Genomic profile analysis of leiomyomas with bizarre nuclei and fumarate hydratase deficient leiomyomas: Strengths, weaknesses, and limitations of array-CGH interpretation.

A close relationship has been demonstrated between genomic complexity and clinical outcome in uterine smooth muscle tumors. We studied the genomic profiles by array-CGH of 28 fumarate hydratase deficient leiomyomas and 37 leiomyomas with bizarre nuclei (LMBN) from 64 patients. Follow-up was available for 46 patients (from three to 249 months, mean 87.3 months). All patients were alive without evidence of disease. For 51 array-CGH interpretable tumors the mean Genomic Index (GI) was 16.4 (median: 9.8; from 1 to 57.8), significantly lower than the mean GI in LMS (mean GI 51.8, p < 0.001). We described three groups: (1) a group with FH deletion (24/58) with low GI (mean GI: 11 vs. 22,4, p = 0.02), (2) a group with TP53 deletion (17/58) with higher GI (22.4 vs. 11 p = 0.02), and (3) a group without genomic events on FH or TP53 genes (17/58) (mean GI:18.3; from 1 to 57.8). Because none of these tumors recurred and none showed morphological features of LMS we concluded that GI at the cut-off of 10 was not applicable in these subtypes of LM. By integration of all those findings, a GI <10 in LMBN remains a valuable argument for benignity. Conversely, in LMBN a GI >10 or alteration in tumor suppressor genes, should not alone warrant a diagnosis of malignancy. Nine tumors were tested with Nanocind CINSARC® signature and all were classified in low risk of recurrence. We propose, based on our observations, a diagnostic approach of these challenging lesions.

Combined prenatal US and post-mortem fetal MRI: can they replace conventional autopsy for fetal body abnormalities?

OBJECTIVES: The acceptance of conventional autopsy (CA), the gold standard method for investigating fetal death, often remains problematic. Post-mortem magnetic resonance imaging (PMMRI) is increasingly advocated, particularly for neurologic malformations. However, PMMRI performances to diagnose non-neurologic malformations remain unclear. We aim to clarify whether a full body CA remains needed after prenatal ultrasound (US) and PMMRI in assessing non-neurologic fetal malformations. METHODS: In this retrospective IRB-approved study, during a 6-year period, all fetuses who underwent PMMRI, prenatal US, and full body CA were included. Body abnormalities were identified in US, PMMRI, and CA reports. US and PMMRI images were all reviewed. All abnormalities were graded as major (2 points) or minor (1 point). Each technique (US, PMMRI, CA) was given a score by adding all grading points. In each fetus, results were compared for both separate and combined US and PMMRI to CA. Sensitivity and specificity were calculated for detecting major abnormalities. RESULTS: Fifty fetuses were included. The score of CA, US, and PMMRI was respectively 53, 37, and 46. Compared with US-PMMRI, CA added information in 2 cases (4%) with major abnormalities and 7 cases (14%) with minor abnormalities. PMMRI and US were concordant in 36/50 (72%) fetuses. Separate US/PMMRI sensitivities and specificities for detecting major body malformations respectively were 80%/80% and 100%/94%. Combined US-PMMRI had a sensitivity of 90% and a specificity of 94%. Two cardiac malformations (2/6) were only described by CA. CONCLUSIONS: After prenatal US and PMMRI, few additional fetal body malformations are discovered with CA. Nevertheless, fetal heart autopsy remains mandatory. CLINICAL RELEVANCE STATEMENT: A cardiac conventional autopsy complemented by prenatal ultrasound and post-mortem MRI allows to detect all major fetal body abnormalities. With this efficient and much less invasive approach, a higher acceptance rate of fetal autopsy can be expected. KEY POINTS: • Excepting cardiac malformations, most major fetal body malformations can reliably be identified by prenatal US combined with post-mortem MRI. • In the post-mortem diagnosis of fetal body malformations, a conventional autopsy limited to the fetal heart might replace a full body autopsy after a well-conducted prenatal US and post-mortem MRI.

Correction: VEGFR1 and VEGFR2 Involvement in Extracellular Galectin-1- and Galectin-3-Induced Angiogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0067029.].

Borderline phyllodes tumour of the breast with eosinophilic inclusion bodies: Case report and molecular sequencing.

