Real-World Data and Clinical Implications of Next-Generation Sequencing (NGS)-Based Analysis in Metastatic Breast Cancer Patients.

Over the last two decades, the use of Next-Generation Sequencing (NGS) in medical oncology has increased the likelihood of identifying druggable mutations that may be potentially susceptible to targeted treatments. The European Society for Medical Oncology (ESMO) currently does not recommend the use of the NGS test to determine the therapeutic course of patients with metastatic breast cancer (mBC) in daily clinical practice. However, the aim of this work is to evaluate the potential contribution of the NGS test in selecting targeted therapies for patients with mBC. Data were retrospectively collected from 101 patients diagnosed with metastatic breast cancer and treated at the Modena Cancer Center between January 2015 and April 2022. A NGS test was performed on the tumor tissue of each patient at the Laboratory of Molecular Pathology of the University Hospital of Modena. This study analyzed the clinical-pathological characteristics and mutational profile of the population using NGS tests, with a focus on actionable mutations that could be targeted in advanced stages of clinical development. The indicator of this study was to quantify the actionable mutations that resulted in a change of cancer treatment. In total, 101 patients with metastatic breast cancer were analyzed, including 86 with luminal phenotype, 10 who were HER2-positive and 5 who were triple-negative. Median age was 52 years. NGS analysis was conducted on 47 samples of primary breast cancer, 52 on metastatic sites of disease and 2 on liquid biopsies. A total of 85 gene mutations were found. The most common mutations were identified in the PIK3CA (47%), FGFR (19%) and ERBB2 genes (12%), and to a lesser extent in other genes. Of the 61 patients with pathogenic mutations, 46 (75%) had at least one actionable mutation. Of these, nine received treatment with a molecular target drug: eight patients with a mutation of the PIK3CA gene were treated with alpelisib and fulvestrant; one patient with FGFR1/2 amplifications received TAS120. Median PFS for these patients was 3.8 months. The study results show that using the NGS test on cancer tissue of metastatic breast cancer could influence the therapeutic choices, considering the small sample size and limited follow-up. About 9% of the study population had their therapy modified based on the results of NGS. The growing number of detectable mutations and increased accessibility of the test may lead to a greater number of potential therapeutic implications for the NGS assay. Perspectives suggest that NGS analysis can be implemented in daily clinical practice, particularly in contexts where a Molecular Tumor Board (MTB) is active.

Clinicopathological, cytogenetic, and molecular profiles of primary cutaneous diffuse large B-cell lymphomas.

We analyzed the clinicopathological, cytogenetic, and molecular features of 18 primary cutaneous diffuse large B-cell lymphomas (PCDLBCLs) and 15 DLBCLs secondarily localized to the skin (SCDLBCLs), highlighting biological similarities and differences between the 2 groups. PCDLBCLs were subclassified after histopathological review as PCDLBCL-leg type (PCDLBCL-LT, 10 cases) and the PCDLBCL-not otherwise specified (PCDLBCL-NOS, 8 cases). Immunohistochemistry for Hans' algorithm markers, BCL2, and MYC was performed. The molecular study included the determination of the cell of origin (COO) by Lymph2Cx assay on NanoString platform, FISH analysis of IgH, BCL2, BCL6, and MYC genes, as well as the mutation analysis of MYD88 gene. In immunohistochemistry analysis, BCL2 and MYC hyperexpression was more frequent in LT than in NOS cases and, according to Hans' algorithm, PCDLBCL-LTs were mostly of the non-GC type (8/10), whereas in PCDLBCL-NOS, the GC type prevailed (6/8). The determination of COO using Lymph2Cx supported and further confirmed these results. In FISH analysis, all but one LT cases versus 5 of 8 PCDLBCL-NOS showed at least one gene rearrangement among IgH, BCL2, MYC, or BCL6. In addition, MYD88 mutations were more frequently present in LT than in NOS subtypes. Interestingly, MYD88-mutated patients were older, with a non-GC phenotype and had worse OS, compared to MYD88 WT cases. Overall, SCDLBCL did not show, at the genetic and expression level, different profiles than PCDLBCL, even if they bear a significantly worse prognosis. At survival analysis, the most important prognostic factors in patients with PCDLBCL were age and MYD88 mutation, whereas relapse and high Ki-67 expression were relevant in patients with SCDLBCL. Our study comprehensively analyzed the clinicopathological and molecular features of PCDLBCL-LT, PCDLBCL-NOS, and SCDLBCL, underlining the differences among them and the importance of properly identifying these entities at the time of diagnosis.

