Oligo-metastatic neoPlasms from the gastro-intestinal tract: iDentIfiCaTIon of cliNical and molecular drivers: the PREDICTION study.

BACKGROUND: Metastatic disease in tumors originating from the gastrointestinal tract can exhibit varying degrees of tumor burden at presentation. Some patients follow a less aggressive disease course, characterized by a limited number of metastatic sites, referred to as "oligo-metastatic disease" (OMD). The precise biological characteristics that define the oligometastatic behavior remain uncertain. In this study, we present a protocol designed to prospectively identify OMD, with the aim of proposing novel therapeutic approaches and monitoring strategies. METHODS: The PREDICTION study is a monocentric, prospective, observational investigation. Enrolled patients will receive standard treatment, while translational activities will involve analysis of the tumor microenvironment and genomic profiling using immunohistochemistry and next-generation sequencing, respectively. The first primary objective (descriptive) is to determine the prevalence of biological characteristics in OMD derived from gastrointestinal tract neoplasms, including high genetic concordance between primary tumors and metastases, a significant infiltration of T lymphocytes, and the absence of clonal evolution favoring specific driver genes (KRAS and PIK3CA). The second co-primary objective (analytic) is to identify a prognostic score for true OMD, with a primary focus on metastatic colorectal cancer. The score will comprise genetic concordance (> 80%), high T-lymphocyte infiltration, and the absence of clonal evolution favoring driver genes. It is hypothesized that patients with true OMD (score 3+) will have a lower rate of progression/recurrence within one year (20%) compared to those with false OMD (80%). The endpoint of the co-primary objective is the rate of recurrence/progression at one year. Considering a reasonable probability (60%) of the three factors occurring simultaneously in true OMD (score 3+), using a significance level of α = 0.05 and a test power of 90%, the study requires a minimum enrollment of 32 patients. DISCUSSION: Few studies have explored the precise genetic and biological features of OMD thus far. In clinical settings, the diagnosis of OMD is typically made retrospectively, as some patients who undergo intensive treatment for oligometastases develop polymetastatic diseases within a year, while others do not experience disease progression (true OMD). In the coming years, the identification of true OMD will allow us to employ more personalized and comprehensive strategies in cancer treatment. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT05806151.

European Real-World Assessment of the Clinical Validity of a CE-IVD Panel for Ultra-Fast Next-Generation Sequencing in Solid Tumors.

Molecular profiling of solid tumors facilitates personalized, targeted therapeutic interventions. The ability to perform next-generation sequencing (NGS), especially from small tissue samples, in a short turnaround time (TAT) is essential to providing results that enable rapid clinical decisions. This multicenter study evaluated the performance of a CE in vitro diagnostic (IVD) assay, the Oncomine Dx Express Test, on the Ion Torrent Genexus System for detecting DNA and RNA variants in solid tumors. Eighty-two archived formalin-fixed paraffin embedded (FFPE) tissue samples from lung, colorectal, central nervous system, melanoma, breast, gastric, thyroid, and soft tissue cancers were used to assess the presence of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variations (CNVs), gene fusions, and splice variants. These clinical samples were previously characterized at the various academic centers using orthogonal methods. The Oncomine Dx Express Test showed high performance with 100% concordance with previous characterization for SNVs, indels, CNVs, gene fusions, and splice variants. SNVs and indels with allele frequencies as low as 5% were correctly identified. The test detected all the expected ALK, RET, NTRK1, and ROS1 fusion isoforms and MET exon 14-skipping splice variants. The average TAT from extracted nucleic acids to the final variant report was 18.3 h. The Oncomine Dx Express Test in combination with the Ion Torrent Genexus System is a CE-IVD-compliant, performant, and multicenter reproducible method for NGS detection of actionable biomarkers from a range of tumor samples, providing results in a short TAT that could support timely decision- making for targeted therapeutic interventions.

Low Impact of Clonal Hematopoiesis on the Determination of RAS Mutations by Cell-Free DNA Testing in Routine Clinical Diagnostics.

