Multitarget Fluorescence In Situ Hybridization Diagnostic Applications in Tumors.

Multitarget fluorescence in situ hybridization (mFISH) is a technique that allows the detection of multiple target sequences on the same sample using spectrally distinct fluorophore labels. The mFISH approach is currently a useful assay in the oncologic field for the detection of predictive, prognostic, and diagnostic biomarkers. In this chapter, we summarize the application of mFISH in the identification of target genetic aberrations in formalin-fixed, paraffin-embedded (FFPE) tissue samples of several tumor types. We discuss the mFISH protocols in FFPE samples, the innovative multitarget probes used, and the critical issues related to their interpretation.

The Role of TLR-2 in Lethal COVID-19 Disease Involving Medullary and Resident Lung Megakaryocyte Up-Regulation in the Microthrombosis Mechanism.

Patients with COVID-19 have coagulation and platelet disorders, with platelet alterations and thrombocytopenia representing negative prognostic parameters associated with severe forms of the disease and increased lethality. METHODS: The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. RESULTS: A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. CONCLUSIONS: Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.

Immunohistochemistry for Cancer Stem Cell Detection: Principles and Methods.

Cancer stem cells (CSCs) are rare immortal cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity. CSCs play a pivotal role in the tumor development, progression, relapse, and resistance of anticancer therapy. The technique of choice to detect CSCs in formalin-fixed and paraffin-embedded (FFPE) samples is immunohistochemistry (IHC) since it is inexpensive and widespread in most laboratories. The main aims of this chapter are the description of the protocols and the automated immunohistochemical systems used for the identification of CSCs. Furthermore, a focus on the most common troubleshooting in CSC IHC is provided. Finally, an overview of the main markers of cancer stem cells in several cancer types will be provided.

Genital and Oral HPV Geno-Prevalence Measured through Urine and Saliva Samples in Young Adults in Italy.

BACKGROUND: The aims of the study were to determine, in the urine and oral samples of young adults, the genotype-specific prevalence of Human Papilloma Virus (HPV) infection, the HPV DNA type-specific prevalence in unvaccinated and vaccinated individuals, and the determinants of HPV infection. METHODS: Selected participants were asked to fill in a self-administered questionnaire and to self-collect urine and saliva samples. RESULTS: Among the 1002 participants, 81 (8.1%) resulted positive for HPV DNA. The most common low-risk genotype was HPV 42 (2.2%), followed by HPV 43 (0.8%), and 40 (0.5%). The HPV 51 was the most common high-risk genotype (1.5%) followed by HPV 66 (1%) and HPV 68 (1%), and no participants were infected with HPV genotypes 18, 33, 45. Females, those who have had one or more occasional sexual partner, those who never/rarely/sometimes used condoms during their sexual activity, those with a previous diagnosis of sexually transmitted infection, and those who were not vaccinated were more likely to be tested positive for HPV infection. CONCLUSIONS: The low prevalence of genital HPV infections has provided evidence of the effectiveness of HPV vaccination both in vaccinated and not yet vaccinated subjects through herd immunity and indicated its decisive role in the changing epidemiology of circulating HPV genotypes in the population.

COVID-19: detection methods in post-mortem samples.

COVID-19 identification is routinely performed on fresh samples, such as nasopharyngeal and oropharyngeal swabs, even if, the detection of the virus in formalin-fixed paraffin-embedded (FFPE) autopsy tissues could help to underlie mechanisms of the pathogenesis that are not well understood.The gold standard for COVID-19 detection in FFPE samples remains the qRT-PCR as in swab samples, contextually other methods have been developed, including immunohistochemistry (IHC), and in situ hybridization (ISH). In this manuscript, we summarize the main data regarding the methods of COVID-19 detection in pulmonary and extra-pulmonary post-mortem samples, and especially the sensitivity and specificity of these assays will be discussed.

The Role of Toll-like Receptor-4 in Macrophage Imbalance in Lethal COVID-19 Lung Disease, and Its Correlation with Galectin-3.

