Cerebellar phenotypes in germline PTEN mutation carriers.

PTEN hamartoma tumour syndrome (PHTS) comprises different hereditary conditions caused by germline PTEN mutations, predisposing to the development of multiple hamartomas in many body tissues and also increasing the risk of some types of cancer. Cerebellar involvement in PHTS patients has been long known due to the development of a pathognomonic cerebellar hamartoma (known as dysplastic gangliocytoma of the cerebellum or Lhermitte-Duclos disease). Recently, a crucial role of the cerebellum has been highlighted in the pathogenesis of autism spectrum disorders, now recognised as a phenotype expressed in a variable percentage of PHTS children. In addition, rare PTEN variants are indeed identified in medulloblastoma as well, even if they are less frequent than other germline gene mutations. The importance of PTEN and its downstream signalling enzymatic pathways, PI3K/AKT/mTOR, has been studied at different levels in both human clinical settings and animal models, not only leading to a better understanding of the pathogenesis of different disorders but, most importantly, to identify potential targets for specific therapies. In particular, PTEN integrity makes an important contribution to the normal development of tissue architecture in the nervous system, including the cerebellum. Thus, in patients with PTEN germline mutations, the cerebellum is an affected organ that is increasingly recognised in different disorders, whereas, in animal models, cerebellar Pten loss causes a variety of functional and histological alterations. In this review, we summarise the range of cerebellar involvement observed in PHTS and its relationships with germline PTEN mutations, along with the phenotypes expressed by murine models with PTEN deficiency in cerebellar tissue.

Prognostic Factors across Poorly Differentiated Neuroendocrine Neoplasms: A Pooled Analysis.

INTRODUCTION: Poorly differentiated neuroendocrine carcinomas (NECs) are characterized by aggressive clinical course and poor prognosis. No reliable prognostic markers have been validated to date; thus, the definition of a specific NEC prognostic algorithm represents a clinical need. This study aimed to analyze a large NEC case series to validate the specific prognostic factors identified in previous studies on gastro-entero-pancreatic and lung NECs and to assess if further prognostic parameters can be isolated. METHODS: A pooled analysis of four NEC retrospective studies was performed to evaluate the prognostic role of Ki-67 cut-off, the overall survival (OS) according to primary cancer site, and further prognostic parameters using multivariable Cox proportional hazards model and machine learning random survival forest (RSF). RESULTS: 422 NECs were analyzed. The most represented tumor site was the colorectum (n = 156, 37%), followed by the lungs (n = 111, 26%), gastroesophageal site (n = 83, 20%; 66 gastric, 79%) and pancreas (n = 42, 10%). The Ki-67 index was the most relevant predictor, followed by morphology (pure or mixed/combined NECs), stage, and site. The predicted RSF response for survival at 1, 2, or 3 years showed decreasing survival with increasing Ki-67, pure NEC morphology, stage III-IV, and colorectal NEC disease. Patients with Ki-67 <55% and mixed/combined morphology had better survival than those with pure morphology. Morphology pure or mixed/combined became irrelevant in NEC survival when Ki-67 was ≥55%. The prognosis of metastatic patients who did not receive any treatment tended to be worse compared to that of the treated group. The prognostic impact of Rb1 immunolabeling appears to be limited when multiple risk factors are simultaneously assessed. CONCLUSION: The most effective parameters to predict OS for NEC patients could be Ki-67, pure or mixed/combined morphology, stage, and site.

Mental health and the effects on methylation of stress-related genes in front-line versus other health care professionals during the second wave of COVID-19 pandemic: an Italian pilot study.

