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AimsSeveral predictive biomarkers of response to specific inhibitors have become mandatory for the therapeutic choice in non-small-cell lung cancer (NSCLC). In most lung cancer patients, the biological materials available to morphological and molecular diagnosis are exclusively cytological samples and minimum tumour wastage is necessary. Multiplex fluorescence in situ hybridisation (mFISH) to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide could be useful in clinical practice to save cytological samples for further molecular analysis. In this study, we aim to validate diagnostic performance of multiplex ALK/ROS1 fluorescence in situ hybridisation (FISH) approach in lung adenocarcinoma cytological series compared with classic single break apart probes.MethodsWe collected a series of 61 lung adenocarcinoma cytological specimens enriched in tumours harbouring ALK-rearrangement and ROS1-rearrangement. ALK and ROS1 status were previously assessed by classic FISH test using single break apart probes and immunohistochemistry. Study population was composed of 6 ALK-positive, 2 ROS1-positive and 53 ALK/ROS1-wild type. All specimens were analysed by multiplex FISH assay using FlexISH ALK/ROS1 DistinguISH Probe Zytovision.ResultsThe dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests on two different slides. 6 ALK-positive and 2 ROS1-positive were confirmed through multiplex FISH test, without false-positive and false-negative results. Multiplex ALK/ROS1 FISH test results agreed with immunohistochemistry assay staining results.ConclusionMultiplex ALK/ROS1 FISH probe test is a useful tool to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide in cytological specimens with a small amount of biomaterial.

– Malignant mesothelioma (MM) is a rare aggressive neoplasm arising from mesothelial lining of body cavities, most commonly pleura and peritoneum. It is characterised by a poor prognosis and limited treatment options. A universally recognised risk factor for the development of MM is exposure to asbestos. However, evidence supporting a genetic susceptibility to the development of MM has been accumulating during the last decades. Intensive research for the identification of MM susceptibility genes has led to the discovery of BAP1 and to the definition of the so-called “BAP1-related tumour predisposition syndrome”. Patients carrying germline BAP1 mutations have an increased risk for the early development of tumours, including MMs, uveal melanomas, cutaneous melanocytic lesions, clear cell renal cell carcinomas and basal cell carcinomas. Furthermore, pathogenic variants in tumour suppressor genes with a role in DNA repair have been recently described in families with clustered MM cases. These genetic alterations seem to confer exaggerate sensitivity to asbestos carcinogenic effect and, arguably, increased response to specific chemotherapeutic strategies. While the translational significance of BAP1 alterations is explored in the research field, the identification of families carrying germline BAP1 mutations is mandatory to start appropriate surveillance programs and guarantee the best clinical management to these patients.

Background: Renal cell carcinoma (RCC) constitutes 3% of all cancers, with a higher incidence in patients with age between 60 and 70 years. RCC frequently present as a metastatic tumor at diagnosis, and bones represent one of the most frequent sites. Many cases, mainly in young patients, includes the Xp11 translocation RCC. The cytological diagnosis of Xp11 translocation RCC in adult population it is rarely performed, likely for the morphological overlap with other adult renal cell carcinoma subtypes. Methods: We retrospectively analyze a series of 92 adult patients with metastatic bone tumors, diagnosed on fine-needle aspiration cytology (FNAC) samples, focusing mainly on the cytological, immunophenotypic and molecular features of Xp11 translocation RCC. Results: In our series 6 of 92 (6.5%) cases were metastatic RCC (mRCC), among them 2 cases were metastasis from Xp11translocation RCC. Those cases showed a bloody background, with several groups of atypical cells arranged in syncytial groups or in papillary groups composed by atypical cells with abundant cytoplasm, with scattered clear cells. TFE3 was positive on immunocytochemical analysis and specific translocation t(Xp11.23) was detected by FISH analysis. Conclusions: In adult patients with mRCC, it is necessary to consider also Xp11 translocation RCC among the diagnostic hypotheses. FNAC represents a valid tool to investigate bone lesions but cytological features of Xp11 translocation RCC are still poorly described and must necessarily be better defined.