To the current data, there have been 6,955,141 COVID-19-related deaths worldwide, reported to WHO. Toll-like receptors (TLRs) implicated in bacterial and virus sensing could be a crosstalk between activation of persistent innate-immune inflammation, and macrophage's sub-population alterations, implicated in cytokine storm, macrophage over-activation syndrome, unresolved Acute Respiratory Disease Syndrome (ARDS), and death. The aim of this study is to demonstrate the association between Toll-like-receptor-4 (TLR-4)-induced inflammation and macrophage imbalance in the lung inflammatory infiltrate of lethal COVID-19 disease. Twenty-five cases of autopsy lung tissues were studied by digital pathology-based immunohistochemistry to evaluate expression levels of TLR-4 (CD 284), pan-macrophage marker CD68 (clone KP1), sub-population marker related to alveolar macrophage Galectin-3 (GAL-3) (clone 9C4), and myeloid derived CD163 (clone MRQ-26), respectively. SARS-CoV-2 viral persistence has been evaluated by in situ hybridation (ISH) method. This study showed TLR-4 up-regulation in a subgroup of patients, increased macrophage infiltration in both Spike-1(+) and Spike-1(-) lungs (p < 0.0001), and a macrophage shift with important down-regulation of GAL-3(+) alveolar macrophages associated with Spike-1 persistence (p < 0.05), in favor of CD163(+) myeloid derived monocyte-macrophages. Data show that TLR-4 expression induces a persistent activation of the inflammation, with inefficient resolution, and pathological macrophage shift, thus explaining one of the mechanisms of lethal COVID-19.

A Critical Issue in Lung Cancer Cytology and Small Biopsies: DNA and RNA Extraction from Archival Stained Slides for Biomarker Detection through Real Time PCR and NGS-The Experience in Pathological Anatomy Unit.

The handling of biomaterials is crucial for precision medicine in advanced-stage lung patients with only cytology or small biopsies available. The main purpose of the study was to evaluate the quantity and quality of nucleic acids extracted from mixed stained slides (MSSs), including H&E, IHC and FISH, compared to the extraction from unstained slides (USs). A series of 35 lung adenocarcinoma surgical samples was selected to set up the method and the technical approach was validated in a series of 15 small biopsies and 38 cytological samples. DNA extracted from MSSs was adequate in all samples and the Real Time PCR was successful in 30/35 surgical samples (86%), 14/15 small biopsies (93%), and 33/38 cytological samples (87%). NGS using DNA extracted from MSSs was successful in 18/35 surgical samples (51%), 11/15 small biopsies (73%), and 26/38 cytological samples (68%). RNA extracted from MSSs was unsatisfactory in all cases showing an inadequate degree of fragmentation. Our technical approach based on the recovery of stained slides could represent a strategic way forward for DNA-based biomarker testing in lung cancer cases without biomaterials. The RNA extracted from MSSs did not represent a successful approach.

Application of CytoMatrix for the diagnosis of melanoma metastases on FNA cytology samples: Performance of a novel cell block method.

BACKGROUND: The management of cytological samples can significantly impact diagnostic interpretation. Cell blocks (CB) are a popular method due to their ability to provide additional morphological information and be used for immunocytochemistry and molecular tests. Recently, a new CB technique called the synthetic matrix CytoMatrix (CM) has been introduced, which can gather and hold cytological material within its three-dimensional structure. METHODS: In this study, 40 cytological samples from patients with melanoma metastases were analyzed to evaluate the diagnostic performance of CM compared to another CB method used in the laboratory. The researchers assessed the morphological adequacy of the two techniques, as well as their performance in immunocytochemical analysis and molecular. RESULTS: This study showed that CM was quicker and equally effective compared to the other method, with the laboratory technician having less of an impact on the CM approach across all passages. Additionally, all CMs were adequate, whereas the other method was adequate in 90% of cases. Immunocytochemistry confirmed the diagnosis of melanoma metastases in all cases, and all 40 CMs and 36 of the other method were adequate for fluorescence in situ hybridization analysis. CONCLUSION: CM is a low-time-consuming technology that is unaffected by technicians during all setup phases, making it simpler to standardize the procedure. Moreover, a low loss of diagnostic cells ensures greater benefits for morphological analysis, immunocytochemistry, and molecular testing. Overall, the study highlights the potential of CM as a valuable technique for managing cytological samples.