Targeted sequencing of circulating cell-free DNA (cfDNA) is used in routine clinical diagnostics for the identification of predictive biomarkers in cancer patients in an advanced stage. The presence of KRAS mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) might represent a confounding factor. We used an amplicon-based targeted sequencing panel, covering selected regions of 52 genes, for circulating cell-free total nucleic acid (cfTNA) analysis of 495 plasma samples from cancer patients. The cfDNA test failed in 4 cases, while circulating cell-free RNA (cfRNA) sequencing was invalid in 48 cases. In the 491 samples successfully tested on cfDNA, at least one genomic alteration was found in 222 cases (45.21%). We identified 316 single nucleotide variants (SNVs) in 21 genes. The most frequently mutated gene was TP53 (74 variants), followed by KRAS (71), EGFR (56), PIK3CA (33) and BRAF (19). Copy number variations (CNVs) were detected in 36 cases, while sequencing of cfRNA revealed 6 alterations. Analysis with droplet digital PCR (ddPCR) of peripheral blood leukocyte (PBL)-derived genomic DNA did not identify any KRAS mutations in 39 cases that showed KRAS mutations at cfDNA analysis. These findings suggest that the incidence of CHIP-associated KRAS mutations is relatively rare in routine clinical diagnostics.

Significance of rare variants in genes involved in the pathogenesis of Lynch syndrome.

The molecular characterization of patients with Lynch syndrome (LS) involves germline testing to detect a deleterious mutation in one of the genes of the mismatch repair (MMR) pathway. To date, however, a large proportion of patients with a clinical suspicion of LS who undergo genetic testing do not show a germline pathogenetic variant in these genes. Germline DNA from 73 patients with a clinical suspicion of LS was examined with next­generation sequencing methods, using a multigene custom panel designed and standardized by our research group, that targets a set of 15 genes. Deleterious variants were identified in 5.6% of index cases, while unclassified variants were identified in 80.3% of probands. To evaluate the pathogenicity of these uncertain variants, the American College of Medical Genetics and Genomics criteria was used, also considering wherever possible the microsatellite instability (MSI) status detected on tumor tissues as pathogenic criterion. In this manner, 8 of these uncertain significance variants were classified as likely pathogenic variants. Notably, some of these likely pathogenetic variants were also identified in the MLH3 gene that is a gene not routinely analyzed for cases with a clinical suspicion of LS. The present study highlighted the importance of verifying the pathogenicity of the numerous variants of unknown significance identified in patients for whom heredity is already clinically confirmed suggesting the importance of considering the MSI­H status on the tumor of patients carrying an uncertain variant to evaluate its pathogenicity. Moreover, the present study also suggested analyzing other MMR genes, such as MLH3, in panels used for the molecular screening of LS.

Challenges in bioinformatics approaches to tumor mutation burden analysis.

Several immune checkpoint inhibitors (ICIs) have already been introduced into clinical practice or are in advanced phases of clinical experimentation. Extensive efforts are being made to identify robust biomarkers to select patients who may benefit from treatment with ICIs. Tumor mutation burden (TMB) may be a relevant biomarker of response to ICIs in different tumor types; however, its clinical use is challenged by the analytical methods required for its evaluation. The possibility of using targeted next-generation sequencing panels has been investigated as an alternative to the standard whole exome sequencing approach. However, no standardization exists in terms of genes covered, types of mutations included in the estimation of TMB, bioinformatics pipelines for data analysis, and cut-offs used to discriminate samples with high, intermediate or low TMB. Bioinformatics serve a relevant role in the analysis of targeted sequencing data and its standardization is essential to deliver a reliable test in clinical practice. In the present study, cultured and formalin-fixed, paraffin-embedded cell lines were analyzed using a commercial panel for TMB testing; the results were compared with data from the literature and public databases, demonstrating a good correlation. Additionally, the correlation between high tumor mutation burden and microsatellite instability was confirmed. The bioinformatics analyses were conducted using two different pipelines to highlight the challenges associated with the development of an appropriate analytical workflow.