CtDNA-guided rechallenge with anti-EGFR therapy in RASwt metastatic colorectal cancer: Evidence from clinical practice.

AIM: To apply extended ctDNA-based RAS genotyping to clinical criteria for improving the selection of patients eligible for anti-EGFR-based rechallenge in a real-world setting. METHODS: ctDNA testing was prospectively applied to RASwt mCRC progressed after a first-line anti-EGFR-containing regimen and at least one other line. The primary endpoint was the objective response rate. RESULTS: Among ten enrolled patients, the anti-EGFR rechallenge resulted in an objective response rate and disease control rate of 70% and 90%. The median progression-free survival was 11.3 months and overall survival was not reached. Compared with a historical cohort retreated with anti-EGFR agents based on clinical criteria, the ctDNA-driven approach resulted in a higher chance of achieving an objective response and longer survival. CONCLUSIONS: Blood-based RASwt status may enrich metastatic colorectal cancer more likely to benefit from anti-EGFR-based rechallenge. RAS genotyping in ctDNA represents a feasible, fast, and cost-effective tool to be implemented in the clinic for advancing precision medicine.

Proteomic profiling of formalin-fixed paraffine-embedded tissue reveals key proteins related to lung dysfunction in idiopathic pulmonary fibrosis.

Introduction: Idiopathic pulmonary fibrosis (IPF) severely affects the lung leading to aberrant deposition of extracellular matrix and parenchymal stiffness with progressive functional derangement. The limited availability of fresh tissues represents one of the major limitations to study the molecular profiling of IPF lung tissue. The primary aim of this study was to explore the proteomic profiling yield of archived formalin-fixed paraffin-embedded (FFPE) specimens of IPF lung tissues. Methods: We further determined the protein expression according to respiratory functional decline at the time of biopsy. The total proteins isolated from 11 FFPE samples of IPF patients compared to 3 FFPE samples from a non-fibrotic lung defined as controls, were subjected to label-free quantitative proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) and resulted in the detection of about 400 proteins. Results: After the pairwise comparison between controls and IPF, functional enrichment analysis identified differentially expressed proteins that were involved in extracellular matrix signaling pathways, focal adhesion and transforming growth factor ß (TGF-ß) signaling pathways strongly associated with IPF onset and progression. Five proteins were significantly over- expressed in the lung of IPF patients with either advanced disease stage (Stage II) or impaired pulmonary function (FVC<75, DLCO<55) compared to controls; these were lymphocyte cytosolic protein 1 (LCP1), peroxiredoxin-2 (PRDX2), transgelin 2 (TAGLN2), lumican (LUM) and mimecan (OGN) that might play a key role in the fibrogenic processes. Discussion: Our work showed that the analysis of FFPE samples was able to identify key proteins that might be crucial for the IPF pathogenesis. These proteins are correlated with lung carcinogenesis or involved in the immune landscape of lung cancer, thus making possible common mechanisms between lung carcinogenesis and fibrosis progression, two pathological conditions at risk for each other in the real life.

Therapeutic Outcomes and Clinical Features of Advanced Non-Small Cell Lung Cancer Carrying KRAS Mutations: A Multicenter Real-life Retrospective Study.