NTRK gene aberrations in triple-negative breast cancer: detection challenges using IHC, FISH, RT-PCR, and NGS.

Triple-negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type-agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan-TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), and RNA-based next-generation sequencing (NGS). Of 83 cases, 16 showed pan-TRK positivity although no cases had NTRK-fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT-PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan-TRK IHC. Our data demonstrate that IHC has a high false-positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.

PATZ1 in Non-Small Cell Lung Cancer: A New Biomarker That Negatively Correlates with PD-L1 Expression and Suppresses the Malignant Phenotype.

Non-small cell lung cancer (NSCLC), the leading cause of cancer death worldwide, is still an unmet medical problem due to the lack of both effective therapies against advanced stages and markers to allow a diagnosis of the disease at early stages before its progression. Immunotherapy targeting the PD-1/PD-L1 checkpoint is promising for many cancers, including NSCLC, but its success depends on the tumor expression of PD-L1. PATZ1 is an emerging cancer-related transcriptional regulator and diagnostic/prognostic biomarker in different malignant tumors, but its role in lung cancer is still obscure. Here we investigated expression and role of PATZ1 in NSCLC, in correlation with NSCLC subtypes and PD-L1 expression. A cohort of 104 NSCLCs, including lung squamous cell carcinomas (LUSCs) and adenocarcinomas (LUADs), was retrospectively analyzed by immunohistochemistry for the expression of PATZ1 and PD-L1. The results were correlated with each other and with the clinical characteristics, showing on the one hand a positive correlation between the high expression of PATZ1 and the LUSC subtype and, on the other hand, a negative correlation between PATZ1 and PD-L1, validated at the mRNA level in independent NSCLC datasets. Consistently, two NSCLC cell lines transfected with a PATZ1-overexpressing plasmid showed PD-L1 downregulation, suggesting a role for PATZ1 in the negative regulation of PD-L1. We also showed that PATZ1 overexpression inhibits NSCLC cell proliferation, migration, and invasion, and that Patz1-knockout mice develop LUAD. Overall, this suggests that PATZ1 may act as a tumor suppressor in NSCLC.

Implementation of BRCA mutations testing in formalin-fixed paraffin-embedded (FFPE) samples of different cancer types.

BRCA1 and BRCA2 are onco-suppressor genes involved in the DNA repair mechanism. The presence of BRCA1/2 mutations confers a higher risk of developing several cancer types. To date, the FDA approved various PARP inhibitors to treat selected BRCA1/2 mutated oncologic patients. At first, PARP inhibitors were approved for patients with ovarian and breast cancers, and subsequently for metastatic pancreatic adenocarcinoma and metastatic castration-resistant prostate cancer after the treatment with chemotherapy. The current guidelines for BRCA testing are very heterogeneous between the different types of tumors regarding the diagnostic algorithm and the type of sample to analyze, such as the blood for the germline mutations and the tumoral tissue for the somatic mutations. Few data have currently been described regarding the detection of BRCA1/2 somatic mutations in formalin-fixed paraffin-embedded (FFPE) samples. In this review, we propose an overview of the BRCA mutations in FFPE samples of several cancers, including breast, ovarian, fallopian tube, primary peritoneal, prostate, and pancreatic cancer. We summarize the types and the frequency of BRCA mutations, the guidelines approved for the test, the molecular assays used for the detection and the PARP inhibitors approved for each tumor type.

To Get the Most out of the Least: BRAF Molecular Evaluation in Melanoma Metastases on Cell Suspension from Fine Needle Aspiration Cytology Needle Rinses.