Healthcare workers experienced high degree of stress during COVID-19. Purpose of the present article is to compare mental health (depressive and Post-Traumatic-Stress-Disorders-PTSD-symptoms) and epigenetics aspects (degree of methylation of stress-related genes) in front-line healthcare professionals versus healthcare working in non-COVID-19 wards. Sixty-eight healthcare workers were included in the study: 39 were working in COVID-19 wards (cases) and 29 in non-COVID wards (controls). From all participants, demographic and clinical information were collected by an ad-hoc questionnaire. Depressive and PTSD symptoms were evaluated by the Patient Health Questionnaire-9 (PHQ-9) and the Impact of Event Scale-Revised (IES-R), respectively. Methylation analyses of 9 promoter/regulatory regions of genes known to be implicated in depression/PTSD (ADCYAP1, BDNF, CRHR1, DRD2, IGF2, LSD1/KDM1A, NR3C1, OXTR, SLC6A4) were performed on DNA from blood samples by the MassARRAY EpiTYPER platform, with MassCleave settings. Controls showed more frequent lifetime history of anxiety/depression with respect to cases (χ2 = 5.72, p = 0.03). On the contrary, cases versus controls presented higher PHQ-9 (t = 2.13, p = 0.04), PHQ-9 sleep item (t = 2.26, p = 0.03), IES-R total (t = 2.17, p = 0.03), IES-R intrusion (t = 2.46, p = 0.02), IES-R avoidance (t = 1.99, p = 0.05) mean total scores. Methylation levels at CRHR1, DRD2 and LSD1 genes was significantly higher in cases with respect to controls (p < 0.01, p = 0.03 and p = 0.03, respectively). Frontline health professionals experienced more negative effects on mental health during COVID-19 pandemic than non-frontline healthcare workers. Methylation levels were increased in genes regulating HPA axis (CRHR1) and dopamine neurotransmission (DRD2 and LSD1), thus supporting the involvement of these biological processes in depression/PTSD and indicating that methylation of these genes can be modulated by stress conditions, such as working as healthcare front-line during COVID-19 pandemic.

Predicting tumor consistency and extent of resection in non-functioning pituitary tumors.

PURPOSE: To (1) identify a radiological parameter to predict non-functioning pituitary tumor (NFPT) consistency, (2) examine the relationship between NFPT consistency and extent of resection (EOR), (3) investigate if tumor consistency predictors can anticipate EOR. METHODS: The ratio (T2SIR) between the T2 min signal intensity (SI) of the tumor and the T2 mean SI of the CSF was the main radiological parameter, being determined through a radiomic-voxel analysis and calculated using the following formula: T2SIR = [(T2 tumor mean SI - SD)/T2 CSF SI]. The tumor consistency was pathologically estimated as collagen percentage (CP). EOR of NFPTs was evaluated by exploiting a volumetric technique and its relationship with the following explanatory variables was explored: CP, Knosp-grade, tumor volume, inter-carotid distance, sphenoidal sinus morphology, Hardy-grade, suprasellar tumor extension. RESULTS: A statistically significant inverse correlation between T2SIR and CP was demonstrated (p = 0.0001), with high diagnostic power of T2SIR in predicting NFPT consistency (ROC curve analysis' AUC = 0.88; p = 0.0001). The following predictors of EOR were identified in the univariate analysis: CP (p = 0.007), preoperative volume (p = 0.045), Knosp grade (p = 0.0001), tumor suprasellar extension (p = 0.044). The multivariate analysis demonstrated two variables as unique predictors of EOR: CP (p = 0.002) and Knosp grade (p = 0.001). The T2SIR was a significant predictor of EOR both in the univariate (p = 0.01) and multivariate model (p = 0.003). CONCLUSION: This study offers the potential to improve NFPT preoperative surgical planning and patient counseling by employing the T2SIR as a preoperative predictor of tumor consistency and EOR. Meanwhile, tumor consistency and Knosp grade were found to play an important role in predicting EOR.

High Filamin a Expression in Adrenocortical Carcinomas Is Associated with a Favourable Tumour Behaviour: A European Multicentric Study.

