Consensus clustering methodology to improve molecular stratification of non-small cell lung cancer.

Recent advances in machine learning research, combined with the reduced sequencing costs enabled by modern next-generation sequencing, paved the way to the implementation of precision medicine through routine multi-omics molecular profiling of tumours. Thus, there is an emerging need of reliable models exploiting such data to retrieve clinically useful information. Here, we introduce an original consensus clustering approach, overcoming the intrinsic instability of common clustering methods based on molecular data. This approach is applied to the case of non-small cell lung cancer (NSCLC), integrating data of an ongoing clinical study (PROMOLE) with those made available by The Cancer Genome Atlas, to define a molecular-based stratification of the patients beyond, but still preserving, histological subtyping. The resulting subgroups are biologically characterized by well-defined mutational and gene-expression profiles and are significantly related to disease-free survival (DFS). Interestingly, it was observed that (1) cluster B, characterized by a short DFS, is enriched in KEAP1 and SKP2 mutations, that makes it an ideal candidate for further studies with inhibitors, and (2) over- and under-representation of inflammation and immune systems pathways in squamous-cell carcinomas subgroups could be potentially exploited to stratify patients treated with immunotherapy.

Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy.

Podocyte injury leading to albuminuria is a characteristic feature of diabetic nephropathy (DN). Hyperglycemia and advanced glycation end products (AGEs) are major determinants of DN. However, the underlying mechanisms of podocyte injury remain poorly understood. The cytosolic protein TNFAIP2/M-Sec is required for tunneling nanotubes (TNTs) formation, which are membrane channels that transiently connect cells, allowing organelle transfer. Podocytes express TNFAIP2 and form TNTs, but the potential relevance of the TNFAIP2-TNT system in DN is unknown. We studied TNFAIP2 expression in both human and experimental DN and the renal effect of tnfaip2 deletion in streptozotocin-induced DN. Moreover, we explored the role of the TNFAIP2-TNT system in podocytes exposed to diabetes-related insults. TNFAIP2 was overexpressed by podocytes in both human and experimental DN and exposre of podocytes to high glucose and AGEs induced the TNFAIP2-TNT system. In diabetic mice, tnfaip2 deletion exacerbated albuminuria, renal function loss, podocyte injury, and mesangial expansion. Moreover, blockade of the autophagic flux due to lysosomal dysfunction was observed in diabetes-injured podocytes both in vitro and in vivo and exacerbated by tnfaip2 deletion. TNTs allowed autophagosome and lysosome exchange between podocytes, thereby ameliorating AGE-induced lysosomal dysfunction and apoptosis. This protective effect was abolished by tnfaip2 deletion, TNT inhibition, and donor cell lysosome damage. By contrast, Tnfaip2 overexpression enhanced TNT-mediated transfer and prevented AGE-induced autophagy and lysosome dysfunction and apoptosis. In conclusion, TNFAIP2 plays an important protective role in podocytes in the context of DN by allowing TNT-mediated autophagosome and lysosome exchange and may represent a novel druggable target.Abbreviations: AGEs: advanced glycation end products; AKT1: AKT serine/threonine kinase 1; AO: acridine orange; ALs: autolysosomes; APs: autophagosomes; BM: bone marrow; BSA: bovine serum albumin; CTSD: cathepsin D; DIC: differential interference contrast; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; HG: high glucose; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PI3K: phosphoinositide 3-kinase; STZ: streptozotocin; TNF: tumor necrosis factor; TNFAIP2: tumor necrosis factor, alpha-induced protein 2; TNTs: tunneling nanotubes; WT: wild type.

Monoclonal/polyclonal PAX-8, PTH and GATA3 immunohistochemistry in parathyroid lesions.

