PD-L1 Overexpression in the Lungs of Subjects Who Died from COVID-19: Are We on the Way to Understanding the Immune System Exhaustion Induced by SARS-CoV-2?

Knowledge of the pathogenic mechanisms of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. The presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. PD-L1 is overexpressed in the pulmonary tissue of subjects died from COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR.

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

Fluorescent in situ hybridization as a screening test for HER2 amplification in G2 and G3 breast cancers of lobular and ductal histotype and metastases.

The aim of the present study was to evaluate the effectiveness of fluorescence in situ hybridisation (FISH), as a screening test, in moderately- (G2) or poorly- (G3) differentiated breast cancers of the ductal (IDC) and lobular (ILC) histotypes and distant metastases. HER2 FISH was performed on 486 G2 and 477 G3 both of IDC and ILC histotypes and in 241 metastases. A significant difference in the HER2 amplification was observed between G2 (14.8%) and G3 (31.9%), with no difference according to the histotype. However, the rate of amplification increased to 36% in the G2/hormone receptor-negative cases as compared to 10.6% in the G2/receptor-positive cases (p<0.0001). HER2 was amplified in 17% of metastases with some differences depending on the location. These data suggest that the HER2 FISH analysis may be an effective screening test in breast cancer metastases and G3 tumors, irrespective of the hormone receptor status or presence of lymphovascular invasion.

Nonpolypoid intestinal mantle cell lymphoma resembling MALT lymphoma with successful response to rituximab.

Two unusual cases of mantle cell lymphoma are reported. They involved the ileum and right colon without multiple lymphomatous polyposis and morphologically resembled an extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type.

Mucinous carcinoma of thyroid gland. Report of a primary and a metastatic mucinous tumour from ovarian adenocarcinoma with immunohistochemical study and review of literature.

Mucinous carcinoma of the thyroid gland is an uncommon tumour that from the histological point of view, resembles mucinous carcinoma of others sites. Although a mucinous appearance has sometimes been reported in association with cases of typical thyroid carcinoma, true mucinous carcinoma is exceptionally rare. We describe two cases of thyroid tumours with mucinous differentiation studied with immunohistochemistry. Both cases disclosed a similar histological appearance, with small nests and sheets of malignant epithelial cells associated with extensive extracellular mucin that substituted and entrapped the follicular parenchyma of the thyroid. Thyroglobulin and focally thyroid transcription factor (TTF) 1 were positive in one case. From these findings, we classified this tumour as primary mucinous thyroid carcinoma. Thyroglobulin and TTF-1 were negative in tumour cells of second case; on the contrary, positivity to the carcinoembryonic antigen and CA-125 was strong and generalized. However, successfully, the patient presented ascites associated to right ovarian mass. In this case, thyroid tumour represents the first clinical sign of an ovarian mucinous adenocarcinoma, and it has not been previously described in literature. Both patients died after few months to diagnosis. In conclusion, primary and secondary mucinous carcinoma are rare and unusual tumours of the thyroid gland that can be a cause of pitfall in differential diagnosis. In these cases, for a correct diagnosis, a complete clinical history, restricted histological criteria and immunohistochemical panel are necessary.

Medicolegal liability in pathology: an international perspective.

An inevitable outcome of modern Medicine in any country is that some patients will experience adverse events, some of which would have been preventable. Different nations have developed various approaches to such cases; their legal efficacies are probably dissimilar and dependent on a number of disparate variables. An international "snapshot" of the results of the interacting forces can be obtained by asking physicians in several countries how they view selected subjective facets of their tort systems. In the U.S., many physicians view the structure of malpractice torts as unfair, and that belief is shared by at least some pathologists. The American Medical Association has declared that a multiregional malpractice "crisis" exists which raises medical costs and threatens access to care. Furthermore, malpractice tort decisions are often flawed scientifically because lay jurors and judges cannot properly evaluate the quality of "expert" testimony given by adversarial witnesses. Despite these factors, there has been little effort to investigate the views of pathologists on malpractice actions outside the U.S. In this paper, the authors have collected the responses of an international group of pathologists to a questionnaire on that topic. The respondents practice in academic centers in 15 countries outside the U.S. As expected, a range of views was represented, with some pathologists reporting that malpractice litigation was uncommon and others noting a worrisome trend toward its growth. Interestingly, so-called "defensive medicine" was found to be relatively common in pathology in many countries.

Unsuspected Xp11 Translocation Renal Neoplasm Associated With Contralateral Clear Cell Carcinoma.

In this report, we present for the first time the coexistence of a conventional renal cell carcinoma (RCC) and an undefined Xp11 translocation renal neoplasm in distinct kidneys, which was difficult to definitively classify as either carcinoma or PEComa (perivascular epithelioid cell tumor). While one of the tumors showed the morphological and immunohistochemical features of clear RCC, the other had an unusual morphology with a prominent nested pattern. Microscopically this tumor showed nests of cells with clear and eosinophilic cytoplasm and nuclei with prominent nucleoli; some hyaline globules were evident. Immunohistochemical panel showed negativity for cytokeratin-pan, cytokeratin-7, PAX8, and CD10 but positive immunostaining for cathepsin K, racemase, Melan-A, and TFE3. A subsequent, metaphase, dual-color fluorescence in situ hybridization confirmed the Xp11 translocation. Attention should be paid to the routine immunohistochemical profile that, in case of negativity of specific RCC markers, may suggest an Xp11 translocation renal tumor. The addition of TFE3 can easily identify the specific subtype.