The insulin-like growth factor 2 (IGF2) promotes cell growth by overactivating the IGF system in an autocrine loop in adrenocortical carcinomas (ACCs). The cytoskeleton protein filamin A (FLNA) acts as a repressor of IGF2 mitogenic signalling in ACC cells. The aims of this study were to test FLNA expression by immunohistochemistry in 119 ACCs and 26 adrenocortical adenomas (ACAs) and to evaluate its relationship with clinicopathological features and outcome in ACCs. We found that 71.4% of ACCs did not express FLNA, whereas FLNA absence was a rare event in ACAs (15.4%, p < 0.001 vs. ACCs). In addition, the expression of FLNA was associated with a less aggressive tumour behaviour in ACCs. Indeed, the subgroup of ACCs with high FLNA showed a lower ENSAT stage, Weiss score, and S-GRAS score compared to ACCs with low FLNA expression (p < 0.05). Moreover, patients with high FLNA had a longer overall survival than those with low FLNA (p < 0.05). In conclusion, our data suggest that FLNA may represent a "protective" factor in ACCs, and the integration of FLNA immunohistochemical expression in ACC tissues along with other clinical and molecular markers could be helpful to improve diagnostic accuracy and prognosis prediction in ACCs.

Mitochondrial DNA content: a new potential biomarker for Sudden Infant Death Syndrome.

BACKGROUND: Sudden Infant Death Syndrome (SIDS) occurs in apparently healthy infants and is unpredictable and unexplained despite thorough investigations and enormous research efforts. The hypothesis tested in this case-control study concerns mitochondrial involvement in SIDS occurrence. METHODS: Mitochondrial DNA content (MtDNAcn) was measured in 24 SIDS cerebral cortex samples and 18 controls using real-time PCR. RESULTS: The median (interquartile range) mtDNAcn in SIDS and controls was 2578 (2224-3838) and 1452 (724-2517) copies per nuclear DNA, respectively (P = 0.0001). MtDNAcn values were higher in SIDS victims born to non-smoking parents (n = 7) 4984 (2832-6908) compared to the controls (n = 5) 2020 (478-2386) (P = 0.006). Increased levels of mtDNAcn have been observed in the SIDS cases with mild defects in nuclei not essential for life compared to those found in SIDS cases with severe alterations of respiratory function (P = 0.034) 3571 (2568-5053) (n = 14) 2356 (1909-3132) (n = 8), respectively. CONCLUSIONS: Our study revealed for the first time higher mtDNAcn in the cerebral cortex of the SIDS cases than the controls, indicating metabolic alterations. MtDNAcn plays an important role in compensatory mechanisms against environmental factors affecting human health. Despite the small sample size, mtDNA may prove to be a potential forensic biomarker for autopsied SIDS victims for gaining new insights into the etiology of SIDS. IMPACT: Mitochondrial DNA content evaluated in cerebral cortex samples is higher in SIDS victims than controls. These results represent a novel line of investigation for the etiology of SIDS and could have a significant role in the compensatory mechanism due to environmental factors affecting human health. These findings suggest that the mitochondria are involved in SIDS: mtDNA content may represent a biomarker of this syndrome.

Histological evaluation of ischemic alterations in donors after cardiac death: A useful tool to predict post-transplant renal function.

Kidneys retrieved from donors after cardiac death (DCD) pose significant challenges from a clinical and technical point of view, undergoing a variable degree of ischemia-reperfusion injury. At present, the utilization of kidneys is assessed according to the Karpinski score, which does not take into account the ischemic insult and does not predict the functional recovery of the organ once transplanted. Therefore, the correlation between biopsy results and post-transplant graft function is still debated. In this study we examined kidney biopsies from DCD donors; we calculated the Karpinski score and subsequently identified and quantified the ischemic lesions in the glomerular, interstitial, and tubular compartments. These same lesions were quantified in kidney biopsies from donors after brain death (DBD) in a case-control analysis. The collected data were correlated with the clinical data of the donors and the post-transplant follow-up. Proximal tubule alterations are crucial in ischemia-reperfusion damage, showing precise histological alterations, which are more frequent in DCD than in DBD donors and are statistically correlated with functional recovery of the organ. Quantification of ischemic tubular lesions in biopsies of kidneys from DCD donors is a useful tool for predicting post-transplant renal function and a valid parameter for assessing the quality of the graft.

Addition of 5% CO2 to Inspiratory Gas Prevents Lung Injury in an Experimental Model of Pulmonary Artery Ligation.