INTRODUCTION: Targeting Kirsten Rat Sarcoma (KRAS) has been deemed impossible for long time, but new drugs have recently demonstrated promising results. Evidence on the outcome of KRAS-mutant advanced-NSCLC treated with new standard regimens are still scarce. Thus, we aimed at assessing the incidence and clinical impact of KRAS mutations in a real-life population of advanced-NSCLC, exploring the prognostic significance of distinct alterations. MATERIALS AND METHODS: The present multicenter retrospective study, conducted by 5 Italian Centers from January 2018 to February 2020, involved 297 advanced KRAS mutant NSCLC. Complete clinico-pathological data were evaluated. RESULTS: Out of 297 patients, 130 carried KRAS_G12C mutation, while 167 presented with mutations other than G12C. Within KRAS_non-G12C group, 73%, 16.8% and 8.9% harboured G12X, codon 13 and Q61H alterations, respectively. No significant differences in survival outcome and treatment response were documented according to KRAS_G12C versus non-G12C, nor KRAS_G12C versus G12X versus other mutations. On univariate analysis ECOG PS, number and sites of metastatic lesions and PD-L1 status significantly impacted on survival. A clear trend towards worse prognosis was apparent in chemotherapy-treated patients, while immunotherapy-based regimens were associated to prolonged survival. Investigating the outcome of PD-L1 ≥ 50% population, we did not detect any significant difference between KRAS_G12C and non-G12C subsets. CONCLUSION: Here, we report on real-life data from a large retrospective cohort of advanced NSCLC harbouring KRAS alterations, with particular attention to G12C mutation. Our study offers useful clues on survival outcome, therapeutic response and clinico-pathological correlations in KRAS-mutant setting, especially in the upcoming era of KRAS G12C targeting therapy.

Multicenter Observational Study on Metastatic Non-Small Cell Lung Cancer Harboring BRAF Mutations: Focus on Clinical Characteristics and Treatment Outcome of V600E and Non-V600E Subgroups.

INTRODUCTION: BRAF mutation involved 2-4% of lung adenocarcinoma. Differences in clinicopathologic features and patient outcome exist between V600E and non-V600E BRAF mutated NSCLC. Thus, we sought to assess the frequency and clinical relevance of BRAF mutations in a real-life population of advanced-NSCLC, investigating the potential prognostic significance of distinct genetic alterations. MATERIALS AND METHODS: The present multicenter Italian retrospective study involved advanced BRAF mutant NSCLC. Complete clinicopathologic data were evaluated for BRAF V600E and non-V600E patients. RESULTS: A total of 44 BRAFmut NSCLC patients were included (V600E, n = 23; non-V600E, n = 21). No significant differences in survival outcome and treatment response were documented, according to V600E vs. non-V600E mutations, although a trend towards prolonged PFS was observed in the V600E subgroup (median PFS = 11.3 vs. 6.0 months in non-V600E). In the overall population, ECOG PS and age significantly impacted on OS, while bone lesions were associated with shorter PFS. Compared to immunotherapy, first-line chemotherapy was associated with longer OS in the overall population, and especially in the BRAF V600E subtype. CONCLUSIONS: Here, we report on real-life data from a retrospective cohort of advanced-NSCLC harboring BRAF alterations. Our study offers relevant clues on survival outcome, therapeutic response, and clinicopathologic correlations of BRAF-mutant NSCLC.

Deepening the Knowledge of ROS1 Rearrangements in Non-Small Cell Lung Cancer: Diagnosis, Treatment, Resistance and Concomitant Alterations.

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1-2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the "single oncogenic driver" paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations.

ROS1 pattern of immunostaining in 11 cases of spitzoid tumour: comparison with histopathological, fluorescence in-situ hybridisation and next-generation sequencing analysis.

