Pathology Laboratory Archives: Conservation Quality of Nucleic Acids and Proteins for NSCLC Molecular Testing.

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids' quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6-8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6-8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond.

Advancing the PD-L1 CPS test in metastatic TNBC: Insights from pathologists and findings from a nationwide survey.

Pembrolizumab has received approval as a first-line treatment for unresectable/metastatic triple-negative breast cancer (mTNBC) with a PD-L1 combined positive score (CPS) of ≥ 10. However, assessing CPS in mTNBC poses challenges. Firstly, it represents a novel analysis for breast pathologists. Secondly, the heterogeneity of PD-L1 expression in mTNBC further complicates the assessment. Lastly, the lack of standardized assays and staining platforms adds to the complexity. In KEYNOTE trials, PD-L1 expression was evaluated using the IHC 22C3 pharmDx kit as a companion diagnostic test. However, both the 22C3 pharmDx and VENTANA PD-L1 (SP263) assays are validated for CPS assessment. Consequently, assay-platform choice, staining conditions, and scoring methods can significantly impact the testing outcomes. This consensus paper aims to discuss the intricacies of PD-L1 CPS testing in mTNBC and provide practical recommendations for pathologists. Additionally, we present findings from a nationwide Italian survey elucidating the state-of-the-art in PD-L1 CPS testing in mTNBC.

Spontaneous intracerebral haemorrhage associated with early-onset cerebral amyloid angiopathy and Alzheimer's disease neuropathological changes five decades after cadaveric dura mater graft.

Cerebral amyloid angiopathy (CAA) is a small vessel disease, causing spontaneous intracerebral hemorrhage (ICH) in the elderly. It is strongly associated with Alzheimer disease (AD), as most CAA patients show deposition of Aß-i.e. the basic component of parenchymal Alzheimer amyloid deposits-in the cerebral vessels. Iatrogenic early-onset CAA has been recently identified in patients with a history of traumatic brain injury or other cerebral as well as extra-cerebral lesions that led to neurosurgery or other medical procedures as intravascular embolization by cadaveric dura mater extracts many years before the first ICH event. In those patients, a transmission of Aß seeds from neurosurgical instruments or from cadaveric dura mater exposure was suggested. We report a 51-year-old woman with unremarkable family history who presented abruptly with aphasia and right hemiparesis. A cerebral left lobar haemorrhagic stroke was documented by neuroimaging. Accurate anamnesis revealed a neurosurgical procedure with cadaveric dura mater graft at the age of 2 years for an arachnoid cyst. The neuropathological examination of the cerebral parietal biopsy showed severe amyloid angiopathy in many leptomeningeal and cortical vessels, as well as abundant parenchymal Aß deposits, neurofibrillary tangles and neuropil threads. The mechanism involved in the human-to-human transmission of the Aß proteinopathy remains to be clarified. In our patient the cadaver derived dura used for grafting is a very strong candidate as the source of the transmission. A systematic monitoring of individuals who have had neurosurgical procedures in early life, especially those involving cadaveric dural grafts, is required to determine the ratio of those affected by CAA many years later and unaffected. Moreover, our report confirms that in addition to vascular and parenchymal Aß pathology, neurofibrillary changes indistinguishable from AD may develop in specific conditions with long latency period from the neurosurgical or embolization procedure.

Exaggerated Placental Site as a Cause of Hysterectomy for Massive Bleeding After First Trimester Voluntary Abortion.

Objectives: Placental implantation anomalies in first-trimester abortions may determine severe bleeding leading to hysterectomy. There are very few cases of urgent hysterectomy post-abortion reported in the literature, related to placenta accreta spectrum, but in any of them is considered association with benign trophoblastic lesions. Methods: We report the case of a woman, who underwent surgical voluntary abortion by vacuum aspiration during first trimester, without any apparent surgical complications. Immediately, after this procedure, the patient had massive vaginal bleeding; an emergency hysterectomy was performed. Histological examination showed an exaggerated placental site (EPS). Results: Morphological findings describe a trophoblastic tumor-like lesion, which differs from placenta accreta and often considered an asymptomatic occasional feature. Proliferative index, evaluated by double immunostain for CK8-18 and Ki-67, was unremarkable (<1%). Conclusions: Only a single report in the literature describes a case of symptomatic EPS 65 after first-trimester abortion. Major attention should be paid to trophoblastic pathology in order to understand a possible relationship with uterine bleeding and to find a clinical, ultrasound or chemical indicator.

Late-onset focal dermal elastosis: Report of a case and review of the literature.

