Molecular aspects of small bowel adenocarcinoma for new therapeutic approaches.

Small Bowel Adenocarcinoma (SBA) is a rare and aggressive neoplastic disease. Several aspects of SBA carcinogenesis still need to be elucidated, but risk factors and histomorphological similarities seem to indicate that SBA can follow a carcinogenetic development similar to that proposed for colorectal cancer. At molecular level, at odds with adenocarcinoma arising in the large intestine, very few, and fragmented, information is available for SBA. In general, it has been suggested that the two models of colorectal carcinogenesis can be valid also for SBA. For that reason, chemotherapies set up for the cancers of the large intestine have been applied also for SBA. Therefore, since recent studies led to the introduction of EGFR-targeted therapies in colorectal cancer, the treatment with anti-EGFR drugs can be proposed also for SBA patients. In particular, in colorectal cancer patients it has been demonstrated that KRAS mutations are correlated with the absence of efficacy of EGFR-targeted therapies, and it has been proposed by few studies to investigate additional markers of the EGFR pathway (EGFR gene copy number, BRAF and PIK3CA mutations, as well as PTEN protein expression), in order to increase the predictive power of the efficacy of anti-EGFR drugs. Information regarding these markers in SBA is quite completely missing. Primary aim of the present work was the evaluation, in the same cohort, of all the alterations involved in colorectal carcinogenesis, in order to shed light more deeply about the molecular similarity between SBA and colorectal cancer. Second aim of the present work was to investigate in the same cohort of SBA the aforementioned markers involved in the EGFR pathway, in order to verify if the pattern of these alterations could justify the possible introduction of these therapies also in patients affected by SBA. To do this, for the first aim we investigated -catenin protein expression by immunohistochemistry (IHC), and KRAS and TP53 mutations by direct sequencing, as well as microsatellite instability (MSI) and allelic imbalance of Chromosome 18q MSI by fragment analysis on genomic DNA extracted from formalin-fixed paraffinembedded tissue sections. For the second aim we investigated EGFR gene status by Fluorescent in situ hybridization (FISH), BRAF and PIK3CA mutational status by direct sequencing, as well as PTEN protein expression by IHC, in the same cohort of SBA. We recruited 40 SBA cases from the Institute of Pathology of Locarno (Canton Tessin, Southern Switzerland) and from three institutions of Northern Italy. First aim. -catenin overexpression was observed in 23.6% at nuclear level and in additional 47.3% of cases only at cytoplasmic level, MSI was found in 23.6% of cases, KRAS mutations in 43.6% of cases, TP53 mutations in 29% of cases and allelic imbalance of Chromosome 18q in 75% of cases. All the percentages of alterations and the types of mutations are in line with those identified in the analysis of colorectal cancer patients. Therefore, by the analysis of all these markers in a same cohort, we can confirm that SBA shares the carcinogenetic development with colorectal cancer also at molecular level. Second aim. We identified a copy number gain of EGFR gene in 57.5% of cases, BRAF and PIK3CA mutations in 2.5% and 10.5% of cases respectively, and PTEN loss of expression in 25.6% of cases. Also for the EGFR pathway analysis, percentages of alterations and types of mutations found in SBA are in line with colorectal cancer, even if we did not detect the classical V600E change in the BRAF gene (where, on the contrary, we found a rare mutation, the G596R change). Taking into account the molecular algorithm proposed for the administration of EGFR-targeted therapies in colorectal cancer patients, if we look only at KRAS mutations, we can propose the administration of EGFR-targeted therapies to about 60% of patients (i.e.: KRAS wildtype cases), and to 23% of cases if we base our evaluation on the whole EGFR pathway (i.e.: cases showing, at the same time, EGFR copy number gain, KRAS, BRAF and PIK3CA wild-type sequences, and PTEN normal expression). The treatment with anti-EGFR therapies of a SBA patient of our cohort who developed a metastatic lesion confirmed the relevance of the molecular characterization of the tumor to predict the response to these therapies. In conclusion, our analyses of SBA confirm the feeling that the mechanisms of carcinogenesis of such disease are superimposable with those proposed for colorectal cancer. Therefore, the hypothesis that therapeutic protocols valid for the large intestine can be applied also to SBA patients is supported. As a consequence, the targeted therapies recently introduced in colorectal cancer can be proposed for SBA patients, pending tumor molecular characterization as demonstrated by our case report

Biomarker characterization in endometrial cancer in Europe: first survey data analysis from 69 pathological academic and hospital labs