BACKGROUND: Parathyroid gland (PG) is an endocrine organ which may display different immunohistochemical stainings with chief cells and oxyphilic cells in normal as well as hyperplasic/tumoral lesions. PURPOSE: In this study, we aimed to identify the demographic properties and diagnostic value of the GATA3 antibody, which is a transcription factor in addition to PTH, and of PAX-8 (monoclonal and polyclonal) antibody. METHODS: We have analyzed in detail the cellular components and staining intensities of 46 adenomas all of which contained parathyroid rims, 12 hyperplasia and 5 adjacent non-neoplastic thyroidectomy materials (63 patients, 114 tissues). RESULTS: While no staining was identified in the thyroid tissue, cytoplasmic PTH immunoreactivity was observed in all (100%) normal parathyroid tissues, rim of PGs and hyperplasia, and in 43/46 cases (93.4%) of adenomas. Adenoma and hyperplasia were less stained than normal PG (p < 0.05). We detected GATA3 staining in all cases except for the thyroid (100%). Weak positivity (1+) was most apparent in adenoma cases (p < 0.05). Monoclonal PAX-8 immunoreactivity was not identified in any normal parathyroid tissue and rim of PG but positive immunoreactivity was detected in 83.3% of hyperplasia cases (10/12), 84.8% of adenoma (39/46) and 100% of thyroid tissues (5/5) (p < 0.05). However, polyclonal PAX-8 immunoreactivity was detected in one normal parathyroid tissue (1/5) and seven (7/46) rim of PGs. In cases of hyperplasia and adenoma, positive immunoreactivity was 75% (9/12) and 74% (34/46), respectively. CONCLUSION: In conclusion, we have observed that PTH and GATA3 constitute a much more reliable and sensitive marker for parathyroid and are stained less in adenomas. While monoclonal PAX-8 (MRQ-50) never stains normal parathyroid and rim of PGs, it may help in the differential diagnosis of proliferated parathyroid lesions as a considerably sensitive and relatively specific marker by staining hyperplasic parathyroid, adenomas and the thyroid.

Outcome and diagnostic reproducibility of the thyroid cytology "indeterminate categories" SIAPEC/SIE 2014 in a consecutive series of 302 cases.

PURPOSE: The clinical impact of the SIAPEC/SIE 2014 classification for thyroid cytology has been addressed in few studies that evaluated the malignancy rate and the relative prevalence of each category. No study analyzed its intra-observer and inter-observer reproducibility, so far. METHODS: We retrospectively collected all "indeterminate" lesions diagnosed before (2011-2014) and after (2015-2018) the application of the SIAPEC/SIE 2014 classification at our Institution. Their relative malignancy risks were calculated based on available histological diagnoses. Cytological and clinical features of TIR3A were compared with the surgical outcome. Finally, a large set of samples was re-evaluated in blind of the original cytological and histological diagnoses by two pathologists, independently. RESULTS: The prevalence of "indeterminate" diagnoses increased in years 2015-2018 (302/1482, 21% with 14% of TIR3A and 7% TIR3B categories) compared to years 2011-2014 (261/1680, 16%). Surgery was performed in 27% TIR3A and in 97% TIR3B cases. Malignancy rates were 40% for TIR3B and 17% for TIR3A, but were greatly influenced by the adoption of the WHO 2017 re-classification of encapsulated follicular-patterned lesions (decreasing to 28% and 6%, respectively). No criteria except for tumor size were associated to malignancy in TIR3A category. Intra-observer agreement of the experienced pathologist was 122/141 (86%), whereas inter-observer agreement between the expert and in-training pathologist was 95/141 (67%). CONCLUSIONS: In this real-life experience, the sub-classification of TIR3A and TIR3B slightly increased the overall prevalence of "indeterminate" diagnoses. Malignancy rates were higher than estimated for both TIR3A and TIR3B categories. Agreement among observers highly depended on pathologist's training.

The IGF2 methylation score for adrenocortical cancer: an ENSAT validation study.