OBJECTIVE: Anaplastic lymphoma kinase (ALK) gene has been demonstrated to be rearranged, mutated or amplified in several haematological and solid tumors. Moreover, the use of ALK inhibitors has recently revolutionized the treatment of ALK-rearranged patients affected by non-small cell lung carcinoma. Herein we review the genetic alterations of ALK in melanocytic neoplasms described in literature, focusing on their potential diagnostic and predictive role. MATERIALS AND METHODS: The Authors reviewed the pertinent literature through research on PubMed server was performed typing the terms "ALK", "Anaplastic lymphoma kinase", "ALKATI", "Melanoma", "Spitz", "Spitzoid". RESULTS: ALK translocations were demonstrated in melanocytic neoplasms, particularly in acral melanoma and spitzoid tumors. ALKATI was described in primary and metastatic melanoma, indicating its early occurrence in oncogenesis, with varying immunohistochemical expression of the protein. CONCLUSIONS: The identification of the specific type of ALK mutations could be interesting for planning biologic therapy of melanoma patients. Further studies are needed to evaluate the possibility to introduce an ALK-targeted therapy in patients affected by malignant melanoma.

Soft tissue tumors comprise a great variety of common and rare entities with overlapping features. Their diagnosis is based on the evaluation of several histological parameters which are difficult to assess on small incisional biopsies. Useful diagnostic markers in the field of soft tissue tumors include: 1) molecular biomarkers detecting pathogenetically relevant, distinctive alterations; 2) immunohistochemical surrogate biomarkers of pathogenetically relevant, distinctive molecular alterations; 3) highly specific immunohistochemical biomarkers indicating tumor differentiation. Their introduction in clinical practice has revolutionized the pre-operative diagnosis of soft tissue tumors. Cytology has long been considered inadequate as a first-line approach in this setting. However, since the implementation of new immunohistochemical and molecular tests with high diagnostic specificity, fine needle aspiration cytology (FNAC) is starting to gain acceptance for the pre-operative assessment of soft tissue tumors. FNAC represents a versatile, poorly expensive and well-tolerated diagnostic strategy with relevant advantages over histological biopsies. Moreover, evidences suggest that, in expert hands, FNAC can also aim at a definite diagnosis, especially if a cell block is prepared, allowing the application of multiple ancillary techniques.

The identification of biomarkers on cancer tissue samples could be obtained through several technologies. In this setting, the immunohistochemistry and in situ hybridization are accessible in most pathology laboratories. Particularly, immunohistochemistry can be used not only for diagnostic issues, but also to define prognostic classes and to define response to specific therapies. Particularly the last applications have been firstly developed in the breast cancer pathology. In addition, the development of molecular classification proposed some prognostic/predictive classes that could be easily defined by immunohistochemistry. Thus, the role of the pathologists has become increasingly important in the definition of prognosis and in the choice therapy, because the immunohistochemical biomarkers are used to guide treatment, to classify breast cancer into biologically and prognostically distinct subtypes. In this review, we will provide information on the current application of the immunohistochemical biomarkers useful in the management of breast cancer patients. Moreover, we consider the application of immunohistochemistry in the definition of the most promising biomarkers derived from molecular studies of the breast cancer, that in the future could integrate the characterization of breast cancer into clinical practice.

Background RAS mutations are the only validated biomarkers in metastatic colorectal cancer (mCRC) for anti-epidermal growth factor receptor (EGFR) therapy. Limited clinical information is available on AXL expression, marker of epithelial to mesenchymal transition, in mCRC. Methods AXL was retrospectively assessed by immunohistochemistry in 307 patients. RAS wild-type (WT) patients (N = 136) received first-line anti-EGFR–based therapy; RAS mutant patients (N = 171) received anti-angiogenic–based regimens. Preclinical experiments were performed using human RAS WT CRC cell lines and xenograft models. AXL RNA levels were assessed in a cohort of patients with available samples at baseline and at progression to anti-EGFR treatment and in the GSE5851 dataset. Results AXL was expressed in 55/307 tumour tissues, correlating with worse survival in the overall population (AXL-positive, 23.7 months; AXL-negative, 30.8 months; HR, 1.455, P = 0.032) and in RAS WT patients (AXL-positive, 23.0 months; AXL-negative, 35.8 months; HR,1.780, P = 0.032). Progression-free survival (PFS) in the RAS WT cohort was shorter in the AXL-positive cohort (6.2 months versus 12.1 months; HR, 1.796, P = 0.013). Three-dimensional cultures obtained from a patient following anti-EGFR therapy resulted AXL-positive, showing resistance to anti-EGFR drugs and sensitivity to AXL inhibition. AXL transfection in CRC cell lines induced AXL overexpression and resistance to the EGFR blockade. At progression to cetuximab, 2/10 SW48-tumour xenograft mice showed AXL expression. Consistently, AXL RNA levels increased in 5/7 patients following anti-EGFR therapy. Moreover, in the GSE5851 dataset higher AXL RNA levels correlated with worse PFS with cetuximab in KRAS-exon2 WT chemorefractory patients. Conclusions AXL is a marker of poor prognosis in mCRC with consistent clinical and preclinical evidences of involvement in primary and acquired resistance to anti-EGFR drugs in RAS WT patients.