INTRODUCTION AND IMPORTANCE: The presence of eosinophilic inclusion bodies in the breast is very rare and fewer than 20 cases were described in the literature. Herein we report the first case of borderline phyllodes tumour associated with this kind of cells. To the best of our knowledge, this is also the first time that a molecular sequencing is made targeting the stroma cells with inclusion bodies. CASE PRESENTATION: A 33-yr-old woman presented a large mass in the right breast. Imaging techniques by mammogram and ultrasonographic examination were performed. After multidisciplinary approach, a breast conserving surgery has been decided. Microscopic analysis, immunohistochemical stains and molecular tests were performed on the lesion. The proposed diagnosis is borderline phyllodes tumour with eosinophilic inclusion bodies. CLINICAL DISCUSSION: Inclusion bodies are typically found in the infantile digital fibromatosis. Finding them in extradigital fibromatosis is rare. Their signification is still unclear. Some studies suggest a disturbance in the metabolism of proliferating myofibroblasts. CONCLUSION: The presence of inclusion bodies in breast tumour do not seem to have a prognosis impact. It might be interesting to perform others molecular tests on lesions with eosinophilic inclusion bodies to discover potential mutations.

SMAD4 Positive Pancreatic Ductal Adenocarcinomas Are Associated with Better Outcomes in Patients Receiving FOLFIRINOX-Based Neoadjuvant Therapy.

BACKGROUND: SMAD4 is inactivated in 50-55% of pancreatic ductal adenocarcinomas (PDACs). SMAD4 loss of expression has been described as a negative prognostic factor in PDAC associated with an increased rate of metastasis and resistance to therapy. However, the impact of SMAD4 inactivation in patients receiving neoadjuvant therapy (NAT) is not well characterized. The aim of our study was to investigate whether SMAD4 status is a prognostic and predictive factor in patients receiving NAT. METHODS: We retrospectively analyzed 59 patients from a single center who underwent surgical resection for primary PDAC after NAT. SMAD4 nuclear expression was assessed by immunohistochemistry, and its relationship to clinicopathologic variables and survival parameters was evaluated. Interaction testing was performed between SMAD4 status and the type of NAT. RESULTS: 49.15% of patients presented loss of SMAD4. SMAD4 loss was associated with a higher positive lymph node ratio (p = 0.03), shorter progression-free survival (PFS) (p = 0.02), and metastasis-free survival (MFS) (p = 0.02), but it was not an independent prognostic biomarker in multivariate analysis. Interaction tests demonstrated that patients with SMAD4-positive tumors receiving FOLFIRINOX-based NAT showed the best outcome. CONCLUSION: This study highlights the potential prognostic and predictive role of SMAD4 status in PDAC patients receiving FOLFIRINOX-based NAT.

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy.

SARS-Cov-2 infection and neuropathological findings: a report of 18 cases and review of the literature.

INTRODUCTION: COVID-19-infected patients harbour neurological symptoms such as stroke and anosmia, leading to the hypothesis that there is direct invasion of the central nervous system (CNS) by SARS-CoV-2. Several studies have reported the neuropathological examination of brain samples from patients who died from COVID-19. However, there is still sparse evidence of virus replication in the human brain, suggesting that neurologic symptoms could be related to mechanisms other than CNS infection by the virus. Our objective was to provide an extensive review of the literature on the neuropathological findings of postmortem brain samples from patients who died from COVID-19 and to report our own experience with 18 postmortem brain samples. MATERIAL AND METHODS: We used microscopic examination, immunohistochemistry (using two different antibodies) and PCR-based techniques to describe the neuropathological findings and the presence of SARS-CoV-2 virus in postmortem brain samples. For comparison, similar techniques (IHC and PCR) were applied to the lung tissue samples for each patient from our cohort. The systematic literature review was conducted from the beginning of the pandemic in 2019 until June 1st, 2022. RESULTS: In our cohort, the most common neuropathological findings were perivascular haemosiderin-laden macrophages and hypoxic-ischaemic changes in neurons, which were found in all cases (n = 18). Only one brain tissue sample harboured SARS-CoV-2 viral spike and nucleocapsid protein expression, while all brain cases harboured SARS-CoV-2 RNA positivity by PCR. A colocalization immunohistochemistry study revealed that SARS-CoV-2 antigens could be located in brain perivascular macrophages. The literature review highlighted that the most frequent neuropathological findings were ischaemic and haemorrhagic lesions, including hypoxic/ischaemic alterations. However, few studies have confirmed the presence of SARS-CoV-2 antigens in brain tissue samples. CONCLUSION: This study highlighted the lack of specific neuropathological alterations in COVID-19-infected patients. There is still no evidence of neurotropism for SARS-CoV-2 in our cohort or in the literature.