Adrenocortical carcinoma (ACC) is diagnosed using the histopathological Weiss score (WS), but remains clinically elusive unless it has metastasized or grows locally invasive. Previously, we proposed the objective IGF2 methylation score as diagnostic tool for ACC. This multicenter European cohort study validates these findings. Patient and tumor characteristics were obtained from adrenocortical tumor patients. DNA was isolated from frozen specimens, where after DMR2, CTCF3, and H19 were pyrosequenced. The predictive value of the methylation score for malignancy, defined by the WS or metastasis development, was assessed using receiver operating characteristic curves and logistic and Cox regression analyses. Seventy-six ACC patients and 118 patients with adrenocortical adenomas were included from seven centers. The methylation score and tumor size were independently associated with the pathological ACC diagnosis (OR 3.756 95% CI 2.224-6.343; OR 1.467 95% CI 1.202-1.792, respectively; Hosmer-Lemeshow test P = 0.903), with an area under the curve (AUC) of 0.957 (95% CI 0.930-0.984). The methylation score alone resulted in an AUC of 0.910 (95% CI 0.866-0.952). Cox regression analysis revealed that the methylation score, WS and tumor size predicted development of metastases in univariate analysis. In multivariate analysis, only the WS predicted development of metastasis (OR 1.682 95% CI 1.285-2.202; P < 0.001). In conclusion, we validated the high diagnostic accuracy of the IGF2 methylation score for diagnosing ACC in a multicenter European cohort study. Considering the known limitations of the WS, the objective IGF2 methylation score could potentially provide extra guidance on decisions on postoperative strategies in adrenocortical tumor patients.

Prognostic Impact of MCPyV and TIL Subtyping in Merkel Cell Carcinoma: Evidence from a Large European Cohort of 95 Patients.

Merkel cell carcinoma is a rare (∼ 2000 cases/year in the USA) but aggressive neuroendocrine neoplasm of the skin. In 2008, the Merkel cell polyomavirus (MCPyV) was found to be clonally integrated in approximately 80% of Merkel cell carcinomas. The remaining 20% have large numbers of UV-associated mutations. Importantly, both the UV-induced neoantigens in virus-negative Merkel cell carcinoma and the Merkel cell polyomavirus oncogenes that are required for virus-positive tumor growth are highly immunogenic. Indeed, antigen-specific T cells detected in patients are frequently "dysfunctional/exhausted," and the inhibitory ligand PD-L1 is often expressed by Merkel cell carcinoma cells. These data led to point our attention on the quantity and the quality of the immune response in Merkel cell carcinoma. Here, we found CD8+ lymphocytes are the only singly evaluated lymphocyte subclass that strongly influenced overall survival and disease-specific survival in Merkel cell carcinoma. In addition, we highlighted as Merkel cell polyomavirus is a strong prognostic factor and as it prompts a host immune response involving various lymphocyte subclasses (CD3, CD8, FoxP3, and PD-L1 positive) in MCC. For this reason, we proposed a novel eye-based "immunoscore" model, obtained by tumor infiltrating lymphocytes subtyping (CD3, CD8, FoxP3, and PD-L1) that could provide additional prognostic information in Merkel cell carcinoma.

Validation of the diagnosis of mesothelioma and BAP1 protein expression in a cohort of asbestos textile workers from Northern Italy.