Rationale: Unilateral ligation of the pulmonary artery may induce lung injury through multiple mechanisms, which might be dampened by inhaled CO2. Objectives: This study aims to characterize bilateral lung injury owing to unilateral ligation of the pulmonary artery in healthy swine undergoing controlled mechanical ventilation and its prevention by 5% CO2 inhalation and to investigate relevant pathophysiological mechanisms. Methods: Sixteen healthy pigs were allocated to surgical ligation of the left pulmonary artery (ligation group), seven to surgical ligation of the left pulmonary artery and inhalation of 5% CO2 (ligation + FiCO2 5%), and six to no intervention (no ligation). Then, all animals received mechanical ventilation with Vt 10 ml/kg, positive end-expiratory pressure 5 cm H2O, respiratory rate 25 breaths/min, and FiO2 50% (±FiCO2 5%) for 48 hours or until development of severe lung injury. Measurements and Main Results: Histological, physiological, and quantitative computed tomography scan data were compared between groups to characterize lung injury. Electrical impedance tomography and immunohistochemistry analysis were performed in a subset of animals to explore mechanisms of injury. Animals from the ligation group developed bilateral lung injury as assessed by significantly higher histological score, larger increase in lung weight, poorer oxygenation, and worse respiratory mechanics compared with the ligation + FiCO2 5% group. In the ligation group, the right lung received a larger fraction of Vt and inflammation was more represented, whereas CO2 dampened both processes. Conclusions: Mechanical ventilation induces bilateral lung injury within 48 hours in healthy pigs undergoing left pulmonary artery ligation. Inhalation of 5% CO2 prevents injury, likely through decreased stress to the right lung and antiinflammatory effects.

Prognostic Role of Mismatch Repair Status, Histotype and High-Risk Pathologic Features in Stage II Small Bowel Adenocarcinomas.

BACKGROUND: Small bowel adenocarcinoma is a relatively rare cancer, often diagnosed in an advanced stage. In localized and resectable disease, surgery alone or in combination with adjuvant chemotherapy is the mainstay of treatment. In the recently published National Comprehensive Cancer Network Clinical Practice guidelines, criteria for selecting patients with stage II small bowel adenocarcinoma to receive adjuvant chemotherapy are provided, and they are mainly extrapolated from studies on colorectal cancer. PATIENTS AND METHODS: In the present study, we aimed to verify whether mismatch repair deficiency phenotype, high-risk pathologic features (including T4, positive resection margins and a low number of lymph nodes harvested), as well as tumor histologic subtype, were associated with cancer-specific survival in 66 stage II non-ampullary small bowel adenocarcinoma patients, collected through the Small Bowel Cancer Italian Consortium. A central histopathology review was performed. Mismatch repair deficiency was tested by immunohistochemistry for MLH1, MSH2, MSH6 and PMS2, and confirmed by polymerase chain reaction for microsatellite instability. RESULTS: We identified mismatch repair deficiency, glandular/medullary histologic subtype, and celiac disease as significant predictors of favorable cancer-specific survival using univariable analysis with retained significance in bivariable models adjusted for pT stage. Among the high-risk features, only T4 showed a significant association with an increased risk of death; however, its prognostic value was not independent of mismatch repair status. CONCLUSIONS: Mismatch repair protein expression, histologic subtype, association with celiac disease, and, in the mismatch repair proficient subset only, T stage, may help identify patients who may benefit from adjuvant chemotherapy.

Combined analysis of PTEN, HER2, and hormone receptors status: remodeling breast cancer risk profiling.