Immune checkpoint inhibitors against programmed cell death protein 1/programmed death-ligand 1 (PD-L1) have proven to be remarkably effective in non-small cell lung cancer. PD-L1 represents a predictive biomarker in lung cancer, although its heterogenous expression represents an emerging challenge for accurate biomarker-based patient selection. Lung adenocarcinomas (ADCs) show a high rate of intratumor morphologic heterogeneity that may reflect a heterogenous molecular and immunophenotypic profile. The aim of our study was to analyze the expression of PD-L1 in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas (mADCs) and adenosquamous lung carcinomas (AdSqLCs). As many as 73 mADCs and 6 AdSqLCs were selected. Comprehensive histologic subtyping was performed, and PD-L1 expression was assessed by immunohistochemistry assay using different primary antibodies and automated immunostainers. Overall, PD-L1 expression was observed in 37 of 79 cases (39.2%) (31 mADCs and all AdSqLCs). PD-L1 expression was heterogenous in 22 of 37 PD-L1-positive cases (23.2% mADC and 83% AdSqLC). PD-L1 expression was observed more frequently in ADC with solid pattern. Heterogeneity of PD-L1 expression was significantly related to the presence of micropapillary (P=0.028) and solid (P=0.017) patterns. All PD-L1-positive cases were epidermal growth factor receptor wild-type, 2 cases harbored concomitantly PD-L1 expression and ALK rearrangement. Our data suggest that PD-L1 expression is quite heterogenous in mADCs and AdSqLCs, partly contributing to explaining the discrepant results between biopsy and surgical resections and discordant clinical effectiveness in regard to PD-L1-positive or negative ADC diagnosed on cytology/small biopsy.

Angiosarcomas are ubiquitous neoplasms involving both cutaneous and soft tissue and visceral locations. Accumulating biomolecular evidences suggest that cutaneous angiosarcomas are distinctive entities with molecular, clinical and pathological peculiarities. Despite several ongoing clinical trials with promising therapeutic agents, the prognosis of cutaneous angiosarcomas is dismal and survival still rely on early diagnosis and surgery. An accurate diagnosis and the knowledge of the underlying molecular landscape are therefore essential to improve the prognosis. We detail the molecular, clinical, dermoscopic, morphological and prognostic features of cutaneous angiosarcoma. Although the molecular landscape of cutaneous angiosarcoma is not completely understood, accumulating evidences suggest that there are characteristic molecular alterations including dysregulation of angiogenesis and several complex molecular pathways. Secondary cutaneous angiosarcomas, arising in correlation with chronic lymphedema and ionizing radiation, have different molecular hallmarks, which are also leading to the first diagnostic applications. The diagnosis of cutaneous angiosarcoma may be challenging, as well-differentiated forms can be hard to distinguish from benign and low-grade vascular neoplasms, while poorly differentiated forms can be easily confounded with other non-vascular high-grade neoplasms. An accurate and early diagnosis, which is mandatory to ensure the best survival for the patients, is mainly based on morphological hallmarks.