Late-onset focal dermal elastosis is a rare cutaneous condition classified as an increased dermal elastic tissue disorder. It is distinguished clinically by multiple papules with a preference for the neck and other flexures, as well as histologically by focally increased elastic fibers in the reticular dermis. Several elastic tissue disorders in the skin have a similar clinical presentation. The distinction between late-onset focal dermal elastosis and other pseudoxanthoma elasticum mimickers is critical because they are not associated with systemic lesions. We present a case of late-onset focal dermal elastosis and conduct a literature review on this unusual condition.

Small Bowel Intussusception Due to Rare Cardiac Intimal Sarcoma Metastasis: A Case Report.

Background: Intimal sarcomas are rare malignant mesenchymal tumors arising from the heart and large blood vessels. Their intraluminal growth leads to vascular obstructive symptoms and peripheral neoplastic embolization. Direct infiltration of the lungs or metastases to the pulmonary system, occur in 40% of cases and extrathoracic spread is frequent, also in presentation. Intussusception is an unusual event in adults, accounting for <5% of bowel obstructions. In most cases it is caused by a malignancy and requires surgical resection. Case Presentation: We describe a rare case of a 50-year-old man suffering of bowel obstruction due to intussusception sustained by a small bowel metastasis of a primary cardiac intimal sarcoma. One year and a half before the onset of abdominal symptoms, a grade II intimal sarcoma was removed from his left atrium and consequently he followed a chemotherapy protocol. Four months later a CT scan revealed local recurrence. Eighteen months after heart surgery he referred to the ER with abdominal pain. CT scan showed an ileal intussusception and the patient was scheduled for surgery. A tract of 10 cm ileus was removed containing an intramural polypoid solid mass. Histological analyses revealed a grade II intimal sarcoma consistent with his first diagnosis. Conclusion: Primary heart tumors are late found and often partially resected, therefore metastatic pathways are to be expected. Adult small bowel intussusception is a rare event and caused by a malignancy in one third of cases. Therefore, our recommendation is to always resect the tract involved in order to perform a proper diagnosis.

PD-L1 Testing and Squamous Cell Carcinoma of the Head and Neck: A Multicenter Study on the Diagnostic Reproducibility of Different Protocols.

Immune checkpoint inhibitors for blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis are now available for squamous cell carcinoma of the head and neck (HNSCC) in relapsing and/or metastatic settings. In this work, we compared the resulting combined positive score (CPS) of PD-L1 using alternative methods adopted in routine clinical practice and determined the level of diagnostic agreement and inter-observer reliability in this setting. The study applied 5 different protocols on 40 tissue microarrays from HNSCC. The error rate of the individual protocols ranged from a minimum of 7% to a maximum of 21%, the sensitivity from 79% to 96%, and the specificity from 50% to 100%. In the intermediate group (1 ≤ CPS < 20), the majority of errors consisted of an underestimation of PD-L1 expression. In strong expressors, 5 out of 14 samples (36%) were correctly evaluated by all the protocols, but no protocol was able to correctly identify all the "strong expressors". The overall inter-observer agreement in PD-L1 CPS reached 87%. The inter-observer reliability was moderate, with an ICC of 0.774 (95% CI (0.651; 0.871)). In conclusion, our study showed moderate interobserver reliability among different protocols. In order to improve the performances, adequate specific training to evaluate PD-L1 by CPS in the HNSCC setting should be coordinated.

Histopathological Study of a Broad Spectrum of Skin Dermatoses in Patients Affected or Highly Suspected of Infection by COVID-19 in the Northern Part of Italy: Analysis of the Many Faces of the Viral-Induced Skin Diseases in Previous and New Reported Cases.

Skin manifestations of COVID-19 infections are diverse and are new to the dermatology community. We had the opportunity to examine the clinical and histopathological features of several patients who were divided into 3 groups. The first group included 8 COVID-19-positive patients who were hospitalized and quarantined at home. The second group included children and young adults who presented with chilblain erythema, erythema multiforme, and urticaria-like lesions. This group of patients was negative for the COVID-19 gene sequences by polymerase chain reaction but had a high risk of COVID-19 infection. The third group included clinically heterogeneous and challenging lesions. These patients were not subject to either polymerase chain reaction tests or serological analyses because they sought dermatological attention only for a dermatosis. The histopathological analysis of these cases showed a wide spectrum of histopathological patterns. What appears to be constant in all skin biopsies was the presence of prominent dilated blood vessels with a swollen endothelial layer, vessels engulfed with red blood cells, and perivascular infiltrates, consisting mainly of cytotoxic CD8+ lymphocytes and eosinophils. In 2 cases, there was diffuse coagulopathy in the cutaneous vascular plexus. In the early phases of the disease, there were numerous collections of Langerhans cells in the epidermis after being activated by the virus. The presence of urticarial lesions, chilblains, targetoid lesions (erythema multiforme-like lesions), exanthema, maculohemorrhagic rash, or chickenpox-like lesions associated with the histopathological features mentioned previously should cause clinical dermatologists to suspect the possibility of COVID-19 infection, especially in patients with fever and cough.