BACKGROUND: Phosphatase and tensin homolog (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing is improved by the analysis of PTEN status for the identification of high-risk breast cancer. METHODS: A cohort of 608 patients with breast cancer was included in this study. Based on the expression on the neoplastic cells compared to the normal internal controls by immunohistochemistry (IHC), cases were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT). The former constituted the study group, while the latter the control group. Analysis of gene expression was performed on publicly available genomic data and included 4265 patients from the METABRIC and MSK cohorts retrieved from cBioPortal. The Shapiro-Wilk test was used to analyze the normal distributions of continuous variables. Relationships between PTEN status and the clinicopathologic and molecular features of the patient population were assessed using Fisher's exact test or Chi-squared/Wilcoxon rank-sum test. Survival curves were built according to the Kaplan-Meier method. RESULTS: Alteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from our group, while genetic aberrations in only 315/4265 (7.4%) cases of the METABRIC and MSK cohorts. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n = 48, 17.1%) compared to PTEN-WT tumors (n = 22, 6.7%; p = 0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival (OS) in PTEN-L but not in PTEN-WT breast cancers (p < .0001). Moreover, PTEN-L protein expression but not gene alterations was related to the outcome, in terms of both OS and disease-free survival (p = 0.002). CONCLUSIONS: The combined analysis of PTEN, HER2, and HR status offers relevant information for a more precise risk assessment of patients with breast cancer.

Ki-67 Index of 55% Distinguishes Two Groups of Bronchopulmonary Pure and Composite Large Cell Neuroendocrine Carcinomas with Distinct Prognosis.

BACKGROUND: Little information is available concerning prognostic factors for bronchopulmonary large cell neuroendocrine carcinomas (BP-LCNECs) and even less is known about combined LCNECs (Co-LCNECs). We investigated whether an integrated morphological, immunohistochemical, and molecular approach could be used for their prognostic evaluation. METHODS: Morphological (including combined features), proliferative (mitotic count/Ki-67 index), immunohistochemical (napsin A, p40, TTF-1, CD44, OTP, SSTR2A, SSTR5, mASH1, p53, RB1, and MDM2), and genomic (TP53, RB1, ATM, JAK2, KRAS, and STK11) findings were analyzed in BP-LCNECs from 5 Italian centers, and correlated with overall survival (OS). The Ki-67 index was expressed as the percentage of positive cells in hot spots as indicated in the WHO 2019 Digestive System Tumors and, for Co-LCNECs, the Ki-67 index was evaluated only in the LCNEC component. RESULTS: A total of 111 LCNECs were distinguished into 70 pure LCNECs, 35 Co-LCNECs (27 with adenocarcinoma [ADC] and 8 with squamous cell carcinoma [SqCC]), and 6 LCNECs with only napsin A immunoreactivity. The Ki-67 index cutoff at 55% evaluated in the neuroendocrine component was the most powerful predictor of OS (log-rank p = 0.0001) in all LCNECs; 34 cases had a Ki-67 index <55% (LCNEC-A) and 77 had a Ki-67 index ≥55% (LCNEC-B). Statistically significant differences in OS (log-rank p = 0.0001) were also observed between pure and Co-LCNECs. A significant difference in OS was found between pure LCNECs-A and Co-LCNECs-A (p < 0.05) but not between pure LCNECs-B and Co-LCNECs-B. Co-LCNEC-ADC and LCNEC napsin A+ cases had longer OS than pure LCNEC and Co-LCNEC-SqCC cases (log-rank p = 0.0001). On multivariable analysis, tumor location, pure versus combined features, and napsin A, but no single gene mutation, were significantly associated with OS after adjustment for Ki-67 index and study center (p < 0.05). CONCLUSIONS: The Ki-67 proliferation index and the morphological characterization of combined features in LCNECs seem to be important tools for predicting clinical outcome in BP-LCNECs.

Unusual Mesenchymal Tumors of the Lower Gastrointestinal Tract: When You Hear Hoofbeats in the Night, Do Not Forget the Zebras.