Human epidermal growth factor receptor 2 (HER2) is a transmembrane growth factor receptor with tyrosine kinase activity regulating cell growth and survival. HER2 amplification is responsible for protein overexpression with a negative impact on prognosis, but HER2-overexpressing patients could be treated with anti-HER2 therapy. In gastric cancer HER2 overexpression is observed in approximately 22% of patients, but prognostic significance in this context is not clear. In colon adenocarcinoma, HER2 gene amplification is reported in a wide range from 0% to 83%. The wide range of reported series could be essentially attributable to a difference in scoring systems for HER2 protein expression. The use of testing score, although measuring the same protein, is different from gastric cancer to colorectal cancer. Herein the different scoring models are reported.

Mitochondria are the powerhouses of energy production and the sites where metabolic pathway and survival signals integrate and focus, promoting adaptive responses to hormone stimulation and nutrient availability. Increasing evidence suggests that mitochondrial bioenergetics, metabolism and signaling are linked to tumorigenesis. AKAP1 scaffolding protein integrates cAMP and src signaling on mitochondria, regulating organelle biogenesis, oxidative metabolism and cell survival. Here, we provide evidence that AKAP1 is a transcriptional target of Myc and supports the growth of cancer cells. We identify Sestrin2, a leucine sensor and inhibitor of the mammalian target of rapamycin (mTOR), as a novel component of the complex assembled by AKAP1 on mitochondria. Downregulation of AKAP1 impaired mTOR pathway and inhibited glioblastoma growth. Both effects were reversed by concomitant depletion of AKAP1 and sestrin2. High levels of AKAP1 were found in a wide variety of high-grade cancer tissues. In lung cancer, AKAP1 expression correlates with high levels of Myc, mTOR phosphorylation and reduced patient survival. Collectively, these data disclose a previously unrecognized role of AKAP1 in mTOR pathway regulation and cancer growth. AKAP1/mTOR signal integration on mitochondria may provide a new target for cancer therapy.

Nonmelanoma skin cancer (NMSC) is the most common cancer observed in the Caucasians, accounting approximately for 80% of basal cell cancer (BCC) and 20% cutaneous squamous cell carcinoma (cSCC) [1]. Differently from BCC, cSCC is related to a relatively high risk of metastasis occurrence. In recent years, a relative increase of cSCC incidence has been recorded, probably due to both higher exposure to sun and more frequent skin examinations [2]. Histopathologic variants show significantly different clinical behaviors [3-6]. They recognize different biological backgrounds with different prognosis. Therefore, their correct classification is critical for both diagnosis and treatment.DNA damage [15]. In details, p53 induction seems to be mainly due to both pyrimidine dimers, and DNA stands break [16]. p53 activation causes essentially cell cycle arrest, with subsequent DNA repair occurring through two major pathways: non-excision repair (NER) and base excision repair (BER). When DNAdamage is too wide, activated p53 induces apoptosis in damaged skin, through induction of various transcriptional factors related to activation of the apoptosomal complex, such as the Bcl-2 family members Bax, p53-upregulated modulator of apoptosis (PUMA), Noxa, p53-upregulated apoptosis-inducing protein 1 (p53AIP1), and PIGa (galectin- 7) [17-19]. Moreover, it has been demonstrated that UVBinduced apoptosis is related also to activation of Fas and Fas ligand, potent activators of pro-caspase 8 cleavage and, at the same time, of the cytochrome c release from mitochondria, followed by activation of the Apaf-1--pro-caspase 9 complex [20]. Chronic UV irradiation causes p53 mutation and loss of Fas--Fas ligand interaction with consequent apoptosis deregulation. p53 mutations seem to be an early genetic event in the UV-induced skin cancers, being present in approximately 53% of premalignant actinic keratosis (AK) lesions [21]. Many reports have recorded p53-mutant cell clones in nonneoplastic cells of skin exposed to sun [21-26]. Not all UV-induced p53 mutations confer a malignant phenotype to epidermal. Some in vivo experiments have demonstrated that the occurrence of the most critical p53 mutations are recorded from 17 to 80 days of exposure to UVB, and skin tumors appear from 30 to 80 weeks later (Figure 1) [27,28]. Regression of a part of precancerous p53-positive clones can be observed after discontinuation of UV irradiation [28,29] although tumor occurrence is only delayed [30]. Other proteins of p53 family, such as p63, are involved in cSCC development, because of its ability to control apoptosis through both extrinsic and intrinsic pathways. In fact, the function of p63 is, at least in part, mediated by IKKa (IkB kinase-a) [31].

Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a low-grade lymphoma comprising 7-8% of all B-cell non-Hodgkin lymphomas. Common sites of involvement include lung, head and neck, ocular adnexa, skin, thyroid and breast, but the gastrointestinal tract is by far the most common site and the stomach is involved in almost two-thirds of all cases. Infection and autoimmune diseases are commonly considered as etiopathogenetic factors, being related to chronic stimulation of B-cell proliferation. The association between Helicobacter pylori infection and gastric MALT lymphoma provides the best evidence of an etiopathogenetic link between lymphoma and infection. Indeed, successful eradication of this microorganism can be followed by lymphoma regression in most cases. In recent years the role of other pathogenetic factors including genetic predisposition, somatic genetic mutations and chemokines activity, has become more evident. Particularly specific genetic abnormalities have been observed in MALT lymphomas, with different distribution accordingly to the site of development.This review, therefore, addresses the major findings obtained in the last few years about MALT lymphoma and summarizes recent advances in its molecular pathogenesis.

Skin adnexal neoplasms comprise a wide spectrum of more than 80 benign and malignant tumours, exhibiting morphological differentiation towards pilosebaceous unit, eccrine and apocrine glands. In addition more than one line of differentiation in this neoplasm could be observed. These tumours generally exhibit a benign behaviour, but malignant histotypes also exist. Clinical diagnosis of specific entity and of their potential malignancy is quite impossible. Thus, the diagnosis always requires surgical excision of skin lesion, and histological features at haematoxylin and eosin-stained sections are considered generally adequate for the correct classification of skin adnexal tumours. The use of histochemical and immunohistochemical stains could further aid in this purpose. Finally, they are usually encountered as single, sporadic tumours, but they may also occasionally be multiple and hereditary, heralding complex genetic syndromes that comprise visceral cancers.

Malignant melanoma (MM) is a highly aggressive neoplasm with a growing worldwide incidence. It is not uncommon that the disease is already metastatic at the time of the first diagnosis. Regional lymph nodes and skin are the first and most common metastatic sites, followed by distant visceral sites (lungs, liver, and central nervous system) and bone. In this clinical setting, fine-needle aspiration (FNA) often represents the first diagnostic approach. FNA is a useful tool to obtain a rapid and accurate diagnosis, in conjunction with ancillary techniques and molecular analysis, as recommended by recent guidelines. The aim of this review was to describe the cytomorphology, immunocytochemical tools, and molecular tools used for the diagnosis of MM metastases on FNA.

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.

Background The high-risk human papillomavirus (HPV) infection represents one of the main etiologic pathways of penile carcinogenesis in approximately 30–50 % of cases. Several techniques for the detection of HPV are currently available including Polymerase chain reaction-based techniques, DNA and RNA in situ hybridization (ISH), p16 immunohistochemistry (IHC). The multiplex HPV RNA ISH/p16 IHC is a novel technique for the simultaneous detection of HPV E6/E7 transcripts and p16INK4a overexpression on the same slide in a single assay. The main aim of this study was to evaluate the discrepancy of p16 IHC expression relatively to HPV RNA ISH in penile cancer tissue. Methods We collected a series of 60 PCs. HPV has been analysed through the RNA ISH, p16 IHC and the multiplex HPV RNA ISH/p16 IHC. Results The multiplex HPV RNA ISH /p16 IHC results in the series were in complete agreement with the previous results obtained through the classic p16 IHC and HPV RNA scope carried out on two different slides. The multiplex HPV RNA ISH /p16 IHC showed that HPV positivity in our series is more frequently in usual squamous cell carcinoma than in special histotypes (19 out of 60 − 15 %- versus 6 out of 60 − 10 %-), in high-grade than in moderate/low grade carcinomas (6 out of 60 − 10 %- versus 4 out of 60 − 6.7 %-). In addition, our data revealed that in 5 out of 20 cases with p16 high intensity expression is not associated with HPV RNA ISH positivity. Conclusions Our findings emphasize that the use of p16 as a surrogate of HPV positivity was unsuccessful in approximatively 8 % of cases analysed in our series. Indeed, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 %. Since HPV positivity could represent a relevant prognostic and predictive value, the correct characterization offered by this approach appears to be of paramount importance.