A Two-Step Diagnostic Approach for NTRK Gene Fusion Detection in Biliary Tract and Pancreatic Adenocarcinomas.

BACKGROUND: It is of interest to determine the incidence and molecular characteristics of NTRK gene fusions in patients with bilio-pancreatic cancers, because of possible treatment with TRK inhibitors for advanced tumors. The aim of the present study was to apply the guidelines for NTRK testing algorithm to a series of patients with bilio-pancreatic cancers. METHODS: Immunohistochemistry screening was applied on formalin-fixed paraffin-embedded archival blocks from surgical resections, biopsies, or cytological samples of biliary tract and pancreatic adenocarcinomas. The presence of at least a weak staining in rare tumor cells led to testing by 2 RNA-based NGS panels. RESULTS: For biliary tract tumors, 153 samples have been selected. A total of 140 samples were suitable to perform IHC, and 17 samples were IHC positive. RNA NGS testing of the 17 IHC-positive samples revealed a single NTRK3 gene fusion (ETV6(4)-NTRK3(14)) that was detected by both NGS panels. In this perihilar cholangiocarcinoma, IHC performed on a biopsy showed a weak focal cytoplasmic and nuclear staining. No other NTRK fusion was detected on the 16 other samples with both panels. Overall in the patients screened by IHC and confirmed by NGS, the percentage of NTRK fusions was 0.7%. For pancreatic cancers, 319 samples have been selected and 297 were suitable to perform IHC. Nineteen samples were IHC positive. No fusion was detected by NGS. CONCLUSION: NTRK gene fusions are rare in bilio-pancreatic cancers but testing is of high interest due to possible treatment with specific TRK inhibitors.

Peritoneal patch in vascular reconstruction during pancreaticoduodenectomy for pancreatic cancer: a single Centre experience.

BACKGROUND: Concomitant venous resection during pancreaticoduodenectomy (PD) for pancreatic adenocarcinoma with mesenterico-portal vein involvement is increasingly performed to achieve oncological resection. This study aims to report a single centre experience in peritoneal patch (PP) as autologous graft for vascular reconstruction (VR) during PD. METHODS: A retrospective analysis of all patients who underwent PD + VR with PP between December 2019 and September 2020 was performed, using a prospective collected database. Postoperative outcome and pathological margins were evaluated. Venous patency was assessed by computed tomography at day 7 and week 12 post surgery. RESULTS: Fifteen patients underwent PD + VR with PP reconstruction for pancreatic cancer, including one total pancreatectomy. VR consisted of lateral (n = 14) or tubular (n = 1) patch. The median PP length was 30 mm [26.3-33.8] and venous clamping time 30 min [27.5-39.0]. Computed tomography showed a patent VR in 93.3% and 53.3% after 7 days and 12 weeks, respectively; venous patency loss was always asymptomatic. The only postoperative VR-related complication was one mesenteric venous thrombosis. Five other patients experienced VR-unrelated complications: septic shock (n = 3), biliary fistula (n = 1) and post-traumatic subdural hematoma (n = 1). Mortality was nihil. At pathology, R0 resection (≥1 mm) was observed in 40.0% (6/15), venous margin was free in 46.7% (7/15), and venous wall was involved in 40.0% (6/15). CONCLUSIONS: Use of PP as venous substitute during PD + VR is safe and feasible with an acceptable postoperative morbidity, and a decreased but asymptomatic venous patency after 12 weeks which should question the role of anticoagulation therapy.

Case report: BRAF A598-T599insV mutation as a potential resistance mechanism to alectinib in ALK-rearranged lung adenocarcinoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have improved the prognosis of advanced-stage non-small cell lung cancer (NSCLC) with ALK rearrangement, but resistance mechanisms limit their efficacy. We describe the case of a 63-year-old man with a stage cIVA ALK-rearranged lung adenocarcinoma who developed a BRAF A598-T599insV mutation as a potential resistance mechanism to alectinib, a second-generation ALK TKI. He was treated with an association of BRAF and MEK inhibitors but death occurred two months after treatment initiation in a context of tumor progression and toxicity. Based on this first report of BRAF A598-T599insV mutation occurring in lung cancer, we discuss resistance mechanisms to ALK TKIs, implications of BRAF mutation in NSCLC, and BRAF A598-T599insV mutation in other cancers.

Molecular pathology testing for non-small cell lung cancer: an observational study of elements currently present in request forms and result reports and the opinion of different stakeholders.