INTRODUCTION: Little information about clinical presentation of mesenchymal tumors of the lower gastrointestinal (GI) tract due to their extreme heterogeneity is available for clinical management. Usually, small solitary asymptomatic polyps are accidently found during a screening colonoscopy performed for hematochezia, abdominal pain, constipation, diarrhea, and bowel obstruction. In this case series, we illustrate our experience with mesenchymal tumors of the lower GI tract, which are a group of unusual and quite challenging lesions. CASE PRESENTATION: We retrospectively collected mesenchymal tumors of the lower GI tract in our institution (Fondazione IRCSS Ca' Granda - Ospedale Maggiore Policlinico di Milano) during the last 10 years. We reviewed the histological slides, and, when necessary, we performed immunohistochemical analyses to better characterize the tumors. A total of 99 cases were identified: 45 GISTs, 42 lipomas, 4 leiomyomas, 3 Kaposi sarcomas, 1 schwannoma, 1 ganglioneuroma, 1 hemangioma, 1 inflammatory fibroid polyp, and 1 challenging case of spindle cell melanoma. We focused on the most rare entities excluding therefore all GISTs and lipomas from re-evaluation. CONCLUSION: Mesenchymal tumors of the lower GI tract represent a highly heterogeneous group of lesions encompassing GISTs, lipomas, smooth muscle tumors (leiomyoma and leiomyosarcoma), GI schwannomas, inflammatory fibroid polyps, solitary fibrous tumors, and other unusual spindle cell tumors. Immunohistochemistry and, in selected cases, molecular biology remain a useful tool which, in addition to a meticulous study of the morphology, helps the pathologist in the tangled jungle of differential diagnosis.

Correction: PD-L1 in small bowel adenocarcinoma is associated with etiology and tumor-infiltrating lymphocytes, in addition to microsatellite instability.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PD-L1 in small bowel adenocarcinoma is associated with etiology and tumor-infiltrating lymphocytes, in addition to microsatellite instability.

Small bowel adenocarcinomas (SBAs) are often associated with poor prognosis and have limited therapeutic options. Programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway blockade is an effective treatment in many microsatellite instability-high (MSI-H) solid tumors. We aimed at investigating PD-L1 and PD-1 expression in non-hereditary, non-ampullary SBAs, associated with celiac disease (CeD), Crohn's disease (CrD), or sporadic, recruited through the Small Bowel Cancer Italian Consortium. We assessed PD-L1 and PD-1 by immunohistochemistry in a series of 121 surgically resected SBAs, including 34 CeD-SBAs, 49 CrD-SBAs, and 38 sporadic SBAs. PD-L1 and PD-1 expression was correlated with several clinico-pathological features, such as the etiology, microsatellite instability status, and tumor-infiltrating lymphocyte (TIL) density. The prevalence of PD-L1 positivity according to combined positive score (CPS) was 26% in the whole cohort of SBAs, with significantly (p = 0.001) higher percentage (35%) in both CeD-SBAs and CrD-SBAs in comparison with sporadic SBAs (5%). CPS ≥ 1 SBAs were significantly (p = 0.013) more frequent in MSI-H cases (41%) than in non-MSI-H ones (18%); however, 15 CPS ≥ 1 microsatellite stable SBAs were also identified. CPS ≥ 1 SBAs showed higher TIL and PD-1+ immune cell density, more frequently medullary histotype, as well as a better outcome in comparison with CPS < 1 cases. This study demonstrates an increased proportion of PD-L1+ cases in both CeD-SBAs and CrD-SBAs in comparison with sporadic SBAs. In addition, the identification of a subset of PD-L1+ microsatellite stable SBAs supports the need to ascertain additional biomarkers of response to immune checkpoint inhibitors along with MSI-H.

Deregulation of miRNAs-cMYC circuits is a key event in refractory celiac disease type-2 lymphomagenesis.

A percentage of celiac disease (CD) patients develop refractory type-2 disease (RCD2), a condition associated with increased risk of enteropathy-associated T-cell-lymphoma (EATL) and without therapeutic option. Therefore, we profiled the miRNome in series of peripheral T-cell lymphomas (PTCLs), CD, RCD1 or 2 and in the murine interleukin-15 (IL15)-transgenic (TG) model of RCD. The transcriptome was analyzed in 18 intestinal T-cell lymphomas (ITLs). Bioinformatics pipelines provided significant microRNA (miRNA) lists and predicted targets that were confirmed in a second set of patients. Our data show that ITLs have a unique miRNA profile with respect to other PTCLs. The c-MYC regulated miR-17/92 cluster distinguishes monomorphic epitheliotropic ITL (MEITL) from EATL and prognosticates EATL outcome. These miRNAs are decreased in IL15-TG mice upon Janus kinase (JAK) inhibition. The random forest algorithm identified a signature of 38 classifier miRNAs, among which, the miR-200 and miR-192/215 families were progressively lost in RCD2 and ITL-CD, whereas miR-17/92 and C19MC miRNAs were up-regulated. Accordingly, SMAD3, MDM2, c-Myc and activated-STAT3 were increased in RCD2 and EATL tissues while JAK inhibition in IL15-TG mice restored their levels to baseline. Our data suggest that miRNAs circuit supports activation of STAT3 and c-Myc oncogenic signaling in RCD2, thus contributing to lymphomagenesis. This novel understanding might pave the way to personalized medicine approaches for RCD and EATL.