Clinical activity and tolerability of FOLFIRI and cetuximab in elderly patients with metastatic colorectal cancer in the CAPRI-GOIM first-line trial.

BACKGROUND: In the cetuximab after progression in KRAS wild-type colorectal cancer patients (CAPRI) trial patients with metastatic colorectal cancer (mCRC) received 5-fluorouracil, folinic acid and irinotecan (FOLFIRI) and cetuximab in first line followed by 5-Fluorouracil, folinic acid, oxaliplatin (FOLFOX) with or without cetuximab until progression. Limited data are available on the efficacy and safety of anti-epidermal growth factor receptor (anti-EGFR) agents on elderly patients with mCRC. In the current study we evaluated the efficacy and safety of FOLFIRI plus cetuximab in age-defined subgroups. METHODS: A post-hoc analysis was performed in CAPRI trial patients; outcomes (progression-free survival (PFS), overall response rate (ORR), safety) were analysed by age-groups and stratified according to molecular characterisation. 3 age cut-offs were used to define the elderly population (≥65; ≥70 and ≥75 years). RESULTS: 340 patients with mCRC were treated in first line with FOLFIRI plus cetuximab. Among those, 154 patients were >65 years, 86 >70 years and 35 >75 years. Next-generation sequencing (NGS) was performed in 182 patients. Among them, 87 patients were >65 years, 46 >70 and 17 >75. 104 of 182 patients were wild type (WT) for KRAS, NRAS, BRAF, PIK3CA genes. In the quadruple WT group, 51 patients were ≥65 years; 29 were ≥70; 9 were ≥75. Median PFS was similar within the age-subgroups in the intention-to-treat population, NGS cohort and quadruple WT patients, respectively. Likewise, ORR was not significantly different among age-subgroups in the 3 populations. Safety profile was acceptable and similarly reported among all age-groups, with the exception of grade ≥3 diarrhoea (55% vs 25%, p=0.04) and neutropaenia (75% vs 37%, p=0.03) in patients ≥75 years and grade ≥3 fatigue (31% vs 20%, p=0.01) in patients <75 years. CONCLUSIONS: Tolerability of cetuximab plus FOLFIRI was acceptable in elderly patients. Similar ORR and PFS were observed according to age-groups. No differences in adverse events were reported among the defined subgroups with the exception of higher incidence of grade ≥3 diarrhoea and neutropaenia in patients ≥75 years and grade ≥3 fatigue in patients <75 years. TRIAL REGISTRATION NUMBER: 2009-014041-81.

Spitz nevus of the tongue with pseudoepitheliomatous hyperplasia: report of three cases of a pseudomalignant condition.

Three cases of Spitz nevus of the tongue associated with pseudoepitheliomatous hyperplasia are reported: two occurring in children and one in an adult. The location at an unusual site and the complex pattern resulting from the intimate admixture of the neoplastic melanocytic component and the hyperplastic keratinocytic component led in each case to consider diagnoses such as malignant melanoma and invasive squamous cell carcinoma. Staining for S-100 protein and keratin was useful to identify and separate the two components. Spitz nevus of the tongue carries some intriguing similarities with granular cell tumor, suggesting a possible histogenetic and pathogenetic relationship.

Pleomorphic carcinomas of the lung show a selective distribution of gene products involved in cell differentiation, cell cycle control, tumor growth, and tumor cell motility: a clinicopathologic and immunohistochemical study of 31 cases.

We investigated 31 cases of pleomorphic carcinomas of the lung, with a double component of neoplastic epithelial cells and of spindle and/or giant cells. To correlate the morphologic diversity of these two cell components with their immunophenotype, we evaluated the expression of several gene products involved in cell differentiation (cytokeratins, epithelial membrane antigen, carcinoembryonic antigen, vimentin, S-100 protein, smooth muscle actin, desmin), cell cycle control and apoptosis (p53, p21Waf1, p27Kip1, FHIT), tumor growth (proliferative fraction, assessed by Ki-67 antigen, and microvascular density, assessed by CD34 immunostaining), and tumor cell motility (fascin). We found the epithelial component to be significantly more immunoreactive for cytokeratins, epithelial membrane antigen, carcinoembryonic antigen, cell cycle inhibitors p21Waf1, p27Kip1 and tumor suppressor gene FHIT, whereas the sarcomatoid component, independent of tumor stage and size, was more immunoreactive for vimentin, fascin, and microvascular density. Accordingly, we suggest a model of tumorigenesis whereby the mesenchymal phenotype of pleomorphic cells is likely induced by the selective activation and segregation of several molecules involved in cell differentiation, cell cycle control, and tumor cell growth and motility. Whether pleomorphic carcinomas of the lung are tumors with a dismal prognosis still remains an unsettled issue. In our series, however, stage I pleomorphic carcinomas have the same clinical behavior as ordinary non-small cell lung cancer, and only a high proliferative index (Ki-67 labeling index >35%) is associated with a worse prognosis in these tumors.