AIMS: Spitzoid tumours have been shown to harbour exclusive kinase fusions. Few studies have analysed substantial numbers of ROS1-rearranged lesions. The aim of the present study was to investigate also their immunohistochemical profile. METHODS AND RESULTS: Among a group of 35 spitzoid tumours, of which 34 were consecutively diagnosed in a 3-year period, we found 11 ROS1 cases that were immunohistochemically positive, from 10 patients, eight of whom were female and two of whom were male, and who were aged 3-52 years (median, 29 years); most lesions (eight) were localized on the lower extremities. Four patterns of immunostaining were observed: cytoplasmic granular diffuse (six cases), sparse cytoplasmic granules (three cases), paranuclear dots (one case), and nuclear (one case). Fluorescence in-situ hybridisation (FISH) analysis showed all cases to be rearranged (cut-off of >15%). RNA next-generation sequencing (NGS) analysis showed specific fusions of ROS1 in four cases: two with PWWP2A, one with PPFIBP1, and one with ZCCHC8. DNA NGS analysis showed in five cases, specific mutations of AKT, EGFR, NRAS, MYC, ALK, and KIT. ROS1 lesions belonged predominantly to the 'atypical Spitz tumour' group, and showed mainly a nested histological pattern. Interestingly, one patient developed two ROS1-positive lesions. CONCLUSIONS: Immunohistochemistry showed 100% sensitivity and specificity as compared with the FISH results, corresponding to ROS1 rearrangement in 31% of cases studied. These observations shed new light on the value of immunohistochemical evaluation of ROS1 in spitzoid tumours. ROS1 patterns of immunostaining probably reflect different subcellular localisations of ROS1 fusions, although no specific correlations were found in the cases studied. Immunohistochemistry and FISH were the most sensitive techniques for detecting ROS1 rearrangement in this subset of neoplasms.

Unexpected tumor response to palliative pelvic radiotherapy in mismatch repair-deficient advanced prostate cancer: a case report.

BACKGROUND: Mismatch-repair-deficiency resulting in microsatellite instability (MSI) may confer increased radiosensitivity in locally advanced/metastatic tumors and thus radiotherapy (RT) potentially might have a changing role in treating this subset of patients, alone or in combination with checkpoint inhibitors. CASE PRESENTATION: We report a 76 year-old Italian male patient presenting with locally advanced undifferentiated prostate cancer (LAPC), infiltrating bladder and rectum. Molecular analysis revealed high-MSI with an altered expression of MSH2 and MSH6 at immunohistochemistry. Two months after 6 chemotherapy cycles with Docetaxel associated to an LHRH analogue, a computed tomography scan showed stable disease. After palliative RT (30 Gy/10 fractions) directed to the tumor mass with a 3D-conformal setup, a follow-up computed tomography scan at 8 weeks revealed an impressive response that remained stable at computed tomography after 9 months, with sustained biochemical response. To our knowledge, this is the first case of such a sustained response to low dose RT alone in high-MSI LAPC. CONCLUSIONS: Routine evaluation of MSI in patients with locally problematic advanced tumors might change treatment strategy and treatment aim in this setting, from a purely palliative approach to a quasi-curative paradigm.

Modulation of Mutational Landscape in HER2-Positive Breast Cancer after Neoadjuvant Chemotherapy.