Laser capture microdissection on formalin-fixed and paraffin-embedded renal transplanted biopsies: Technical perspectives for clinical practice application.

Renal biopsy (RBx) is an essential tool in the diagnostic and therapeutic process of most native kidney diseases and in the renal transplanted graft. Laser capture microdissection (LCM), combined with molecular biology, might improve the diagnostic power of RBx. However, the limited amount of available renal tissue is often an obstacle for achieving a satisfactory qualitative and quantitative analysis. In our work we present a method which allows us to obtain good quality and quantity of RNA from formalin-fixed and paraffin-embedded (FFPE) renal tissue derived from RBx performed in transplanted patients. Histology, immunohistochemistry, LCM, pre-amplify system and qRT-PCR of biomarkers related to tubular damage, inflammation and fibrosis on FFPE RBx were performed. Glomeruli, tubules and interstitium of three RBx (RB-A: no alteration; RB-B and -C: the progressive rise of creatinine) were compared. The method proposed, could well be useful in future clinical practice. It is quick, easy to perform and allows the analyses of many biomarkers. In addition, it could be extended to all types of RBx without any limitation on the sample amount. Nevertheless, the need for a higher number of well-trained technicians might represent some limitation, counterbalanced by the opportunity to elaborate more accurate diagnosis and, consequently, more targeted therapies.

PTEN Expression as a Complementary Biomarker for Mismatch Repair Testing in Breast Cancer.

Mismatch repair (MMR) analysis in breast cancer may help to inform immunotherapy decisions but it lacks breast-specific guidelines. Unlike in other neoplasms, MMR protein loss shows intra-tumor heterogeneity and it is not mirrored by microsatellite instability in the breast. Additional biomarkers can improve MMR clinical testing. Phosphatase and tensin homolog (PTEN) inactivation is an early oncogenic event that is associated with MMR deficiency (dMMR) in several tumors. Here, we sought to characterize the diagnostic utility of PTEN expression analysis for MMR status assessment in breast cancer. A total of 608 breast cancers were profiled for their MMR and PTEN status. Proteins expression and distribution were analyzed by immunohistochemistry (IHC) on tissue microarrays and confirmed on full sections; PTEN copy number alterations were detected using a real-time PCR assay. Overall, 78 (12.8%) cases were MMR-heterogeneous (hMMR), while all patterns of PTEN expression showed no intra-tumor heterogeneity. Wild-type PTEN expression was observed in 15 (18.5%) dMMR tumors (p < 0.0001). Survival analyses revealed significant correlations between MMR-proficient (pMMR), PTEN expression, and a better outcome. The positive predictive value of PTEN-retained status for pMMR ranged from 94.6% in estrogen receptor (ER)+/HER2- tumors to 100% in HER2-amplified and ER-/HER2- cases. We propose a novel diagnostic algorithm where PTEN expression analysis can be employed to identify pMMR breast cancers.

Unproductive Effects of ALK Gene Amplification and Copy Number Gain in Non-Small-Cell Lung Cancer. ALK Gene Amplification and Copy Gain in NSCLC.

