence-based treatment information and risk estimates for predisposition to breast and/or other cancer types. The use of germline findings for treatment decision making expands the indication of genetic testing to include individuals that could benefit from targeted treatments.

Analysis of hereditary cancer syndromes by using a panel of genes: novel and multiple pathogenic mutations.

BACKGROUND: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single-gene analysis of specific high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. The application of Next Generation Sequencing (NGS) technology has facilitated multigene panel analysis and is widely used in clinical practice, for the identification of individuals with cancer predisposing gene variants. The purpose of this study was to investigate the extent and nature of variants in genes implicated in hereditary cancer predisposition in individuals referred for testing in our laboratory. METHODS: In total, 1197 individuals from Greece, Romania and Turkey were referred to our laboratory for genetic testing in the past 4 years. The majority of referrals included individuals with personal of family history of breast and/or ovarian cancer. The analysis of genes involved in hereditary cancer predisposition was performed using a NGS approach. Genomic DNA was enriched for targeted regions of 36 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated by computational analysis and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A pathogenic variant was identified in 264 of 1197 individuals (22.1%) analyzed while a variant of uncertain significance (VUS) was identified in 34.8% of cases. Clinically significant variants were identified in 29 of the 36 genes analyzed. Concerning the mutation distribution among individuals with positive findings, 43.6% were located in the BRCA1/2 genes whereas 21.6, 19.9, and 15.0% in other high, moderate and low risk genes respectively. Notably, 25 of the 264 positive individuals (9.5%) carried clinically significant variants in two different genes and 6.1% had a LGR. CONCLUSIONS: In our cohort, analysis of all the genes in the panel allowed the identification of 4.3 and 8.1% additional pathogenic variants in other high or moderate/low risk genes, respectively, enabling personalized management decisions for these individuals and supporting the clinical significance of multigene panel analysis in hereditary cancer predisposition.

Tumor molecular profiling of NSCLC patients using next generation sequencing.

Non­small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with a broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using next generation sequencing (NGS) technology, has become a key tool for facilitating treatment decisions and the clinical management of NSCLC patients. The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq™ technology, was evaluated for the analysis of tumor DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Furthermore, the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. The mutation spectrum of the tumors was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. The panel used for tumor DNA analysis in this study exhibited high rates (100%) of sensitivity, specificity and reproducibility at a mutation allelic frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer-driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 23% presented a mutation in a gene associated with approved or emerging targeted therapy. More specifically, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK, ROS1) and 9.4% (47/502) had an alteration in a gene related to emerging targeted therapies (ERBB2, BRAF, MET and RET). Furthermore, 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or indicative for access to a clinical trial. Thus, the targeted NGS panel used in this study is a reliable approach for tumor molecular profiling and can be applied in personalized treatment decision making for NSCLC patients.

A unique gene expression signature is significantly differentially expressed in tumor-positive or tumor-negative sentinel lymph nodes in patients with melanoma.

The purpose of this study was to learn whether molecular characterization through gene expression profiling of node-positive and node-negative sentinel lymph nodes (SLNs) in patients with clinical stage I and II melanoma may improve the understanding of mechanisms of metastasis and identify gene signatures for SLNs/SLNs that correlate with diagnosis or clinical outcome. Gene expression profiling was performed on SLN biopsies of 48 (24 SLN and 24 SLN) patients (T3a/b-T4a/b) who underwent staging of SLNs using transcriptome profiling analysis on 5 µm sections of fresh SLNs. U133A 2.0 Affymetrix gene chips were used. Significance analysis of microarrays was used to test the association between gene expression level and SLN status. Genes with fold change more than 1.5 and q value less than 0.05 were considered differentially expressed. Pathway analysis was performed using Ingenuity Pathway Analysis. The Benjamini and Hochberg method was used to adjust for multiple testing in pathway analysis. We identified 89 probe sets that were significantly differentially expressed (1.5-27-fold; q<0.05). Upon performing the pathway analysis, it was found that 25 genes were common among the most significant and biologically relevant canonical pathways. The molecules and pathways that achieved differential expression of highest statistical significance were notably related to melanoma and its microenvironment and to signaling pathways implicated in immunosuppression and development of cancer. A 25-gene signature is significantly differentially expressed between SLN and SLN and is related to melanoma oncogenesis and immunosuppression. The identified expression profile provides a signature of melanoma nodal involvement. These findings warrant further investigation into the mechanisms of metastasis, melanoma metastasis diagnosis, and prediction of outcome.

Phase 1/2 study of rilotumumab (AMG 102), a hepatocyte growth factor inhibitor, and erlotinib in patients with advanced non-small cell lung cancer.

BACKGROUND: Activation of the mesenchymal-epidermal transition factor (MET) tyrosine kinase and its ligand, hepatocyte growth factor (HGF), is implicated in resistance to epidermal growth factor receptor (EGFR) inhibitors. In this phase 1/2 trial, rilotumumab (an anti-HGF antibody) combined with erlotinib was evaluated in patients with metastatic, previously treated non-small cell lung cancer. METHODS: In phase 1, a dose de-escalation design was adopted with rilotumumab starting at 15 mg/kg intravenously every 3 weeks and oral erlotinib 150 mg daily. In phase 2, the disease control rate (DCR) (according to Response Evaluation Criteria in Solid Tumors) of the combination was evaluated using a Simon 2-stage design. The biomarkers examined included 10 plasma-circulating molecules associated with the EGFR and MET pathways. RESULTS: Without indications for de-escalation, the recommended phase 2 dose was dose level 0. Overall, 45 response-evaluable patients were enrolled (13 with squamous carcinoma, 32 with adenocarcinoma; 2 had confirmed EGFR mutations, 33 had confirmed wild-type [WT] EGFR, and 7 had KRAS mutations). The DCR for all patients was 60% (90% confidence interval [CI], 47.1%-71.3%). Median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 6.6 months (90% CI, 5.6-8.9 months). Among patients with WT EGFR, the DCR was 60.6% (90% CI, 46.3%-73.3%), median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 7.0 months (90% CI, 5.6-13.4 months). Elevated baseline levels of neuregulin 1 were associated with longer progression-free survival (hazard ratio, 0.41; 95% CI, 0.19-0.87), whereas elevated amphiregulin levels were associated with more rapid progression (hazard ratio, 2.14; 95% CI, 1.48-3.08). CONCLUSIONS: Combined rilotumumab and erlotinib had an acceptable safety profile, and the DCR met the prespecified criteria for success. In the EGFR WT group, the DCR exceeded published reports for erlotinib alone. High circulating levels of neuregulin 1 may indicate sensitivity to this combination. Cancer 2017;123:2936-44. © 2017 American Cancer Society.