MSH2 Overexpression Due to an Unclassified Variant in 3'-Untranslated Region in a Patient with Colon Cancer.

BACKGROUND: The loss or low expression of DNA mismatch repair (MMR) genes can result in genomic instability and tumorigenesis. One such gene, MSH2, is mutated or rearranged in Lynch syndrome (LS), which is characterized by a high risk of tumor development, including colorectal cancer. However, many variants identified in this gene are often defined as variants of uncertain significance (VUS). In this study, we selected a variant in the 3' untranslated region (UTR) of MSH2 (c*226A > G), identified in three affected members of a LS family and already reported in the literature as a VUS. METHODS: The effect of this variant on the activity of the MMR complex was examined using a set of functional assays to evaluate MSH2 expression. RESULTS: We found MSH2 was overexpressed compared to healthy controls, as determined by RTqPCR and Western blot analyses of total RNA and proteins, respectively, extracted from peripheral blood samples. These results were confirmed by luciferase reporter gene assays. CONCLUSIONS: We therefore speculated that, in addition to canonical inactivation via a gene mutation, MMR activity may also be modulated by changes in MMR gene expression.

Genomic Profiling of KRAS/NRAS/BRAF/PIK3CA Wild-Type Metastatic Colorectal Cancer Patients Reveals Novel Mutations in Genes Potentially Associated with Resistance to Anti-EGFR Agents.

Previous findings suggest that metastatic colorectal carcinoma (mCRC) patients with KRAS/NRAS/BRAF/PIK3CA wild-type (quadruple-wt) tumors are highly sensitive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs). However, additional molecular alterations might be involved in the de novo resistance to these drugs. We performed a comprehensive molecular profiling of 21 quadruple-wt tumors from mCRC patients enrolled in the "Cetuximab After Progression in KRAS wild-type colorectal cancer patients" (CAPRI-GOIM) trial of first line FOLFIRI plus cetuximab. Tumor samples were analyzed with a targeted sequencing panel covering single nucleotide variants (SNVs), insertions/deletions (Indels), copy number variations (CNVs), and gene fusions in 143 cancer-related genes. The analysis revealed in all 21 patients the presence of at least one SNV/Indel and in 10/21 cases (48%) the presence of at least one CNV. Furthermore, 17/21 (81%) patients had co-existing SNVs/Indels in different genes. Quadruple-wt mCRC from patients with the shorter progression free survival (PFS) were enriched with peculiar genetic alterations in KRAS, FBXW7, MAP2K1, and NF1 genes as compared with patients with longer PFS. These data suggest that a wide genetic profiling of quadruple-wt mCRC patients might help to identify novel markers of de novo resistance to anti-EGFR MoAbs.

The Presence of Concomitant Mutations Affects the Activity of EGFR Tyrosine Kinase Inhibitors in EGFR-Mutant Non-Small Cell Lung Cancer (NSCLC) Patients.

Recent findings suggest that a fraction of EGFR-mutant non-small-cell lung cancers (NSCLC) carry additional driver mutations that could potentially affect the activity of EGFR tyrosine kinase inhibitors (TKIs). We investigated the role of concomitant KRAS, NRAS, BRAF, PIK3CA, MET and ERBB2 mutations (other mutations) on the outcome of 133 EGFR mutant patients, who received first-line therapy with EGFR TKIs between June 2008 and December 2014. Analysis of genomic DNA by Next Generation Sequencing (NGS) revealed the presence of hotspot mutations in genes other than the EGFR, including KRAS, NRAS, BRAF, ERBB2, PIK3CA, or MET, in 29/133 cases (21.8%). A p.T790M mutation was found in 9/133 tumour samples (6.8%). The progression free survival (PFS) of patients without other mutations was 11.3 months vs. 7 months in patients with other mutations (log-rank test univariate: p = 0.047). In a multivariate Cox regression model including the presence of other mutations, age, performance status, smoking status, and the presence of p.T790M mutations, the presence of other mutations was the only factor significantly associated with PFS (Hazard Ratio 1.63, 95% CI 1.04⁻2.58; p = 0.035). In contrast, no correlation was found between TP53 mutations and patients' outcome. These data suggest that a subgroup of EGFR mutant tumours have concomitant driver mutations that might affect the activity of first-line EGFR TKIs.