Introduction. Endometrial carcinoma (EC) is the commonest gynecological cancer affecting women in Western populations. To predict patient risk, the 2020 edition of the World Health Organization (WHO) Classification of Tumors of the Female Genital Tract stressed the importance of integrated histo-molecular classification of the disease. This survey analysis poses attention on the most frequently used immunohistochemical and molecular markers adopted in daily categorization of ECs in European laboratories. Methods. We analyzed data collected through questionnaires administered to 40 Italian, 20 Spanish, 3 Swiss and 6 United Kingdom (UK) laboratories. We collected information regarding daily practice in EC evaluation, specifically concerning mismatch repair status (MMR) and microsatellite instability (MSI). Summary and descriptive statistical analyses were carried out to evaluate the current practice of each laboratory. Results. The results show that MMR status is mainly evaluated by using immunohistochemistry (IHC) on most EC samples. The most frequent approach for the analysis of MMR status is IHC of four proteins (PMS2, MSH6, MSH2, MLH1). MSI analysis by molecular methods is uncommon but useful as a supplemental tool in specific conditions. MLH1 promoter hypermethylation and BRAF V600 mutations analysis are performed in case of negative expression of MLH1/PMS2. Other markers (mainly p53 followed by POLE and PTEN) are investigated in particular in Spain and Switzerland in a consistent number of cases. Conclusion. Guidelines consultation and standardization of laboratory procedures are efficient means for EC prognostic risk stratification and improving the quality of care

Prognostic and predictive role of EGFR pathway alterations in biliary cancer patients treated with chemotherapy and anti-EGFR

The association of anti-EGFR to gemcitabine and oxaliplatin (GEMOX) chemotherapy did not improve survival in biliary tract carcinoma (BTC) patients. Multiple mechanisms might be involved in the resistance to anti-EGFR. Here, we explored the mutation profile of EGFR extracellular domain (ECD), of tyrosine kinase domain (TKD), and its amplification status. EGFR mutational status of exons 12, 18-21 was analyzed in 57 tumors by Sanger sequencing. EGFR amplification was evaluated in 37 tumors by Fluorescent In Situ Hybridization (FISH). Kaplan-Meier curves were calculated using the log-rank test. Six patients had mutations in exon 12 of EGFR ECD and 7 in EGFR TKD. Neither EGFR ECD nor TKD mutations affected progression free survival (PFS) or overall survival (OS) in the entire population. In the panitumumab plus GEMOX (P-GEMOX) arm, ECD mutated patients had a worse OS, while EGFR TKD mutated patients had a trend towards shorter PFS and OS. Overall, the presence of mutations in EGFR or in its transducers did not affect PFS or OS, while the extrahepatic cholangiocarcinoma (ECC) mutated patients had a worse prognosis compared to WT. Nineteen out of 37 tumors were EGFR amplified, but the amplification did not correlate with survival. ECC EGFR amplified patients had improved OS, whereas the amplification significantly correlated with poor PFS (p = 0.03) in gallbladder carcinoma patients. The high molecular heterogeneity is a predominant feature of BTC: the alterations found in this work seem to have a prognostic impact rather than a predictive role towards anti-EGFR therapy

One-instrument, objective microsatellite instability analysis using high-resolution melt

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers.The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type.The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status.The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance.Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors.Novel technologies have been applied and concepts such as tumor mutation burden have been introduced.However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator.In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue.The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles.The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study.The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.</p

Reporting of somatic variants in clinical cancer care: recommendations of the Swiss Society of Molecular Pathology

Somatic variant testing through next-generation sequencing (NGS) is well integrated into Swiss molecular pathology laboratories and has become a standard diagnostic method for numerous indications in cancer patient care. Currently, there is a wide variation in reporting practices within our country, and as patients move between different hospitals, it is increasingly necessary to standardize NGS reports to ease their reinterpretation. Additionally, as many different stakeholders-oncologists, hematologists, geneticists, pathologists, and patients-have access to the NGS report, it needs to contain comprehensive and detailed information in order to answer the questions of experts and avoid misinterpretation by non-experts. In 2017, the Swiss Institute of Bioinformatics conducted a survey to assess the differences in NGS reporting practices across ten pathology institutes in Switzerland. The survey examined 68 reporting items and identified 48 discrepancies. Based on these findings, the Swiss Society of Molecular Pathology initiated a Delphi method to reach a consensus on a set of recommendations for NGS reporting. Reports should include clinical information about the patient and the diagnosis, technical details about the sample and the test performed, and a list of all clinically relevant variants and variants of uncertain significance. In the absence of a consensus on an actionability scheme, the five-class pathogenicity scheme proposed by the ACMG/AMP guideline must be included in the reports. The Swiss Society of Molecular Pathology recognizes the importance of including clinical actionability in the report and calls on the European community of molecular pathologists and oncologists to reach a consensus on this issue