BACKGROUND: For patients with non-small cell lung cancer (NSCLC), targeted therapies are becoming part of the standard treatment. It is of question which information the clinicians provide on test requests and how the laboratories adapt test conclusions to this knowledge and regulations. METHODS: This study consisted of two components; 1) checking the presence of pre-defined elements (administrative and key for therapy-choice) on completed requests and corresponding reports in Belgian laboratories, both for tissue- and liquid biopsy (LB)-testing and b) opinion analysis from Belgian pathologists/molecular biologists and clinicians during national pathology/oncology meetings. RESULTS: Data from 4 out of 6 Belgian laboratories with ISO-accreditation for LB-testing were analyzed, of which 75% were university hospitals. On the scored requests (N = 4), 12 out of 19 ISO-required elements were present for tissue and 11 for LB-testing. Especially relevant patient history, such as line of therapy (for LB), tumor histology and the reason for testing were lacking. Similarly, 11 and 9 out of 18 elements were present in the reports (N = 4) for tissue and LB, respectively. Elements that pathologists/molecular biologists (N = 18) were missing on the request were the initial activating mutation, previous therapies, a clinical question and testing-related information. For reporting, an item considered important by both groups is the clinical interpretation of the test result. In addition, clinicians (N = 28) indicated that they also wish to read the percentage of neoplastic cells. CONCLUSIONS: Communication flows between the laboratory and the clinician, together with possible pitfalls were identified. Based on the study results, templates for complete requesting and reporting were proposed.

The Role of SMAD4 Inactivation in Epithelial-Mesenchymal Plasticity of Pancreatic Ductal Adenocarcinoma: The Missing Link?

Pancreatic ductal adenocarcinoma (PDAC) presents a five-year survival rate of 10% and its incidence increases over the years. It is, therefore, essential to improve our understanding of the molecular mechanisms that promote metastasis and chemoresistance in PDAC, which are the main causes of death in these patients. SMAD4 is inactivated in 50% of PDACs and its loss has been associated with worse overall survival and metastasis, although some controversy still exists. SMAD4 is the central signal transducer of the transforming growth factor-beta (TGF-beta) pathway, which is notably known to play a role in epithelial-mesenchymal transition (EMT). EMT is a biological process where epithelial cells lose their characteristics to acquire a spindle-cell phenotype and increased motility. EMT has been increasingly studied due to its potential implication in metastasis and therapy resistance. Recently, it has been suggested that cells undergo EMT transition through intermediary states, which is referred to as epithelial-mesenchymal plasticity (EMP). The intermediary states are characterized by enhanced aggressiveness and more efficient metastasis. Therefore, this review aims to summarize and analyze the current knowledge on SMAD4 loss in patients with PDAC and to investigate its potential role in EMP in order to better understand its function in PDAC carcinogenesis.

First Case Report of a Uterine Angiolipoleiomyoma With KRAS and KIT Mutations.

Angiolipoleiomyoma is a very rare lesion of the uterus. To the best of our knowledge, only 20 cases have been described in the literature. It is an insufficiently defined entity, which is not included in the WHO classification. This lesion may be therefore underdiagnosed and underestimated. We describe here a case of a 58-yr-old woman who underwent routine gynecological examination. Ultrasonography revealed a heterogeneous myometrial mass, while magnetic resonance imaging showed a voluminous corporeal and fundic myometrial mass protruding into the uterine cavity. A total hysterectomy was performed. The macroscopic examination revealed an intramural solitary round mass with a heterogeneous cut-surface. Microscopically, the lesion consisted of an admixture of smooth muscle, adipose tissue, and blood vessels. Desmin was positive, while HMB45 was negative in the tumor. Molecular tests were performed and revealed, for the first time to our knowledge, a case of an angiolipoleiomyoma harboring KRAS and KIT mutations.

Implementation of fetal clinical exome sequencing: Comparing prospective and retrospective cohorts.