INTRODUCTION: Cytological samples from cutaneous melanoma (CM) metastases may be the only biomaterial available for diagnostic and predictive purpose in the clinical practice. BRAF evaluation from cytological samples actually implies the loss of one or more diagnostic smears, or the execution of one or more passes to obtain dedicated cytological samples. We tested BRAF molecular evaluation in CM metastases on cell suspension obtained from fine needle aspiration cytology (FNAC) needle rinses. METHODS: Forty-two patients with lymph node enlargements and a previous CM were enrolled. Patients were submitted to FNAC, and direct smears and cell-block were prepared for diagnostic purpose. The needle was carefully flushed in a vial containing 350 µL of nuclease-free water and a cell suspension was obtained for BRAF molecular evaluation. Molecular evaluation was also performed on histological samples for statistics. RESULTS: The series included 35 CM metastases and 7 reactive lymphadenopathies. Three cases resulted inadequate and adequacy was 92.9%. BRAF V600E/Ec mutations were found in 7 out of 32 (21.9%) CM metastases cases. BRAF mutations other than V600E/Ec were found in 2 out of 32 (6.25%) cases. Sensitivity, specificity, positive predictive value, and negative predictive value resulted 100%. CONCLUSION: BRAF molecular evaluation in CM metastases on cell suspension obtained from FNAC needle rinses is a time-sparing and accurate technique allowing to spare biomaterial in the clinical setting.

The prognostic value of histopathology in invasive lung adenocarcinoma: a comparative review of the main proposed grading systems.

INTRODUCTION: An accurate histological evaluation of invasive lung adenocarcinoma is essential for a correct clinical and pathological definition of the tumour. Different grading systems have been proposed to predict the prognosis of invasive lung adenocarcinoma. AREAS COVERED: Invasive non mucinous lung adenocarcinoma is often morphologically heterogeneous, consisting of complex combinations of architectural patterns with different proportions. Several grading systems for non-mucinous lung adenocarcinoma have been proposed, being the main based on architectural differentiation and the predominant growth pattern. Herein we perform a thorough review of the literature using PubMed, Scopus and Web of Science and we highlight the peculiarities and the differences between the main grading systems and compare the data about their prognostic value. In addition, we carried out an evaluation of the proposed grading systems for less common histological variants of lung adenocarcinoma, such as fetal adenocarcinoma and invasive mucinous adenocarcinoma. EXPERT OPINION: The current IASLC grading system, based on the combined score of predominant growth pattern plus high-grade histological pattern, shows the stronger prognostic significance than the previous grading systems in invasive non mucinous lung adenocarcinoma.

Expression of c-MET in Estrogen Receptor Positive and HER2 Negative Resected Breast Cancer Correlated with a Poor Prognosis.

Introduction: The mesenchymal-epithelial transition factor (c-MET) receptor is overexpressed in about 14−54% of invasive breast cancers, but its prognostic value in clinical practice is still unclear. Methods: In order to investigate the relationship between c-MET expression levels and prognosis, we retrospectively reviewed the clinical features and outcomes of 105 women with estrogen receptor positive HER2 negative (ER+/HER2-) resected breast cancer. We used the Kaplan Meier method to estimate Disease Free Survival (DFS) and Breast Cancer Specific Survival (BCSS) in the subgroups of patients with high (≥50%) and low (<50%) c-MET expression. Univariate and multivariate Cox proportional regression models were performed to assess the prognostic impact of clinicopathological parameters for DFS an BCSS. Results: High c-MET values significantly correlated with tumor size, high Ki67 and low (<20%) progesterone receptor expression. At a median follow up of 60 months, patients with high c-MET tumor had significantly worse (p = 0.00026) and BCSS (p = 0.0013). Univariate analysis showed a significant association between large tumor size, elevated Ki67, c-MET values and increased risk of recurrence or death. The multivariate COX regression model showed that tumor size and high c-MET expression were independent predictors of DFS (p = 0.019 and p = 0.022). Moreover, large tumor size was associated with significantly higher risk of cancer related death at multivariate analysis (p = 0.017), while a trend towards a poorer survival was registered in the high c-MET levels cohort (p = 0.084). Conclusions: In our series, high c-MET expression correlated with poor survival outcomes. Further studies are warranted to validate the clinical relevance and applicability of c-MET as a prognostic factor in ER+/HER2- early BC.

TRK Protein Expression in Merkel Cell Carcinoma Is Not Caused by NTRK Fusions.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.