AimsThe diagnosis of metastatic cutaneous melanoma (CM) on lymph node fine needle aspiration samples may be challenging and usually requires confirmation by immunocytochemistry. However, the cytological material could be too scant to order a broad panel of markers. In this case, the pathologist is forced to choose the most advantageous antibodies. The most commonly used melanocytic markers include S100, Melan-A, HMB45 and SOX10 but their diagnostic yield on cytological samples has been poorly studied. The current work aimed to evaluate the diagnostic performance of melanocytic markers when applied to cell blocks obtained from fine needle aspiration cytology (FNAC) of lymph node metastases from CM.MethodsS100, Melan-A, HMB45 and SOX10 were tested on cell block sections of 38 lymphnode metastases from CM diagnosed by cytology. A combined score was built to evaluate each immunostaining, considering the intensity of the staining and the percentage of stained neoplastic cells.ResultsS100 and SOX10 revealed a higher sensitivity (100%) than Melan-A and HMB45 for the diagnosis of metastatic CM. Furthermore, SOX10 emerged as the melanocytic marker with the best staining performance.ConclusionSOX10 has a 100% detection rate and the most easily interpretable staining pattern compared with other melanocytic markers. Therefore, it is strongly recommended that SOX10 is included in the minimal immunocytochemical panel for the diagnostic evaluation of lymph node FNAC in patients with suspected CM metastasis.

BackgroundPenile cancer (PC) is an extremely rare malignancy, and the patients at advanced stages have currently limited treatment options with disappointing results. Immune checkpoint inhibitors anti-programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) are currently changing the treatment of several tumors. Furthermore, the microsatellite instability (MSI) and the deficient mismatch repair system (dMMR) proteins represent predictive biomarkers for response to immune checkpoint therapy. Until present, few data have been reported related to PD-L1 expression and MSI in PC. The main aim of our study was the evaluation of PD-L1 expression in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) in immune cells and the analysis of dMMR/MSI status in a large series of PCs.MethodsA series of 72 PC, including 65 usual squamous cell carcinoma (USCC), 1 verrucous, 4 basaloid, 1 warty, and 1 mixed (warty-basaloid), was collected. Immunohistochemistry (IHC) was performed to assess PD-L1 expression using two different anti-PD-L1 antibodies (clone SP263 and SP142 Ventana) and MMR proteins expression using anti-MLH1, anti-PMS2, anti-MSH2, and anti-MSH6 antibodies. PCR analysis was performed for the detection of MSI status.ResultsOf the 72 PC cases analyzed by IHC, 45 (62.5%) cases were TC positive and 57 (79%) cases were combined positive score (CPS) using PDL1 SP263. In our cohort, TILs were present in 62 out of 72 cases (86.1%), 47 (75.8%) out of 62 cases showed positivity to PDL1 clone SP142. In our series, 59 cases (82%) had pMMR, 12 cases (16.7%) had lo-paMMR, and only 1 case (1.3%) had MMR. PCR results showed that only one case lo-paMMR was MSI-H, and the case dMMR by IHC not confirmed MSI status.ConclusionOur findings showed that PD-L1 expression and MSI status represent frequent biological events in this tumor suggesting a rationale for a new frontier in the treatment of patients with PC based on the immune checkpoint inhibitors.

(1) Background: Fine-needle aspiration cytology is often used for the pre-operative diagnosis of melanoma metastases. The diagnosis may not be confidently established based on morphology alone, and immunocytochemistry is mandatory. The choice of the most advantageous immunocytochemical antibodies is critical, as the sample may be scant, and the presence of pigmented histiocytes may be confounding. However, the diagnostic performance of melanocytic markers in this setting is poorly investigated. Moreover, PRAME (preferentially expressed antigen in melanoma) recently emerged as a novel marker for the diagnosis of melanoma. The current work aimed to evaluate the sensitivity and specificity of PRAME for the diagnosis of melanoma metastases in cytological samples, compared to other melanocytic markers. (2) Methods: PRAME, S100, Melan-A, HMB45 and SOX10 were tested on cell block sections of 48 cases of melanoma metastases diagnosed from cytological samples, and 20 cases of reactive lymphadenopathy. (3) Results: S100 and SOX10 showed the highest sensitivity (100%), while the sensitivity of PRAME was 85.4%. PRAME, Melan-A, SOX10 and HMB45 showed a specificity of 100%, while the specificity of S100 was lower (85%), as it marked some histiocytes. (4) Conclusion: PRAME immunocytochemistry is highly specific for the diagnosis of melanoma metastasis from a cytological sample, but is less sensitive compared with other melanocytic markers.