Suppression of Lymphocyte Functions by Plasma Exosomes Correlates with Disease Activity in Patients with Head and Neck Cancer.

Purpose: Head and neck cancers (HNCs) often induce profound immunosuppression, which contributes to disease progression and interferes with immune-based therapies. Body fluids of patients with HNC are enriched in exosomes potentially engaged in negative regulation of antitumor immune responses. The presence and content of exosomes derived from plasma of patients with HNC are evaluated for the ability to induce immune dysfunction and influence disease activity.Experimental Design: Exosomes were isolated by size-exclusion chromatography from plasma of 38 patients with HNC and 14 healthy donors. Morphology, size, numbers, and protein and molecular contents of the recovered exosomes were determined. Coculture assays were performed to measure exosome-mediated effects on functions of normal human lymphocyte subsets and natural killer (NK) cells. The results were correlated with disease stage and activity.Results: The presence, quantity, and molecular content of isolated, plasma-derived exosomes discriminated patients with HNC with active disease (AD) from those with no evident disease (NED) after oncologic therapies. Exosomes of patients with AD were significantly more effective than exosomes of patients with NED in inducing apoptosis of CD8+ T cells, suppression of CD4+ T-cell proliferation, and upregulation of regulatory T-cell (Treg) suppressor functions (all at P < 0.05). Exosomes of patients with AD also downregulated NKG2D expression levels in NK cells.Conclusions: Exosomes in plasma of patients with HNC carry immunosuppressive molecules and interfere with functions of immune cells. Exosome-induced immune suppression correlates with disease activity in HNC, suggesting that plasma exosomes could be useful as biomarkers of HNC progression. Clin Cancer Res; 23(16); 4843-54. ©2017 AACR.

Anticancer Cytokines: Biology and Clinical Effects of Interferon-α2, Interleukin (IL)-2, IL-15, IL-21, and IL-12.

Efforts over nearly four decades have focused on ways to use cytokines to manipulate the host immune response towards cancer cell recognition and eradication. Significant advances were achieved with interleukin-2 (IL-2) and interferon-α (IFN-α), primarily in the treatment of patients with melanoma and renal cell carcinoma. However, the utility of other cytokines showing promise in the preclinical setting has not been established largely because of toxicity, the complex functionality of each cytokine and the difficulty mimicking in preclinical models the human environment. Here, we review the basic biology and the clinical experiences with IFN-α, IL-2, IL-15, IL-21, and IL-12. We will also review ongoing clinical trials and discuss future directions including potential use of cytokines in combination with other effective immunotherapy approaches that have come of age in recent years.

Combination chemotherapy with docetaxel, vinorelbine and estramustine phosphate in metastatic androgen-resistant prostate cancer: a single institution experience.

UNLABELLED: The aim of this study was to evaluate the activity and toxicity of docetaxel, vinorelbine and oral estramustine in androgen-resistant prostate cancer (ARPC). PATIENTS AND METHODS: Fifty-two eligible patients were treated with docetaxel at 30 mg/m2 (day 1 and 8), vinorelbine at 20 mg/m2 (day 1 and 8), and oral estramustine of 280 mg p.o. (daily on days 1 to 7) every 3 weeks for 12 cycles. Patients with osseous metastases received zoledronic acid of 4 mg every 3 weeks. Low molecular weight heparin was administered on a prophylaxis basis to all patients. RESULTS: A prostate-specific antigen (PSA) response > or = 50% from baseline was obtained in 29 (56%; 95% confidence interval [CI], 42-70%) patients. Objective responses among the 25 patients with measurable disease were observed in 48% (95% CI, 27-69%), including 1 patient with complete response (CR) and 11 patients with partial response (PR). Patients with extraosseous only, skeletal only, and extraosseous and skeletal metastases showed different PSA responses (87% vs. 44% vs. 59%, respectively, p = 0.094). Furthermore, patients with soft tissue disease only showed insignificantly better PSA response than those with skeletal metastases (response rate: 87% vs. 50%, p = 0.064). The median progression-free survival was 7.6 months (95% CI, 6.7-8.4 months) and the median overall survival was 18.2 months (95% CI, 15.5-20.8 months). The only parameters which were found to have an impact on survival were the extent of disease and the baseline levels of PSA. Toxicity was generally mild except for myelotoxicity. Neutropenia grade 3/4 was recorded in 33% of patients and 6% experienced febrile neutropenia. Anemia and thrombocytopenia grade 3 or 4 were not a problem. Three patients (6%) developed grade 3 sensory neuropathy and 2 patients (4%) developed grade 3 fatigue. Edema grade 3 occurred in 1 (2%) patient and thromboembolism grade 3 occurred in 2 (4%) patients. CONCLUSION: The combination of docetaxel, vinorelbine and oral estramustine is a well-tolerated regimen with high biochemical and objective response rates in patients with ARPC.