Multisite analysis of high-grade serous epithelial ovarian cancers identifies genomic regions of focal and recurrent copy number alteration in 3q26.2 and 8q24.3.

High-grade serous epithelial ovarian cancer (HGS-EOC) is a systemic disease, with marked intra and interpatient tumor heterogeneity. The issue of spatial and temporal heterogeneity has long been overlooked, hampering the possibility to identify those genomic alterations that persist, before and after therapy, in the genome of all tumor cells across the different anatomical districts. This knowledge is the first step to clarify those molecular determinants that characterize the tumor biology of HGS-EOC and their route toward malignancy. In our study, -omics data were generated from 79 snap frozen matched tumor biopsies, withdrawn before and after chemotherapy from 24 HGS-EOC patients, gathered together from independent cohorts. The landscape of somatic copy number alterations depicts a more homogenous and stable genomic portrait than the single nucleotide variant profile. Genomic identification of significant targets in cancer analysis identified two focal and minimal common regions (FMCRs) of amplification in the cytoband 3q26.2 (region α, 193 kb long) and 8q24.3 (region ß, 495 kb long). Analysis in two external databases confirmed regions α and ß are features of HGS-EOC. The MECOM gene is located in region α, and 15 genes are in region ß. No functional data are yet available for the genes in the ß region. In conclusion, we have identified for the first time two FMCRs of amplification in HGS-EOC, opening up a potential biological role in its etiopathogenesis.

Expression of calretinin in high-grade hormone receptor-negative invasive breast carcinomas: correlation with histological and molecular subtypes.

Calretinin expression has been reported in neoplasms arising in various organs, including the breast. We investigated the relationship of calretinin expression with different histological and molecular subtypes of invasive breast carcinomas (IBCs) and its prognostic significance in high-grade female hormone receptor-negative IBCs. A total of 196 cases of IBCs of different histological subtypes were analyzed for immunohistochemical expression of calretinin, human epidermal growth factor receptor 2 (HER2), basal-like (BL), apocrine, and proliferative markers and grouped in different molecular subtypes. We found significant morphological differences in the group of formally classified invasive ductal carcinoma of no special type (IDC-NST), which we further subdivided into two types (type I IDC-NST and type II IDC-NST) according to their morphology. Calretinin expression was found in 55.1% of the IBCs and was strongly associated with carcinoma with medullary features (P = 0.014) and type II IDC-NST (P < 0.001), while type I IDC-NST correlated (P < 0.001) with a lack of calretinin expression. Among the molecular subtypes of IBC, calretinin expression was identified in a significant portion of BL breast cancers (BLBCs), while expression was poor in HER2-overexpressing and molecular-apocrine (MA) HER2-negative subtypes and even less in MA/HER2+ ones. Calretinin expression was significantly associated with high (≥50) Ki-67 (P = 0.02), but not with parameters like age, tumor size, lymph node status, overall survival (OS), and disease-free survival. Calretinin expression is most common in high-grade IBCs with histological medullary features, type II IDC-NST and BL phenotype, and is associated with high neoplastic proliferative index.

[Molecular biology as a diagnostic tool in the newborn Emergency department: a rare case of idiopathic infantile arterial calcification]. / La biologia molecolare diventa strumento diagnostico nell'emergenza pediatrica: un raro caso di arteriopatia calcifica infantile idiopatica.

A Turkish female infant of 96 days was admitted to the pediatric emergency room because of inconsolable crying, persistent cough, and difficulty in feeding during the previous day. She was conscious and did not show any signs or symptoms of multiorgan failure. A few minutes afterwards, the child experienced cardiac arrest with an initial cardiac rhythm of asystole and died 75 minutes later following cardiopulmonary resuscitation maneuvers. As the pathological cause of death, autopsy findings revealed a rare type of idiopathic infantile arterial calcification resulting from a mutation in the gene encoding for the ENPP1 enzyme.

Robotic Shaving Technique in 25 Patients Affected by Deep Infiltrating Endometriosis of the Rectovaginal Space.