AXL and MET Tyrosine Kinase Receptors Co-Expression as a Potential Therapeutic Target in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly lethal malignancy that unfortunately cannot benefit from molecularly targeted therapies. Although previous results showed the pivotal role of various receptor tyrosine kinases (RTKs) in MPM tumorigenesis, the treatment with a single inhibitor targeting one specific RTK has been shown to be ineffective in MPM patients. The main aim of the present study was to investigate the potential role of AXL and MET receptors in MPM and the possible efficacy of treatment with AXL and MET multitarget inhibitors. Immunohistochemical and FISH analyses were performed in a wide series of formalin-fixed paraffin-embedded MPM samples to detect the expression of two receptors and the potential gene amplification. In vitro studies were performed to evaluate putative correlations between the target's expression and the cell sensitivity to AXL-MET multitarget inhibitors. In our series, 10.4% of cases showed a co-expression of AXL and MET, regardless of their ligand expression, and the gene amplification. Furthermore, our in vitro results suggest that the concomitant pharmacological inhibition of AXL and MET may affect the proliferative and aggressiveness of MPM cells. In conclusion, the subset of MPM patients with AXL-MET co-activation could benefit from treatment with specific multitarget inhibitors.

Triple blockade of Ido-1, PD-L1 and MEK as a potential therapeutic strategy in NSCLC.

BACKGROUND: Despite the recent progress in the treatment and outcome of Non Small Cell Lung Cancer (NSCLC), immunotherapy has still significant limitations reporting a significant proportion of patients not benefiting from therapy, even in patients with high PD-L1 expression. We have previously demonstrated that the combined inhibition of MEK and PD-L1 in NSCLC patients derived three dimensional cultures exerted significant synergistic effect in terms of immune-dependent cancer cell death. However, subsequent experiments analyzing the expression of Indoleamine 2,3-dioxygenase-1 (Ido-1) gene expression demonstrated that Ido-1 resulted unaffected by the MEK inhibition and even increased after the combined inhibition of MEK and PD-L1 thus representing a potential escape mechanism to this combination. METHODS: We analyzed transcriptomic profile of NSCLC lung adenocarcinoma cohort of TCGA (The Cancer Genome Atlas), stratifying tumors based on EMT (Epithelial mesenchymal Transition) score; in parallel, we investigated the activation of Ido-1 pathway and modulation of immune cytokines productions both in NSCLC cells lines, in peripheral blood mononuclear cells (PBMCs) and in ex-vivo NSCLC spheroids induced by triple inhibition with an anti-PD-L1 monoclonal antibody, the MEK inhibitor and the Ido-1 inhibitor. RESULTS: In NSCLC lung adenocarcinoma patient cohort (from TCGA) Ido-1 gene expression was significantly higher in samples classified as mesenchymal according EMT score. Similarly, on a selected panel of NSCLC cell lines higher expression of MEK and Ido-1 related genes was detected in cells with mesenchymal phenotype according EMT score, thus suggesting a potential correlation of co-activation of these two pathways in the context of EMT, with cancer cells sustaining an immune-suppressive microenvironment. While exerting an antitumor activity, the dual blockade of MEK and PD-L1 enhances the secretion of pro-inflammatory cytokines (IFNγ, TNFα, IL-12 and IL-6) and, consequently, the expression of new immune checkpoints such as Ido-1. The triple inhibition with an anti-PD-L1 monoclonal antibody, the MEK inhibitor and the Ido-1 inhibitor demonstrated significant antiproliferative and proapoptotic activity on ex-vivo NSCLC samples; at the same time the triple combination kept increased the levels of pro-inflammatory cytokines produced by both PBMCs and tumor spheroids in order to sustain the immune response and simultaneously decreased the expression of other checkpoint (such as CTLA-4, Ido-1 and TIM-3) thus promoting an immune-reactive and inflamed micro-environment. CONCLUSIONS: We show that Ido-1 activation is a possible escape mechanism to immune-mediated cell death induced by combination of PD-L1 and MEK inhibitors: also, we show that triple combination of anti-PD-L1, anti-MEK and anti-Ido-1 drugs may overcome this negative feedback and restore anti-tumor immune response in NSCLC patients' derived three dimensional cultures.

Preliminary Experience of Liquid Biopsy in Lung Cancer Compared to Conventional Assessment: Light and Shadows.