We aimed to overcome intratumoral heterogeneity in clear cell renal cell carcinoma (clearRCC). One hundred cases of clearRCC were sampled. First, usual standard sampling was applied (1 block/cm of tumor); second, the whole tumor was sampled, and 0.6 mm cores were taken from each block to construct a tissue microarray; third, the residual tissue, mapped by taking pieces 0.5 × 0.5 cm, reconstructed the entire tumor mass. Precisely, six randomly derived pieces of tissues were placed in each cassette, with the number of cassettes being based on the diameter of the tumor (called multisite 3D fusion). Angiogenic and immune markers were tested. Routine 5231 tissue blocks were obtained. Multisite 3D fusion sections showed pattern A, homogeneous high vascular density (10%), pattern B, homogeneous low vascular density (8%) and pattern C, heterogeneous angiogenic signatures (82%). PD-L1 expression was seen as diffuse (7%), low (33%) and absent (60%). Tumor-infiltrating CD8 scored high in 25% (pattern hot), low in 65% (pattern weak) and zero in 10% of cases (pattern desert). Grading was upgraded in 26% of cases (G3–G4), necrosis and sarcomatoid/rhabdoid characters were observed in, respectively, 11 and 7% of cases after 3D fusion (p = 0.03). CD8 and PD-L1 immune expressions were higher in the undifferentiated G4/rhabdoid/sarcomatoid clearRCC subtypes (p = 0.03). Again, 22% of cases were set to intermediate to high risk of clinical recurrence due to new morphological findings of all aggressive G4, sarcomatoid/rhabdoid features by using 3D fusion compared to standard methods (p = 0.04). In conclusion, we propose an easy-to-apply multisite 3D fusion sampling that negates bias due to tumor heterogeneity.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology. Supplementary Information The online version contains supplementary material available at 10.1007/s00428-021-03262-8.

Background. Triple negative breast cancer (TNBC) is a heterogeneous group of tumors with early relapse, poor overall survival, and lack of effective treatments. Hence, new prognostic biomarkers and therapeutic targets are needed. Methods. The expression profile of all twenty-five human selenoproteins was analyzed in TNBC by a systematic approach.In silicoanalysis was performed on publicly available mRNA expression datasets (Cancer Cell Line Encyclopedia, CCLE and Library of Integrated Network-based Cellular Signatures, LINCS). Reverse transcription quantitative PCR analysis evaluated selenoprotein mRNA expression in TNBC versus non-TNBC and normal breast cells, and in TNBC tissues versus normal counterparts. Immunohistochemistry was employed to study selenoproteins in TNBC tissues. STRING and Cytoscape tools were used for functional and network analysis. Results.GPX1, GPX4, SELENOS, TXNRD1 and TXNRD3 were specifically overexpressed in TNBC cells, tissues and CCLE/LINCS datasets. Network analysis demonstrated that SELENOS-binding valosin-containing protein (VCP/p97) played a critical hub role in the TNBCselenoproteins sub-network, being directly associated with SELENOS expression. The combined overexpression of SELENOS and VCP/p97 correlated with advanced stages and poor prognosis in TNBC tissues and the TCGA dataset. Conclusion. Combined evaluation of SELENOS and VCP/p97 might represent a novel potential prognostic signature and a therapeutic target to be exploited in TNBC.

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.