Background: The Anaplastic Lymphoma Kinase (ALK) gene is known to be affected by several genetic alterations, such as rearrangement, amplification and point mutation. The main goal of this study was to comprehensively analyze ALK amplification (ALK-A) and ALK gene copy number gain (ALK-CNG) in a large cohort of non-small-cell lung cancer (NSCLC) patients in order to evaluate the effects on mRNA and protein expression. Methods: ALK locus number status was evaluated in 578 NSCLC cases by fluorescence in situ hybridization (FISH). In addition, ALK immunohistochemistry and ALK mRNA in situ hybridization were performed. Results: Out of 578 cases, 17 cases showed ALK-A. In addition, 14 cases presented ALK-CNG and 72 cases presented chromosome 2 polyploidy. None of those carrying ALK-A and -CNG showed either ALK immunohistochemical expression or ALK mRNA expression through in situ hybridization. We observed a high frequency of extra copies of the ALK gene. Conclusions: Our findings demonstrated that ALK-A is not involved in mRNA production and consequently is not involved in protein production; these findings support the hypothesis that ALK-A might not play a role in the pathogenesis of NSCLC, underlining the absence of a specific clinical application.

Sudden intrauterine unexplained death: time to adopt uniform postmortem investigative guidelines?

BACKGROUND: Worldwide approximately 2.6 million are stillborn, mostly occurring in developing countries. In the great part these deaths are inexplicable. The evenness and standardisation of the diagnostic criteria are prerequisites to understand their pathogenesis. The core goal of this article is to propose new evidence based investigative post-mortem guidelines that should be adopted in all the Institutions especially when a fetal death, after a routine autopsy procedure, is diagnosed as "unexplained". The proposed protocol is mainly focused on the anatomopathological examination of the autonomic nervous system and in particular of the brainstem where the main centers that control vital functions are located. METHODS: Updated investigative guidelines for the examination of unexplained stillbirths, prevalently focused on the histological examination of the brainstem, where the main centers that are involved in monitoring the vital functions are located, are here presented. A section of this protocol concerns the Immunohistochemical evaluation of specific functional markers such as the neuronal nuclear antigen, nicotinic acetylcholine receptors, serotonin, orexin, apoptosis and gliosis. The important role of risk factors, having regard in particular to maternal smoking and air pollution is also contemplated in these guidelines. RESULTS: Specific morphological and/or functional alterations of vital brainstem structures have been found with high incidence in over 100 cases of unexplained fetal death sent to the "Lino Rossi Research Center" of the Milan University according to the Italian law. These alterations were rarely detected in a group of control cases. CONCLUSIONS: We hope this protocol can be adopted in all the Institutions notably for the examination of unexplained fetal deaths, in order to make uniform investigations. This will lead to identify a plausible explanation of the pathogenetic mechanism behind the unexplained fetal deaths and to design preventive strategies to decrease the incidence of these very distressing events for both parents and clinicians. TRIAL REGISTRATION: not applicable for this study.

Neuropathology of Early Sudden Infant Death Syndrome-Hypoplasia of the Pontine Kolliker-Fuse Nucleus: A Possible Marker of Unexpected Collapse during Skin-to-Skin Care.

OBJECTIVE: To find a possible pathogenetic mechanism of the early sudden infant death occurring in newborns during the skin-to-skin care (SSC), through the examination of neuronal centers regulating the vital activities. STUDY DESIGN: This is an in-depth examination of the brain stem in 22 healthy term newborns, suddenly died in the first hour of life without the identification of a cause at autopsy (early sudden infant death syndrome [eSIDS]), 12 of them concomitantly with SSC, and 10 with age-matched controls died of known pathology. RESULTS: Developmental alterations of neuronal structures of the brain stem were highlighted in 19 of the 22 eSIDS, but not in control. The hypoplasia of the pontine Kölliker-Fuse nucleus (KFN), an important respiratory center, was diagnosed at the histological examination, validated by morphometric quantifications, in 11 of the 12 eSIDS while they were placed on the mother's chest and in 2 of the 10 SSC unrelated neonatal deaths. CONCLUSION: The delayed development of the KFN could represent a specific finding of eSIDS occurring during SSC. Therefore, it is necessary to point out that the SSC represents a further risk factor that must be added to others already known for sudden infant death syndrome. Then this practice needs appropriate monitoring strategies of the infant's conditions.