INTRODUCTION: In early-stage HER2 positive breast cancer (BC) patients, tumor response to neoadjuvant chemotherapy (NACT) predict survival outcomes. Patients achieving less than pathological complete response (pCR) have a worse prognosis, however, this group is heterogeneous. Nowadays limited data on predictive/prognostic biomarkers in patients with residual cancer disease are available. METHODS: Using next-generation sequencing technology, we evaluated a panel of 21 cancer genes in a group of HER2 positive BC patients with residual disease after NACT. A control group of patients who achieved the pCR was selected too. The BC mutational profile was analyzed on both the tumor diagnostic biopsy and matched residual disease. RESULTS: Overall, the detection rate of mutations was 79% in the No-pCR group versus 90% in the pCR cohort and 98% in the residual BC. The most mutated genes were TP53 and PIK3CA. No correlations between single gene mutations and survival outcomes were found. In no-pCR cohort, 52% of patients had different mutational profile after NACT, 69% of them had an increased in the number of mutated genes. Mutational profile changes from diagnostic biopsy to residual BC were a negative prognostic factor in term of relapse free survival: recurrence probability in different gene profile sub-group was 42% vs 0% in the same profile one (P = .019). CONCLUSIONS: Treatment selective pressure on tumor cells due to NACT changed the gene mutational profile in more than half of BC patient with residual tumor disease. Treatment-induced gene mutations significantly increase the risk of relapse. Profiling primary and residual BC is a major step in order to further personalized adjuvant treatment strategy.

Detection of EGFR-Activating and T790M Mutations Using Liquid Biopsy in Patients With EGFR-Mutated Non-Small-Cell Lung Cancer Whose Disease Has Progressed During Treatment With First- and Second-Generation Tyrosine Kinase Inhibitors: A Multicenter Real-Life Retrospective Study.

BACKGROUND: In advanced epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients whose disease has progressed during treatment with first- and second-generation tyrosine kinase inhibitors (TKIs), liquid biopsy (LB) is routinely used to evaluate the presence of EGFR T790M as an acquired resistance mechanism. The objective of this study was to assess a real-life picture of EGFR T790M detection in LB. MATERIALS AND METHODS: Liquid biopsies performed between June 2016 and October 2018 for advanced EGFR-mutated NSCLC at disease progression during treatment with first- and second-generation TKIs were retrospectively evaluated in 5 Italian centers. Circulating tumor DNA was extracted from plasma and tested with different commercial kits. The detection rate in LBs and the patients' characteristics were correlated. RESULTS: We enrolled 120 consecutive patients. The overall T790M detection rate observed using LB was 25.8%. Fifty-four of 89 (60.7%) patients with negative LB results underwent tissue rebiopsy, and 56% were positive for T790M. The overall rate of T790M positivity in the study cohort was 49.2%. LB performed before formal tumor progression according to Response Evaluation Criteria In Solid Tumors criteria was negative for T790M in all patients (n = 21; P = .012). T790M positivity was statistically significantly higher in cases of disease progression at extrathoracic metastatic sites (P = .008) and, specifically, in the case of worsening bone disease (P = .003). CONCLUSION: Our study shows that the detection of T790M-positive patients whose disease progressed during treatment with first- and second-generation TKIs in real life was according to the literature. However, this result was obtained with a specific clinical course (repeat LBs and tissue rebiopsy), thus implying the necessity for multidisciplinary management.

Overall survival in patients with lung adenocarcinoma harboring "niche" mutations: an observational study.

Objective: In addition to the most common somatic lung cancer mutations (i. e., KRAS and EGFR mutations), other genes may harbor mutations that could be relevant for lung cancer. We defined BRAF, c-MET, DDR2, HER2, MAP2K1, NRAS, PIK3CA, and RET mutations as "niche" mutations and analyzed. The aim of this retrospective cohort study was to assess the differences in the overall survival (OS) of patients with lung adenocarcinoma harboring niche somatic mutations. Results: Data were gathered for 252 patients. Mutations were observed in all genes studied, except c-MET, DDR2, MAP2K1, and RET. The multivariable analysis showed that 1) niche mutations had a higher mortality than EGFR mutations (HR = 2.3; 95% CI = 1.2-4.4; p = 0.009); 2) KRAS mutations had a higher mortality than EGFR mutations (HR = 2.5; 95% CI = 1.4-4.5; p = 0.003); 3) niche mutations presented a similar mortality to KRAS mutations (HR = 0.9; 95% CI = 0.6-1.5; p = 0.797). Methods: Three cohorts of mutations were selected from patients with lung adenocarcinoma and their OS was compared. Mutations that were searched for, were 1) BRAF, c-MET, DDR2, HER2, MAP2K1, NRAS, PIK3CA, and RET; 2) K-RAS; and 3) EGFR. Differences in OS between these three cohorts were assessed by means of a multivariable Cox model that adjusted for age, sex, smoking habits, clinical stages, and treatments. Conclusions: Niche mutations exhibited an increased risk of death when compared with EGFR mutations and a similar risk of death when compared with KRAS mutations.