Mutational Profile of GNAQQ209 in Human Tumors

BACKGROUND: Frequent somatic mutations have recently been identified in the ras-like domain of the heterotrimeric G protein alpha-subunit (GNAQ) in blue naevi 83%, malignant blue naevi (50%) and ocular melanoma of the uvea (46%). The mutations exclusively affect codon 209 and result in GNAQ constitutive activation which, in turn, acts as a dominant oncogene. METHODOLOGY: To assess if the mutations are present in other tumor types we performed a systematic mutational profile of the GNAQ exon 5 in a panel of 922 neoplasms, including glioblastoma, gastrointestinal stromal tumors (GIST), acute myeloid leukemia (AML), blue naevi, skin melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, and thyroid carcinomas. PRINCIPAL FINDINGS: We detected the previously reported mutations in 6/13 (46%) blue naevi. Changes affecting Q209 were not found in any of the other tumors. Our data indicate that the occurrence of GNAQ mutations display a unique pattern being present in a subset of melanocytic tumors but not in malignancies of glial, epithelial and stromal origin analyzed in this study

P1245 Polymorphic Variants of HSD3B1 Gene Confer Different Outcome in Specific Subgroups of Patients Infected With SARS-CoV-2

Introduction: Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the androgen receptor (AR), through ACE2 receptor and TMPRSS2, to enter nasal and upper airways epithelial cells. Genetic analyses revealed that HSD3B1 P1245C polymorphic variant increases dihydrotestosterone production and upregulation of TMPRSS2 with respect to P1245A variant, thus possibly influencing SARS-CoV-2 infection. Our aim was to characterize the HSD3B1 polymorphism status and its potential association with clinical outcomes in hospitalized patients with COVID-19 in Southern Switzerland. Materials and Methods: The cohort included 400 patients hospitalized for COVID-19 during the first wave between February and May 2020 in two different hospitals of Canton Ticino. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue blocks, and HSD3B1 gene polymorphism was evaluated by Sanger sequencing. Statistical associations were verified using different test. Results: HSD3B1 polymorphic variants were not associated with a single classical factor related to worse clinical prognosis in hospitalized patients with SARS-CoV-2. However, in specific subgroups, HSD3B1 variants played a clinical role: intensive care unit admission was more probable in patients with P1245C diabetes compared with P1245A individuals without this comorbidity and death was more associated with hypertensive P1245A>C cases than patients with P1245A diabetes without hypertension. Discussion: This is the first study showing that HSD3B1 gene status may influence the severity of SARS-CoV-2 infection. If confirmed, our results could lead to the introduction of HSD3B1 gene status analysis in patients infected with SARS-CoV-2 to predict clinical outcome. Keywords: HSD3B1 gene polymorphism; Likelihood-ratio tests; SARS-CoV-2; androgen receptor; direct sequencing

Multi-Determinants Analysis of Molecular Alterations for Predicting Clinical Benefit to EGFR-Targeted Monoclonal Antibodies in Colorectal Cancer

KRAS mutations occur in 35-45% of metastatic colorectal cancers (mCRC) and preclude responsiveness to EGFR-targeted therapy with cetuximab or panitumumab. However, less than 20% patients displaying wild-type KRAS tumors achieve objective response. Alterations in other effectors downstream of the EGFR, such as BRAF, and deregulation of the PIK3CA/PTEN pathway have independently been found to give rise to resistance. We present a comprehensive analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression in mCRC patients treated with cetuximab or panitumumab, with the aim of clarifying the relative contribution of these molecular alterations to resistance.We retrospectively analyzed objective tumor response, progression-free (PFS) and overall survival (OS) together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumors from cetuximab or panitumumab treated mCRC patients. Among the 106 non-responsive patients, 74 (70%) had tumors with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with > or =2 alterations (p<0.0001). Accordingly, PFS and OS were increasingly worse for patients with tumors harboring none, 1, or > or =2 molecular alteration(s) (p<0.001).When expression of PTEN and mutations of KRAS, BRAF and PIK3CA are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to anti-EGFR therapies can be identified. We propose to define as 'quadruple negative', the CRCs lacking alterations in KRAS, BRAF, PTEN and PIK3CA. Comprehensive molecular dissection of the EGFR signaling pathways should be considered to select mCRC patients for cetuximab- or panitumumab-based therapies