Background: Diagnosis of mesothelioma based on death certificate is subject to misclassification, which may bias the results of epidemiology studies. A high proportion of mesothelioma harbor mutations in the BRCA1-associated protein 1 (BAP1) gene. Methods: We searched medical and pathology records and specimens for 127 workers from a textile-asbestos factory in Italy who died during 1963-2013 with a diagnosis of pleural or peritoneal neoplasm or mesothelioma on death certificate, to confirm the diagnosis with immunohistochemistry markers. We calculated the odds ratio of confirmation by selected characteristics and asbestos exposure variables. When sufficient pathology material was available, we analyzed BAP1 protein expression. Results: The diagnosis of mesothelioma was histologically confirmed for 35 cases (27.6%); 5 cases were classified as non-mesothelioma (3.9%), for 33 cases a mention of mesothelioma was found on record but no sufficient material was available for revision (26.0%); no records were available for 54 cases (death-certificate-only 42.5%). Diagnostic confirmation was not associated with sex, location of the neoplasm, age, or duration of employment; however, there was a significant association with time since first employment (P for linear trend 0.04). An association between duration of employment and time since first employment was observed for confirmed cases but not for death-certificate-only cases. BAP1 protein was lost in 18/35 cases (51.4%), without an association with sex, location, age, indices of asbestos exposure, or survival. Conclusions: We were able to confirm by immunohistochemistry a small proportion of mesothelioma diagnoses on certificates of deceased asbestos workers, and confirmation correlated with latency of asbestos exposure but not other characteristics. BAP1 protein loss is a frequent event in mesothelioma of asbestos-exposed workers, but does not correlate with exposure.

The Third Italian Consensus Conference for Malignant Pleural Mesothelioma: State of the art and recommendations.

Malignant Pleural Mesothelioma (MPM) remains a relevant public health issue, and asbestos exposure is the most relevant risk factor. The incidence has considerably and constantly increased over the past two decades in the industrialized countries and is expected to peak in 2020-2025. In Italy, a standardized-rate incidence in 2011 among men was 3.5 and 1.25 per 100,000 in men and women, respectively, and wide differences are noted among different geographic areas. The disease remains challenging in terms of diagnosis, staging and treatment and an optimal strategy has not yet been clearly defined. The Third Italian Multidisciplinary Consensus Conference on Malignant Pleural Mesothelioma was held in Bari (Italy) in January 30-31, 2015. This Consensus has provided updated recommendations on the MPM management for health institutions, clinicians and patients.

CDKN2A and BAP1 germline mutations predispose to melanoma and mesothelioma.

BAP1 germline mutations predispose to a cancer predisposition syndrome that includes mesothelioma, cutaneous melanoma, uveal melanoma and other cancers. This co-occurrence suggests that these tumors share a common carcinogenic pathway. To evaluate this hypothesis, we studied 40 Italian families with mesothelioma and/or melanoma. The probands were sequenced for BAP1 and for the most common melanoma predisposition genes (i.e. CDKN2A, CDK4, TERT, MITF and POT1) to investigate if these genes may also confer susceptibility to mesothelioma. In two out of six families with both mesothelioma and melanoma we identified either a germline nonsense mutation (c.1153C > T, p.Arg385*) in BAP1 or a recurrent pathogenic germline mutation (c.301G > T, p.Gly101Trp) in CDKN2A. Our study suggests that CDKN2A, in addition to BAP1, could be involved in the melanoma and mesothelioma susceptibility, leading to the rare familial cancer syndromes. It also suggests that these tumors share key steps that drive carcinogenesis and that other genes may be involved in inherited predisposition to malignant mesothelioma and melanoma.

Does nephrectomy during radical adrenalectomy for stage II adrenocortical cancer affect patient outcome?

PURPOSE: To evaluate if including nephrectomy in the standard surgical approach to stage II adrenocortical cancer (i.e., adrenalectomy) might modify oncologic outcome of patients. METHODS: We performed a retrospective analysis involving 41 patients with stage II adrenocortical cancer (ACC) who had undergone radical surgery. Patients were divided into two groups according to the surgical procedure: group A = radical adrenalectomy alone, group AN = radical adrenalectomy + radical nephrectomy. Oncologic effectiveness of the procedures was tested comparing the recurrence-free and overall survival of patients of A vs AN groups. RESULTS: The group A consisted of 25 patients and group AN of 16 patients. No differences were noted between the two groups in terms of demographic data and ACC characteristics. During follow-up, 15/25 (60 %) patients of group A vs 14/16 (87.5 %) patients of group AN experienced a recurrence, after a median of 36 months in group A and 10 months in group AN (p = 0.06); a significant impairment of renal function was recorded in patients of AN group with respect to those of group A. Finally, 13/25 (52 %) patients of group A and 10/16 (62.5 %) patients of group AN died due to ACC-related causes. No differences in survival times were noted (p = 0.3). CONCLUSION: Our study suggests that adjunctive nephrectomy does not modify the oncologic results of adrenalectomy in the treatment of stage II ACC in terms of recurrence-free and overall survival. Thus, when there are no signs of ACC local invasion, surgeon should make every effort to preserve the kidney.