Surrogate Molecular Classification of Invasive Breast Carcinoma: A Comparison Between Core Needle Biopsy and Surgical Excision, With and Without Neoadjuvant Therapy.

Surrogate molecular classification identifies different subtypes of invasive breast carcinoma on the basis of their immunohistochemical markers. The purpose of the study is to verify whether the immunohistochemical markers and surrogate molecular subtypes can be correctly assessed on the core needle biopsy (CNB) when compared with the corresponding surgical excision (SE), with or without neoadjuvant treatment (NAT). Cases with invasive carcinomas identified on both CNB and SE were retrospectively selected. With immunohistochemistry for estrogen receptors (ER), progesterone receptors (PgR), Ki67, human epidermal growth factor receptor 2 (Her2), and molecular analysis for Her2, surrogate molecular classification was determined in 4 and 5 groups, according to the 2013 St Gallen consensus. A total of 1067 cases was considered and complete data for surrogate molecular classification were available for 988 cases (655 without NAT, 333 with NAT). Without NAT, concordance was strong for ER and Her2, moderate for PgR, and weak for Ki67; concordance for surrogate molecular classification was moderate. After NAT, lower concordance rates were recorded, with significant reduction of PgR (P<0.001) and Ki67 (P<0.001). Without NAT, the surrogate molecular subtypes of breast carcinoma can be reliably assessed on CNB; Ki67 and/or PgR may be repeated on SE when values are close to cutoffs to avoid tumor subtype misclassification. After NAT, it seems advisable to repeat at least Ki67 and PgR.

Quick assessment of cell-free DNA in seminal fluid and fragment size for early non-invasive prostate cancer diagnosis.

BACKGROUND: Liquid biopsy consists in the quantification and qualification of circulating cell-free DNA (cfDNA) and tumor-derived DNA (ctDNA) for cancer recognition. Recently, the characterization of seminal cfDNA (scfDNA) has been reported as a possible biomarker for prostate cancer (PCa) diagnosis. METHODS: Thirty patients with histologically proven PCa, 33 with benign prostate hyperplasia (BPH) and 21 healthy controls were enrolled. cfDNA was extracted from seminal fluid samples. cfDNA quantification and analysis were performed using Qubit ssDNA Kit and Agilent 2100 Bioanalyzer. Statistical analysis included: Levene's test, Shapiro-Wilk, Kolmogorov-Smirnov and Kruskal Wallis tests. RESULTS: Median cfDNA was significantly higher in PCa patients 428.45 ng/mL (173.93-1159.62) compared to BPH patients 77.4 ng/mL (18.23-501) and healthy controls 25.4 ng/mL (15.37-76.62). scfDNA fragments longer than 1000 base-pairs were more common in patients with PCa compared to those with BPH and controls. CONCLUSIONS: scfDNA concentration and fragment size differed significantly in the three groups of PCa, BPH and healthy controls. Both parameters are potential clinical biomarkers for PCa and can be used in both early diagnosis and follow-up. Using automated systems for high-throughput cfDNA quantification could improve the reproducibility of the method and facilitate the implementation of liquid biopsies in the clinical setting.

Differential molecular pathways expression in HER2 positive early breast cancer according to hormone receptor status.