PURPOSE: We compared the diagnostic yield of fetal clinical exome sequencing (fCES) in prospective and retrospective cohorts of pregnancies presenting with anomalies detected using ultrasound. We evaluated factors that led to a higher diagnostic efficiency, such as phenotypic category, clinical characterization, and variant analysis strategy. METHODS: fCES was performed for 303 fetuses (183 ongoing and 120 ended pregnancies, in which chromosomal abnormalities had been excluded) using a trio/duo-based approach and a multistep variant analysis strategy. RESULTS: fCES identified the underlying genetic cause in 13% (24/183) of prospective and 29% (35/120) of retrospective cases. In both cohorts, recessive heterozygous compound genotypes were not rare, and trio and simplex variant analysis strategies were complementary to achieve the highest possible diagnostic rate. Limited prenatal phenotypic information led to interpretation challenges. In 2 prospective cases, in-depth analysis allowed expansion of the spectrum of prenatal presentations for genetic syndromes associated with the SLC17A5 and CHAMP1 genes. CONCLUSION: fCES is diagnostically efficient in fetuses presenting with cerebral, skeletal, urinary, or multiple anomalies. The comparison between the 2 cohorts highlights the importance of providing detailed phenotypic information for better interpretation and prenatal reporting of genetic variants.

Pancreatic medullary carcinoma developed on a pancreatic intraductal papillary mucinous neoplasm with loss of MSH2 and MSH6 expression: a case report.

BACKGROUND: Pancreatic medullary carcinoma (PMC) is a rare pancreatic tumor, usually showing the presence of microsatellite instability, mostly MLH1 silencing, and a wild-type KRAS mutation status. We report here a PMC arising from a Pancreatic Intraductal Papillary Mucinous Neoplasm (IPMN), both having KRAS and TP53 mutations. CASE PRESENTATION: We report the case of a 73-year-old woman presenting with right iliac fossa pain. MRI revealed a 16 mm diameter mass in the pancreas, leading to a pancreatic duct stricture and upstream a dilatation of the distal pancreatic duct of Wirsung. A fine needle aspiration was performed, and pathology analysis revealed malignant glandular cells. The patient underwent distal pancreatectomy. Gross examination revealed an12 mm indurated white lesion, adjacent to a cystic lesion extending into the rest of the pancreatic body. Microscopically, the cystic area represented a mixed (gastric-type and pancreatobiliary-type) IPMN, involving the main and secondary pancreatic ducts with low-grade and high-grade dysplasia. In the periphery of this IPMN, a 14mm associated invasive carcinoma was observed, characterized by focal gland formation and by poorly differentiated cells with a syncytial appearance, associated with a dense lymphoplasmocytic and neutrophilic infiltrate. Immunohistochemical analyses showed loss of MSH2 and MSH6 expression. Microsatellite instability was confirmed by molecular test. Molecular analysis was performed both on the invasive carcinoma and on the high-grade dysplasia IPMN, revealing the same mutation profile with KRAS and TP53 mutations. The proposed diagnosis was mixed IPMN with associated invasive medullary carcinoma that presented loss of MSH2 and MSH6 expression. CONCLUSIONS: The present case reports for the first time, at the best of our knowledge, the coexistence of IPMN lesions and PMC, both having the same molecular alterations. It also describes the second case of PMC with microsatellite instability, MSH2 and MSH6 silenced.

Immunohistochemistry as an accurate tool for the assessment of BRAF V600E and TP53 mutations in primary and metastatic melanoma.

Metastatic melanoma is a fatal disease with poor prognosis. Ever since targeted therapy against oncogenic BRAF was approved, molecular profiling has become an integral part of the management of such patients. While molecular testing is not available in all pathology laboratories, immunohistochemistry (IHC) is a reliable screening option. The major objective of the present study was to evaluate whether IHC detection of BRAF and the tumor (suppressor) protein 53 gene (TP53) are reliable surrogates for mutation detection. Formalin-fixed paraffin-embedded samples of melanomas for which molecular data were previously obtained by targeted next-generation sequencing (NGS) between January 2014 and February 2019 were immunostained with BRAF V600E and p53 antibodies. A blinded evaluation of the IHC slides was performed by two pathologists in order to evaluate inter-observer concordance (discordant cases were reviewed by a third observer). The associations between the results of IHC and molecular profiling were evaluated. The study included a series of 37 cases of which 15 harbored a BRAF mutation and five a TP53 mutation. IHC had an overall diagnostic accuracy of 93.9% for BRAF V600E and 68.8% for TP53 compared to NGS. A statistically significant association between the two diagnostic methods was obtained for BRAF V600E (P=0.0004) but not for p53 (P=0.3098) IHC. The κ coefficient for IHC assessment of p53 was 0.55 and that for BRAF V600E was 0.72. In conclusion, the present results evidenced that IHC staining is a reliable surrogate for NGS in identifying the BRAF V600E mutation, which may become an efficient screening tool. Aberrant expression of p53 on IHC is at times associated with TP53 mutations but it was not possible to establish a direct link.