Molecular and Histological Changes in Post-Treatment Biopsies of Non-Squamous Non-Small Cell Lung Cancer: A Retrospective Study.

BACKGROUND: Recently, in advanced non-small cell lung cancer (NSCLC), standard chemotherapy was flanked by biological agents directed against genomic abnormalities, including EGFR and ALK alterations, that significantly improved patient outcome. Despite these achievements, tumour progression almost always occurs and a reassessment of the tumour genetic profile may contribute to modulating the therapeutic regimen. Resampling may provide tissue for additional tests to detect acquired resistance and/or new genetic alterations, but the currently available information is limited. PATIENTS AND METHODS: Histological and genetic reassessments of biopsy or surgical tissue samples from 50 non-squamous NSCLC patients before and after at least one systemic treatment were performed. EGFR, KRAS, BRAF, PIK3CA and HER2 mutations were sequenced, p.T790M was identified with real-time PCR, and ALK and MET genomic alterations by fluorescence in situ hybridization. RESULTS: Overall in baseline biopsies, 37/50 (74 %) tumours had genetic alterations, either single (52 %) or multiple (22 %). Among them, 16 were EGFR mutations and 6 ALK rearrangements. In the second tissue sampling, 54 % of cases had additional genomic changes, including newly acquired alterations (81 %) or losses (18 %). The commonest changes were MET amplification and p.T790M mutation. One case had a histological shift from adenocarcinoma to small cell carcinoma. CONCLUSIONS: The remarkable number of molecular changes following systemic therapy and the genetic complexity of some cases underline the value of histological and molecular re-evaluation of lung cancer to tailor the most appropriate therapy during disease progression.

Survival prognostic factors of gastro-enteric-pancreatic neuroendocrine tumors after primary tumor resection in a single tertiary center: Comparison of gastro-enteric and pancreatic locations.

AIM: This study aimed to evaluated prognostic factors of patients with GEP-NETs after primary tumor resection comparing pancreatic and gastro-enteric locations. METHODS: Patients undergone surgery for primary GEP-NETs between 01/2000 and 03/2012 were considered. All specimens were reclassified according to the WHO 2010 scheme. RESULTS: A total of 83 patients were considered: 37 pancreatic NETs (pNET) and 46 gastroenteric NETs (GE-NET). The two groups were similar in terms of age, sex and tumors size. A higher rate of patients with pNETs had Ki67 score ≥3 (64.8% vs. 39%, p = 0.027) while the rates of Mitotic Index ≥2x10HPF (62% pNET vs. 50% GE-NET, p = 0.374) and diagnosis of neuroendocrine carcinoma NEC (16.2% pNET vs. 17.3% GE-NET, p = 0.100) were similar. The rates of distant metastases (GE-NETs 30.4% vs. p-NETs 29.7%, p = 0.944) and liver metastases (19.5% GE-NET vs. 27% pNET, p = 0.421) were comparable. Radical resection was achieved in a similar proportion in both groups [33 patients (89.1%) pNET vs. 36 (78.2%) GE-NET, p = 0.393]. After a median follow-up of 47.1 months overall 3, 5 and 10-years survival rates of whole patients were 88.1%, 81.2% and 76.7%. There was not difference on 5-years overall survival between pNET (81.4%) and GE-NET (81%, p = 0.901). At multivariate analysis age ≥70 [OR 4.177 (CI 95% 1.26-13.8), p = 0.019] and NEC [OR 5.932 (CI 95% 1.81-19.40), p < 0.001] were negative prognostic factors of survival. CONCLUSION: Overall survival of GEP-NET after resection of primary tumors seems to be correlated to patient's age and WHO 2010 staging system but not to primary tumor site.