PURPOSE: Hormone receptors (HR) status in HER2 + breast cancer (BC) is a recognized stratification factor with relevant clinical implication. According to HR expression, HER2 + BC show different clinical characteristics, treatment sensitivity and prognosis. The interaction between HR and HER2 pathways remains incompletely understood. METHODS: Thirty-four HER2 + BC were included: 18 tumors with HER2+/HR + and 16 with HER2+/HR-. The expression of 770 genes and 13 molecular pathways were evaluated using Nanostring PanCancer Pathway panel performed on FFPE BC biopsies. RESULTS: Gene expression analysis identified 127 genes with significantly different expression between the two cohorts. 83% of these genes were overexpressed in HER2+/HR- cohort. Globally, 23% of them belonged to PI3K pathway (41 genes), 15% to Trascriptional regulation (26 genes) and 12% to MAPK (22 genes). Regarding pathway expression, PI3K, MAPK and NOTCH were significantly differently expressed between the two groups (p = 0.003, p = 0.0018 and p = 0.02, respectively), all of them were overexpressed in HER2+/HR- tumors. CONCLUSIONS: According to HR status, HER2 + tumors express different pathways profiles: the overexpression of PI3K, MAPK and NOTCH pathways in HER2+/HR- group could justify different survival outcomes and treatment sensitivity. The identification of tumor driver pathways may be a useful instrument for individualized pathway-directed therapies. Further clinical implications are warranted.

Beyond the Beyond: A Case of an Extraordinary Response to Multiple Lines of Therapy in a de novo Metastatic HER2-Negative Gastric Cancer Patient.

BACKGROUND: Gastric cancer is the fourth cause of cancer-related death in Europe and the prognosis of these patients remains dismal. It has been demonstrated that chemotherapy improved survival compared with best supportive care and recently, subsequent lines of therapy, also with new drugs, obtained positive results. SUMMARY: We present the case of a patient diagnosed with a de novo metastatic gastric cancer who experienced an extraordinary long response to multiple lines of chemotherapy (FOLFOX6, paclitaxel plus ramucirumab, FOLFIRI, rechallenge with FOLFOX6). KEY MESSAGE: Gastric cancer therapy should be considered as the result of a strategy based on the patient's condition, and tolerance and response to various therapies. The emerging evidence of the role of subsequent lines of therapy, along with the recognition of the pivotal role of nutritional support and the availability of new drugs, should help clinicians in the management of patients with gastric cancer. PRACTICAL IMPLICATIONS: We propose a practical therapeutic algorithm in order to help clinicians who deal with patients with gastric cancer.

Mutational Profile of Metastatic Breast Cancer Tissue in Patients Treated with Exemestane Plus Everolimus.

BACKGROUND: Everolimus has been shown to overcome endocrine resistance in hormone receptor positive advanced breast cancer patients. Predictive biomarkers of everolimus efficacy have been investigated in primary breast cancer tissue without finding univocal results. The goal of this study was to investigate the mutational burden in the metastatic site of endocrine-resistant tumors treated with everolimus plus exemestane. PATIENTS AND METHODS: Mass Array Sequenom platform was used to analyse genetic status of 18 cancer-related genes in 25 archival tumor specimens from metastatic lesions and available primary matched breast cancer tissue of patients treated with everolimus and exemestane for advanced disease. An exploratory analysis of everolimus efficacy in terms of progression free survival benefit and single gene mutation was carried out. RESULTS: The overall detection rate of mutation was 30% and 38% from metastatic and primary breast cancer samples, respectively. AKT1E17K was the most frequent mutated gene. No primary breast cancer and matched relapse maintained the same mutation profile. Considering molecular pathways, the most of the genes belong to PI3K pathway (AKT1E17K, PI3KCAE545K, and KITG565R,S709F). In patients with detected mutations in breast and/or recurrence tissue the median PFS was 5,6 months while in the subgroup of patients with no mutations the median PFS was 7,5 months. CONCLUSIONS: The mutational status of breast cancer recurrence allows the identification of some genes potentially correlating tumor response/resistance to everolimus. The most frequently mutated genes were involved in the PI3K/AKT/mTOR pathway highlighting that the deregulation of this pathway in the relapse plays a crucial role in the mechanisms of everolimus resistance/sensitivity. Owing to the small sample size and the retrospective nature of the study, these correlations need to be validated in a large prospective study.