Purpose: To assess the qualitative relationship between liquid biopsy and conventional tissue biopsy. As a secondary target, we evaluated the relationship between the liquid biopsy results and the T stage, N stage, M stage, and compared to grading. Methods: The Local Ethics Committee of the "Università degli Studi della Campania Luigi Vanvitelli", with the internal resolution number 24997/2020 of 12.11.2020, approved this spontaneous prospective study. According to the approved protocol, patients with lung cancer who underwent Fine-Needle Aspiration Cytology (FNAC), CT-guided biopsy, and liquid biopsy were enrolled. A Yates chi-square test was employed to analyze differences in percentage values of categorical variables. A p-value < 0.05 was considered statistically significant. Data analysis was performed using the Matlab Statistic Toolbox (The MathWorks, Inc., Natick, MA, USA). Results: When a genetic mutation is present on the pathological examination, this was also detected on the liquid biopsy. ROS1 and PDL1 mutations were found in 2/29 patients, while EGFR Exon 21 was identified in a single patient. At liquid biopsy, 26 mutations were identified in the analyzed samples. The mutations with the highest prevalence rate in the study populations were: ALK (Ile1461Val), found in 28/29 patients (96.6%), EML4 (Lys398Arg), identified in 16/29 (55.2%) patients, ALK (Asp1529Glu), found in 14/29 (48.3%) patients, EGFR (Arg521Lys), found in 12/29 (41.4%) patients, ROS (Lys2228Gln), identified in 11/29 (37.9%) patients, ROS (Arg167Gln) and ROS (Ser2229Cys), identified in 10/29 (34.5%) patients, ALK (Lys1491Arg) and PIK3CA (Ile391Met), identified in 8/29 (27.6%) patients, ROS (Thr145Pro), identified in 6/29 (20.7%) patients, and ROS (Ser1109Leu), identified in 4/29 (13.8%) patients. No statistically significant differences can be observed in the mutation rate between the adenocarcinoma population and the squamous carcinoma population (p > 0.05, Yates chi-square test). Conclusions: We showed that, when a genetic mutation was detected in pathological examination, this was always detected by liquid biopsy, demonstrating a very high concordance rate of genomic testing between tissues and their corresponding mutations obtained by liquid biopsy, without cases of false-negative results. In addition, in our study, liquid biopsy highlighted 26 mutations, with the prevalence of ALK mutation in 96.6% of patients, supporting the idea that this approach could be an effective tool in cases with insufficient tumor tissue specimens or in cases where tissue specimens are not obtainable.

Microsatellite Instability: From the Implementation of the Detection to a Prognostic and Predictive Role in Cancers.

Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.

Microsatellite Status Detection in Gastrointestinal Cancers: PCR/NGS Is Mandatory in Negative/Patchy MMR Immunohistochemistry.

BACKGROUND: Microsatellite instability (MSI) is a predictive biomarker for immune checkpoint inhibitors. The main goal was to investigate the discordance between IHC and PCR/NGS for MSI testing in gastrointestinal cancers. METHODS: Two series were analyzed through IHC for mismatch-repair-system proteins (MMRP) and PCR, with one series of 444 colorectal cancers (CRC) and the other of 176 gastric cancers (GC). All cases with discordant results between IHC and PCR were analyzed by NGS. IHC staining was evaluated as follows: proficient MMR (pMMR), with all MMR positive; deficient MMR (dMMR), with the loss of one heterodimer; and cases with the loss/patchy expression of one MMR (lo-paMMR). Cases with instability in at least two markers by PCR were MSI-high (MSI-H) and with instability in one marker, MSI-low (MSI-L). Cases without instability were evaluated as microsatellite-stable (MSS). RESULTS: In the CRC cohort, 15 out of 444 cases were dMMR and 46 lo-paMMR. Among the 15 dMMR, 13 were MSI-H and 2 MSS. Among the 46 lo-paMMR, 13 were MSI-H and 33 were MSS. In the GC cohort, 13 out of 176 cases were dMMR and 6 cases lo-paMMR. Among the 13 dMMR, 12 were MSI-H and only 1 was MSS. All six lo-paMMR cases were MSS. All NGS results were in agreement with PCR. CONCLUSIONS: In clinical practice, MMR-IHC could be used as a screening test and additional molecular analysis is mandatory exclusively in cases carrying loss/patchy MMR-IHC.