Complete Response to Afatinib of an EGFR Exon 18 delE709_T710insD-Mutated Stage IV Lung Adenocarcinoma.

INTRODUCTION: Epidermal growth factor receptor (EGFR) mutations are frequently found in patients with lung adenocarcinomas, 90% being deletions in exon 19 or point mutation in exon 21. Three generations of tyrosine kinase inhibitors (TKIs) targeting EGFR mutations are available and have changed patient prognosis but less data is available on exon 18 mutations and their sensitivity to TKI therapy. Exon 18 delE709_T710insD accounts for 0.06% (16/27,294) of all EGFR mutations and is an oncogenic driver. Several partial responses to afatinib have been described. CASE DESCRIPTION: We report the first case, to the best of our knowledge, of the complete response to afatinib of a 57-year-old patient with stage IV lung adenocarcinoma with a delE709_T710insD mutation in the EGFR gene detected by next-generation sequencing. Oral afatinib was prescribed and despite treatment interruptions and dosage tapering due to cutaneous adverse events, a complete response was achieved 12 months after treatment initiation and is currently maintained at 17 months. CONCLUSION: When EGFR mutation is suspected, complete DNA sequencing of exons 18 to 21 should be carried out and we suggest that afatinib should be the first-line treatment for exon 18 delE709_T710insD-mutated advanced lung adenocarcinomas. LEARNING POINTS: Rare EGFR gene mutations are not detected by standard diagnostic kits.DNA sequencing is required to diagnose rare mutations of the EGFR gene.delE709_T710insD-mutated stage IV lung adenocarcinomas respond to afatinib.

Analyses of DNA Methylation Profiling in the Diagnosis of Intramedullary Astrocytomas.

Intramedullary astrocytomas (IMAs) consist of a heterogeneous group of rare central nervous system (CNS) tumors associated with variable outcomes. A DNA methylation-based classification approach has recently emerged as a powerful tool to further classify CNS tumors. However, no DNA methylation-related studies specifically addressing to IMAs have been performed yet. In the present study, we analyzed 16 IMA samples subjected to morphological and molecular analyses, including DNA methylation profiling. Among the 16 samples, only 3 cases were classified in a reference methylation class (MC) with the recommended calibrated score (≥0.9). The remaining cases were either considered "no-match" cases (calibrated score <0.3, n = 7) or were classified with low calibrated scores (ranging from 0.32 to 0.53, n = 6), including inconsistent classification. To obtain a more comprehensive tool for pathologists, we used different unsupervised analyses of DNA methylation profiles, including our data and those from the Heidelberg reference cohort. Even though our cohort included only 16 cases, hypotheses regarding IMA-specific classification were underlined; a potential specific MC of PA_SPINE was identified and high-grade IMAs, probably consisting of H3K27M wild-type IMAs, were mainly associated with ANA_PA MC. These hypotheses strongly suggest that a specific classification for IMAs has to be investigated.

Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease.

OBJECTIVE: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. DESIGN: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. RESULTS: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. CONCLUSION: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Genome Sequencing from Post-Mortem Formalin-Fixed, Paraffin-Embedded Lung Tissues.

Implementation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the daily practice of pathology laboratories requires procedure adaptation to formalin-fixed, paraffin-embedded (FFPE) samples. So far, one study reported the feasibility of SARS-CoV-2 genome sequencing on FFPE tissues with only one contributory case of two. This study optimized SARS-CoV-2 genome sequencing using the Ion AmpliSeq SARS-CoV-2 Panel on 22 FFPE lung tissues from 16 deceased coronavirus disease 2019 (COVID-19) patients. SARS-CoV-2 was detected in all FFPE blocks using a real-time RT-qPCR targeting the E gene with crossing point (Cp) values ranging from 16.02 to 34.16. Sequencing was considered as contributory (i.e. with a uniformity >55%) for 17 FFPE blocks. Adapting the number of target amplification PCR cycles according to the RT-qPCR Cp values allowed optimization of the sequencing quality for the contributory blocks (i.e. 20 PCR cycles for blocks with a Cp value <28 and 25 PCR cycles for blocks with a Cp value between 28 and 30). Most blocks with a Cp value >30 were non-contributory. Comparison of matched frozen and FFPE tissues revealed discordance for only three FFPE blocks, all with a Cp value >28. Variant identification and clade classification was possible for 13 patients. This study validates SARS-CoV-2 genome sequencing on FFPE blocks and opens the possibility to explore correlation between virus genotype and histopathologic lesions.