The role of circulating free DNA in the management of NSCLC.

INTRODUCTION: Circulating cell-free DNA (cfDNA) testing has emerged as an alternative to tumor tissue analyses for the management of metastatic non-small-cell lung cancer (NSCLC) patients. Analysis of cfDNA is a minimally invasive procedure that might better reflect tumor heterogeneity and allows repeated testing over the time. Areas covered: This review article covers the different applications of cfDNA testing in NSCLC: early diagnosis of the disease; detection of minimal residual disease in early lung cancer; identification of predictive and prognostic markers in advanced NSCLC patients; monitoring the response to therapy; assessment of tumor mutation burden. Expert commentary: The use of liquid biopsy is rapidly expanding to different applications. The combination of different circulating biomarkers (cfDNA, protein, miRNA) might improve the sensitivity and specificity of this approach in patients with low tumor burden. cfDNA testing is representing a valid source for molecular profiling in management of metastatic NSCLC patients and is providing important knowledge on tumor heterogeneity. Clinical trials are needed in order to transfer the information deriving from liquid biopsy testing in new therapeutic strategies.

Limits and potential of targeted sequencing analysis of liquid biopsy in patients with lung and colon carcinoma.

The circulating free tumor DNA (ctDNA) represents an alternative, minimally invasive source of tumor DNA for molecular profiling. Targeted sequencing with next generation sequencing (NGS) can assess hundred mutations starting from a low DNA input. We performed NGS analysis of ctDNA from 44 patients with metastatic non-small-cell lung carcinoma (NSCLC) and 35 patients with metastatic colorectal carcinoma (CRC). NGS detected EGFR mutations in 17/22 plasma samples from EGFR-mutant NSCLC patients (sensitivity 77.3%). The concordance rate between tissue and plasma in NSCLC was much lower for other mutations such as KRAS that, based on the allelic frequency and the fraction of neoplastic cells, were likely to be sub-clonal. NGS also identified EGFR mutations in plasma samples from two patients with EGFR wild type tumor tissue. Both mutations were confirmed by droplet digital PCR (ddPCR) in both plasma and tissue samples. In CRC, the sensitivity of the NGS plasma analysis for RAS mutations was 100% (6/6) in patients that had not resection of the primary tumor before blood drawing, and 46.2% (6/13) in patients with primary tumor resected before enrollment. Our study showed that NGS is a suitable method for plasma testing. However, its clinical sensitivity is significantly affected by the presence of the primary tumor and by the heterogeneity of driver mutations.

Human lung adenocarcinoma cell cultures derived from malignant pleural effusions as model system to predict patients chemosensitivity.