Minimally invasive surgery represents the gold standard for the management of deep infiltrating endometriosis (DIE) involving the rectovaginal septum (RVS). This analysis aimed to evaluate the feasibility of robotic-assisted laparoscopy (RAL) and clinical outcomes in terms of long-term complications, pain relief, and recurrence rate for the treatment of DIE of the RVS. A prospective cohort study of robotic procedures was performed between October 2010 and July 2014, including removal of endometriotic nodules from the RVS with rectal shaving alone or in combination with accessory procedures. In all cases, the revised American Society for Reproductive Medicine (rASRM) score for endometriosis was >40 points (stage IV). Twenty-five consecutive patients underwent RAL, with a successful complete nodule debulking by the wall shaving technique. Pathology confirmed the adequacy of the surgical specimen and the median largest endometriotic nodule was of 21 mm (range, 10-60 mm), with free margins in all cases. The median operative time from skin opening to closure was 174 minutes (range, 75-300 minutes), and blood loss was close to 0 mL. The median revised Enzian score for location A (RVS) was 2 (range, 1-3). The most frequent Enzian class was A2B0C0 (48%), followed by A3B0C0 (12%). In 3 cases (12%), partial vaginal resection was required to remove endometriotic nodules of the RVS (1 each in classes A3B0C1FI, A3B0C0FO, and A3B0C0). No intraoperative complications occurred. This series has a median long-term follow up of 22 months (range, 6-50 months) currently available with an optimal operative time, demonstrating good long-term outcomes. Our data support robotics as a safe and attractive alternative for comprehensive surgical treatment of DIE.

Linear and micronodular neuroendocrine cell hyperplasia in an ovarian mucinous cystadenoma.

We report a rare case of an ovarian mucinous cystadenoma in which there were peculiar neuroendocrine micronests composed of gastrin-immunoreactive cells. There was no clinical evidence of hypergastrinemia. The mucinous component of the neoplasm was represented by columnar cells mostly expressing a gastric phenotype with MUC5AC and claudin 18 positivity, which was consistent with the presence of interspersed gastrin cells. The tumoral stroma displayed areas of luteinization with cells intensely positive for α-inhibin, MART-1 and calretinin.

Primary pleural epithelioid angiosarcoma. A case report and review of the literature.

Malignant vascular tumors are uncommon sarcomas that arise from endothelial cells of small blood vessels and may affect every organ. Pleural localization is very exceptional, and only 48 cases have been reported in the English literature to date. Even if etiological factors implicated in the development of vascular sarcomas are still unclear, the strongest association with the disease was a history of chronic tuberculous pyothorax, observed only in Japanese patients, while prior radiotherapy and occupational exposure to asbestos have been reported in few Western cases. The mean age at diagnosis was 58 years, and the male to female ratio was 6:1. The overall prognosis was poor, and most of the patients died of disease soon after diagnosis. Histological features and clinical presentation often cause several problems in the differential diagnosis, particularly with mesothelioma and metastasis from poorly differentiated carcinomas. Immunohistochemistry plays an important role in identifying these rare entities, confirming the endothelial origin of the neoplasm with the expression of at least one of the vascular markers CD31, CD34, or factor VIII-related antigen. We report herein a further case of a 62-year-old man who presented with progressive dyspnea and bilateral massive hemothorax. The overall pathological and immunohistochemical features of the pleural specimens supported the diagnosis of epithelioid angiosarcoma.

The high frequency of de novo promoter methylation in synchronous primary endometrial and ovarian carcinomas.

PURPOSE: The methylation status of hMLH1, CDKN2A, and MGMT was investigated in a panel of synchronous cancers of the ovary and endometrium, fulfilling the clinicopathologic criteria for independent primary tumors to define the possible role of epigenetic mechanisms in the development of these cancers. EXPERIMENTAL DESIGN: Bisulfite-converted DNA from 31 tumors (13 endometrial and 18 ovarian carcinomas) and from matched normal tissue of 13 patients was analyzed by a methylation-specific PCR assay at the CpG-rich 5' regions of all three genes. In all tumors, we also investigated the presence of microsatellite instability and hMLH1 immunohistochemical expression in relation to hMLH1 hypermethylation status. RESULTS: Methylation of hMLH1, CDKN2A, and MGMT was detected in 39%, 41%, and 48% of endometrial and ovarian tumors, respectively. hMLH1 hypermethylation was observed in all tumors of five patients, and it was invariably associated with loss of hMLH1 protein and presence of microsatellite instability. CDKN2A and MGMT methylation was randomly detected among both endometrial (45% and 24% of cases, respectively) and ovarian carcinomas (39% and 39% of cases, respectively). Concordant methylation at two or three genes was observed in 35% of cases. CONCLUSIONS: Epigenetic inactivation of hMLH1, CDKN2A, and MGMT may be a common and early event in the development of synchronous primary endometrial and ovarian carcinomas and may qualify as a marker of a field cancerization encompassing the ovary and endometrium. Detection of MGMT hypermethylation may be useful to define a set of gynecologic malignancies with a specific sensitivity to alkylating chemotherapy.