Clinical and molecular predictors of long-term response in HER2 positive metastatic breast cancer patients.

BACKGROUND: HER2+ metastatic breast cancer (MBC) is a poor prognosis disease, unusually curable. To date, no predictive factors have been clearly correlated with long-term response to anti-HER2 agents. METHODS: 54 HER2+ MBC patients treated with HER2 targeted therapy as first line treatment were analysed: 40 with a time to progression longer than 3 years in Long Responders (LR) group and 14 with a progression disease within one year of anti-HER2 therapy in a control group named Early Progressors (EP). The expression of 770 genes and 13 molecular pathways were evaluated using Nanostring PanCancer pathway panel performed on FFPE BC tissues. RESULTS: Considering baseline patients and tumor characteristics, EP women had more CNS spread and more metastatic burden of disease compared to LR (p > 0.05). Gene expression analysis identified 30 genes with significantly different expression in the two cohorts; five were driver genes (BRCA1, PDGFRA, AR, PHF6 and MSH2). The majority of these genes were over-expressed, mainly in LR patients, and encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. Only four genes were down regulated, all in EP group (TNFSF10, CACNG1, IL20RB and BRCA1). Most of these genes were involved in MAPK and PI3K pathways. MAPK pathway was differently expressed between LR and EP (p = 0.05). PI3K was the only pathway overexpressed in EP patients. CONCLUSIONS: Whole genome expression analysis comparing LR vs. EP identified a group of genes that may predict more favourable long-term outcomes. Up-regulation of MAPK and down-regulation of PI3K pathway could be a positive predictive factors. Further clinical implications are warranted. ABBREVIATIONS: BC: breast cancer; MBC: metastatic breast cancer; LR: long responder; EP: early progressor; FFPE: formalin-fixed paraffin-embedded; CNS: central nervous system; PFS: progression free survival; OS: overall survival.

Clinical Impact and Prognostic Role of KRAS/BRAF/PIK3CA Mutations in Stage I Colorectal Cancer.

Stage I colorectal carcinoma has excellent prognosis, with 5-year survival rate up to 95%. The occurrence of lymphovascular invasion, tumor budding, high number of PDC, or lymph node micrometastases is associated with tumor progression. The aim of this study was to evaluate the mutational status of 62 stage I colorectal carcinomas (CRC) (taken from 37 patients surviving more than five years since the initial diagnosis and from 25 patients who died of disease) and to correlate it with histopathological features and the clinical outcome. Mutations of KRAS, NRAS, BRAF, and PIK3CA genes were analyzed through Myriapod Colon Status Kit, using the high-throughput genotyping platform Sequenom MassARRAY System. Mutations in those genes were found in 31 cases (50%) and mainly in those with poor prognosis. The most frequent mutations occurred at codons 12 and 13 of the KRAS gene (40% of cases). We found concomitant PIK3CA mutations in 5 cases (8%). The presence of PIK3CA mutations was mainly observed in tumors with poor prognosis and with unfavorable histopathological prognostic features. High PDC grade (P = 0.0112), the presence of tumor budding (P = 0.0334), LVI (P < 0.0001), KRAS mutations (P = 0.0228), PIK3CA mutations (P = 0.0214), multiple genetic mutations in KRAS and PIK3CA genes (P = 0.039), and nodal micrometastases (P < 0.0001) were significant prognostic variables for CSS. The presence of LVI was the only independent and statistically significant prognostic variable for CSS in our cohort of pTNM stage I CRCs. The analysis of KRAS/PIK3CA mutational status may be used to identify patients with stage I CRC at high risk of bad outcome and who may need additional treatments, including biological therapies.