BACKGROUND: Lung cancer is the leading cause of cancer related deaths and Malignant Pleural Effusion (MPE) is a frequent complication. Current therapies suffer from lack of efficacy in a great percentage of cases, especially when cancer is diagnosed at a late stage. Moreover patients' responses vary and the outcome is unpredictable. Therefore, the identification of patients who will benefit most of chemotherapy treatment is important for accurate prognostication and better outcome. In this study, using malignant pleural effusions (MPE) from non-small cell lung cancer (NSCLC) patients, we established a collection of patient-derived Adenocarcinoma cultures which were characterized for their sensitivity to chemotherapeutic drugs used in the clinical practice. METHODS: Tumor cells present in MPEs of patients with NSCLC were isolated by density gradient centrifugation, placed in culture and genotyped by next generation sequencing. In a subset of cases patient derived xenografts (PDX) were obtained upon tumor cell inoculation in rag2/IL2 knock-out mice. Isolated primary cultures were characterized and tested for drug sensitivity by in vitro proliferation assays. Additivity, antagonism or synergy for combinatorial treatments were determined by analysis with the Calcusyn software. RESULTS: We have optimized isolation procedures and culture conditions to expand in vitro primary cultures from Malignant Pleural Effusions (MPEs) of patients affected by lung adenocarcinomas, the most frequent form of non small cell lung cancer. Using this approach we have been able to establish 16 primary cultures from MPEs. Cells were banked at low passages and were characterized for their mutational pattern by next generation sequencing for most common driver mutations in lung cancer. Moreover, amplified cultures were shown to engraft with high efficiency when injected in immunocompromised mice. Cancer cell sensitivity to drugs used in standard chemotherapy regimens was assessed either individually or in combination. Differential chemosensitivity and different mutation profiles were observed which suggests that this isolation method could provide a platform for predicting the efficacy of chemotherapy in the clinical setting. Most importantly for six patients it was possible to establish a correlation between drug response in vitro and response to therapy in the clinic. CONCLUSIONS: Results obtained using primary cultured cells from MPEs underscore the heterogeneity of NSCLC in advanced stage as indicated by drug response and mutation profile. Comparison of data obtained from in vitro assays with patients' responses to therapy leads to the conclusion that this strategy may provide a potentially useful approach for evaluating individual chemosensitivity profile and tailor the therapy accordingly. Furthermore, combining MPE-derived primary cultures with their genomic testing allows to identify patients eligible to trials with novel targeted agents.

EGFR mutations in lung cancer: from tissue testing to liquid biopsy.

ABSTRACT The presence of EGFR mutations predicts the sensitivity to EGF receptor (EGFR)-tyrosine kinase inhibitors in a molecularly defined subset of non-small-cell lung carcinoma (NSCLC) patients. For this reason, EGFR testing of NSCLC is required to provide personalized treatment options and better outcomes for NSCLC patients. As surgery specimens are not available in the majority of NSCLC, other currently available DNA sources are small biopsies and cytological samples, providing however limited and low-quality material. In order to address this issue, the use of surrogate sources of DNA, such as blood, serum and plasma samples, which often contains circulating free tumor DNA or circulating tumor cells, is emerging as a new strategy for tumor genotyping.

Maintenance Treatment with Cetuximab and BAY86-9766 Increases Antitumor Efficacy of Irinotecan plus Cetuximab in Human Colorectal Cancer Xenograft Models.

PURPOSE: The use of cetuximab in the treatment of metastatic colorectal cancer is limited by development of resistance. EXPERIMENTAL DESIGN: We have investigated in three models of highly epidermal growth factor receptor (EGFR)-dependent colorectal cancer xenografts, the effect of maintenance therapy with different kinase inhibitors alone or in combination with cetuximab, after cytotoxic treatment induction with irinotecan plus cetuximab. RESULTS: SW48, LIM 1215, and GEO colorectal cancer cell lines were engrafted into nude mice and treated for 3 weeks with irinotecan and/or cetuximab. The combined treatment induced a significant reduction of tumor size. A subsequent experiment was performed in all three xenograft models in which after an induction treatment with irinotecan plus cetuximab, mice were randomly assigned to one of the following treatments: control, cetuximab, regorafenib, a selective PIK3CA inhibitor (PIK3CAi), a selective MEK inhibitor (MEKi), and/or the combination of each inhibitor with cetuximab. The cetuximab plus MEKi treatment determined the best antitumor activity with suppression of tumor growth. This effect was prolonged for 13 to 15 weeks after cessation of therapy and was accompanied by prolonged survival. Antitumor activity was accompanied by inhibition of the MAPK and MEK pathways. Moreover, in the cetuximab plus MEKi-treated SW48 xenograft group, KRAS mutations as a mechanism of acquired resistance were detected in 25% of cases compared with 75% KRAS mutations in the MEKi-treated group. CONCLUSIONS: A possible strategy to prevent and/or overcome resistance to anti-EGFR inhibitors in metastatic colorectal cancer is a maintenance therapy with cetuximab plus MEKi after an initial treatment with irinotecan plus cetuximab.

Molecular typing of lung adenocarcinoma on cytological samples using a multigene next generation sequencing panel.

Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.