Rationale About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. Objective To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. Methods and results We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. Conclusions The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

The diagnosis of cutaneous melanoma and melanocytic neoplasms in general is one of the most challenging fields in pathology, and the reported interobserver diagnostic agreement in the evaluation of melanocytic lesions is poor. Nevertheless, a correct histopathological diagnosis is crucial to ensure a good clinical management of the patients. The institution of multidisciplinary teams has recently modified the approach to the patients with cutaneous melanoma. Patients referred to a multidisciplinary melanoma unit after receiving a diagnosis of melanoma elsewhere are encouraged to have their histopathological diagnosis confirmed by a second opinion from the experienced pathologist of the team before any treatment is initiated. We performed a retrospective analysis on a series of 121 histopathological revisions required for melanocytic neoplasms in the context of a multidisciplinary team, in order to evaluate the effects of second diagnostic opinion (SDO) on the clinical management of the patients. We defined three types of diagnostic discrepancies between the first diagnosis and the second opinion, according to the greatness of their clinical impact. Overall, the incidence of diagnostic discrepancies of any type was quite high in our series (56%). Interestingly, the SDO determined relevant changes in the clinical management of the patients in 33 out of 121 (27.3%) cases. This study confirms that SDO by expert pathologists significantly affects the course of treatment of melanoma patients and helps improving the diagnostic accuracy and clinical outcome.

RNA is considered as an indicator of the dynamic genetic expression changes in a cell. RNAScope is a commercially available in situ hybridization assay for the detection of RNA in formalin-fixed paraffin-embedded tissue. In this work, we describe the use of RNAScope as a sensitive and specific method for the evaluation of c-KIT messenger RNA (mRNA) in canine mast cell tumor. We investigated the expression of c-KIT mRNA with RNAscope in 60 canine mast cell tumors (MCTs), comparing it with the histological grade and KIT immunohistochemical expression patterns. Our results showed an overall good expression of c-KIT mRNA in neoplastic cells if compared with control probes. We also observed a statistically significant correlation between histological grade and c-KIT mRNA expression. No correlations were found between KIT protein immunohistochemical distribution pattern and c-KIT mRNA expression or histological grade. Our results provide a reference basis to better understand c-KIT mRNA expression in canine MCTs and strongly encourage further studies that may provide useful information about its potential and significant role as a prognostic and predictive biological marker for canine MCTs clinical outcome.

Background: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. Methods: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. Results: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. Conclusion: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods. This work resulted from projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), “GenomePT” (POCI-01-0145-FEDER-022184), “Advancing cancer research: from knowledge to application” (NORTE-01-0145-FEDER-000029) supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Norte Portugal Regional Programme (NORTE 2020), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT - Fundação para a Ciência e a Tecnologia (PTDC/DTP-PIC/2500/2014). This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to N. Normanno (Grant number: IG17135).

Background: Purpose of this study was to evaluate the contribution of the Extracellular-regulated protein kinase (ERK)-1/2 pathway to oncogenic signaling elicited by the tyrosine kinase receptor HER2 in Non-Small Cell Lung Cancer (NSCLC) and to assess the prognostic value of these oncoproteins in NSCLC patients. Methods: Immunohistochemistry was performed to determine expression and activation of HER2 and ERK1/2 (detected by phosphorylation of Y1248 and T202/Y204, respectively) using Tissue Micro Arrays (TMA) containing matched normal and neoplastic tissues from 132 NSCLC patients. Survival analysis was carried out using the Kaplan-Meier method. Univariate and multivariate analysis were used to evaluate the prognostic value of pERK1/2, pHER2 and a combination thereof with clinical-pathological parameters such as age, lymph node status (N), size (T), stage (TNM) and grade. Results: We found that HER2 was overexpressed in 33/120 (27%) and activated in 41/114 (36%) cases; ERK1/2 was activated in 44/102 (43%) cases. A direct association was found between pERK1/2 and pHER2 (23/41; p=0.038). In addition, patients positive for pERK1/2 and for both pHER2 and pERK1/2 showed significantly worse overall survival (OS) and disease-free survival (DFS) compared with negative patients. Univariate and multivariate analysis of patients' survival revealed that positivity for pHER2-pERK1/2 and for pERK1/2 alone were independent prognostic factors of poor survival in NSCLC patients. In particular, this association was significantly important for DFS in stage I+II patients. Conclusion: This study provides evidence that activated ERK1/2 and/or the combined activation of HER2 and ERK1/2 are good indicators of poor prognosis in NSCLC patients, not only in unselected patients but also in early stage disease.