Large cell carcinoma of the lung: clinically oriented classification integrating immunohistochemistry and molecular biology.

This study aimed at challenging pulmonary large cell carcinoma (LLC) as tumor entity and defining different subgroups according to immunohistochemical and molecular features. Expression of markers specific for glandular (TTF-1, napsin A, cytokeratin 7), squamous cell (p40, p63, cytokeratins 5/6, desmocollin-3), and neuroendocrine (chromogranin, synaptophysin, CD56) differentiation was studied in 121 LCC across their entire histological spectrum also using direct sequencing for epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and FISH analysis for ALK gene translocation. Survival was not investigated. All 47 large cell neuroendocrine carcinomas demonstrated a true neuroendocrine cell lineage, whereas all 24 basaloid and both 2 lymphoepithelioma-like carcinomas showed squamous cell markers. Eighteen out of 22 clear cell carcinomas had glandular differentiation, with KRAS mutations being present in 39 % of cases, whereas squamous cell differentiation was present in four cases. Eighteen out of 20 large cell carcinomas, not otherwise specified, had glandular differentiation upon immunohistochemistry, with an exon 21 L858R EGFR mutation in one (5 %) tumor, an exon 2 KRAS mutation in eight (40 %) tumors, and an ALK translocation in one (5 %) tumor, whereas two tumors positive for CK7 and CK5/6 and negative for all other markers were considered adenocarcinoma. All six LCC of rhabdoid type expressed TTF-1 and/or CK7, three of which also harbored KRAS mutations. When positive and negative immunohistochemical staining for these markers was combined, three subsets of LCC emerged exhibiting glandular, squamous, and neuroendocrine differentiation. Molecular alterations were restricted to tumors classified as adenocarcinoma. Stratifying LCC into specific categories using immunohistochemistry and molecular analysis may significantly impact on the choice of therapy.

Grading the neuroendocrine tumors of the lung: an evidence-based proposal.

Lung neuroendocrine tumors are catalogued in four categories by the World Health Organization (WHO 2004) classification. Its reproducibility and prognostic efficacy was disputed. The WHO 2010 classification of digestive neuroendocrine neoplasms is based on Ki67 proliferation assessment and proved prognostically effective. This study aims at comparing these two classifications and at defining a prognostic grading system for lung neuroendocrine tumors. The study included 399 patients who underwent surgery and with at least 1 year follow-up between 1989 and 2011. Data on 21 variables were collected, and performance of grading systems and their components was compared by Cox regression and multivariable analyses. All statistical tests were two-sided. At Cox analysis, WHO 2004 stratified patients into three major groups with statistically significant survival difference (typical carcinoid vs atypical carcinoid (AC), P=0.021; AC vs large-cell/small-cell lung neuroendocrine carcinomas, P<0.001). Optimal discrimination in three groups was observed by Ki67% (Ki67% cutoffs: G1 <4, G2 4-<25, G3 ≥25; G1 vs G2, P=0.021; and G2 vs G3, P≤0.001), mitotic count (G1 ≤2, G2 >2-47, G3 >47; G1 vs G2, P≤0.001; and G2 vs G3, P≤0.001), and presence of necrosis (G1 absent, G2 <10% of sample, G3 >10% of sample; G1 vs G2, P≤0.001; and G2 vs G3, P≤0.001) at uni and multivariable analyses. The combination of these three variables resulted in a simple and effective grading system. A three-tiers grading system based on Ki67 index, mitotic count, and necrosis with cutoffs specifically generated for lung neuroendocrine tumors is prognostically effective and accurate.