Proliferating pilar tumors: a clinicopathologic study of 76 cases with a proposal for definition of benign and malignant variants.

We studied proliferating pilar tumors (PPTs) to establish histologic criteria that could predict behavior. We reviewed all cases in our consultation files (1989-2000) and evaluated 76 cases with meaningful follow-up information. Histologic examination involved attention to tumor silhouette, degree of nuclear atypia, mitotic activity, necrosis, and perineurial or angiolymphatic invasion. Tumors were stratified as follows: group 1 PPTs, circumscribed silhouettes with "pushing " margins, modest nuclear atypia, and an absence of pathologic mitoses, necrosis, and invasion of nerves or vessels; group 2 PPTs, similar to group 1 but manifested irregular, locally invasive silhouettes with involvement of the deep dermis and subcutis; group 3, invasive growth patterns, marked nuclear atypia, pathologic mitotic forms, and geographic necrosis, with or without involvement of nerves or vascular structures. Recurrence occurred in none of 48 group 1 PPTs; 3 (15%) of 20 group 2 PPTs had local regrowth; 4 (50%) of 8 of group 3 PPTs recurred and/or metastasized to regional lymph nodes. The differences between groups 1 and 3 and between 2 and 3 were statistically significant (P = .0002, P < .05, respectively). It seems justifiable to regard group 1 PPTs as benign, group 2 as having potential for locally aggressive growth, and group 3 also as having metastatic potential. The latter 2 categories might be equated with low and high grades of malignancy among PPTs of the skin.

Ectopic neural and neuroendocrine neoplasms.

Although neural and neuroendocrine tissues are distributed virtually ubiquitously throughout the body, the occurrence of selected neoplasms related to those lineages is extremely uncommon in some topographic sites. This review considers the clinicopathologic characteristics of heterotopic pituitary adenomas; neuroendocrine carcinomas in non-organ-based locations; ectopic (extraneuraxial) meningiomas and gliomas; visceral neuroblastic neoplasms and primitive neuroectodermal tumors; and paragangliomas arising outside the sympathoadrenal neural network. Practical approaches to differential diagnosis are emphasized.

Extramedullary hematopoietic proliferations, extraosseous plasmacytomas, and ectopic splenic implants (splenosis).

Hematopoietic proliferations are well known to present ectopically outside the bone marrow, either in benign or malignant form. As such, they present a distinct problem with respect to morphologic interpretation because of their uncommonality in extramedullary sites and their capacity to simulate other lesions histologically. This review considers extramedullary myeloid tumors ("granulocytic sarcoma," "erythroblastic sarcoma," "megakaryocytic sarcoma"), tumefactive extramedullary hematopoiesis, and the peculiar condition known as "splenosis," with consideration of their clinical, microscopic, and cytohistochemical chararacteristics.

Multicystic mesothelioma of the liver with secondary involvement of peritoneum and inguinal region.

A case of multicystic mesothelioma of the liver with secondary involvement of the pelvic peritoneum and the inguinal region is presented. The case is of interest because of its unusual location and peculiar biological behavior.

EGFR and KRAS mutations detection on lung cancer liquid-based cytology: a pilot study.

In advanced non-small-cell lung carcinomas epidermal growth factor receptor (EGFR) and KRAS testing is often performed on cytology. Liquid-based cytology (LBC), which eliminates the need for slide preparation by clinicians, may be very useful. In 42 LBC DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after smear preparation and laser capture microdissection (LCM) of Papanicolaou-stained cells. EGFR and KRAS mutational analyses were performed by direct sequencing. On CytoLyt-derived DNA four EGFR (9%) and five KRAS (12%) gene mutations were found. When direct sequencing was performed after LCM, the rate of cases that displayed either EGFR or KRAS mutations increased from 21% to 40%. Although time-consuming, LCM makes direct sequencing highly sensitive even on LBC preparations containing only a few cells.

EGFR mutations detection on liquid-based cytology: is microscopy still necessary?

Currently, there is a trend towards an increasing use of liquid-based cytology (LBC) to diagnose non-small cell lung cancer. In this study, to detect epidermal growth factor receptor mutations, different molecular techniques were applied to LBC samples with and without laser capture microdissection (LCM). In 58 LBCs, DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after slide preparation and LCM of Papanicolaou-stained cells. The rate of mutant cases obtained by direct sequencing was discordant between CytoLyt-derived (10.3%) and LCM-derived (17.2%) DNA. However, the same mutant rate (17.2%) was achieved on the matched samples by high-resolution melting analysis, fragment and TaqMan assays. Thus, LCM and direct sequencing may be replaced by more sensitive non-sequencing methods directly performed on CytoLyt-derived DNA, an easier and faster approach to improve epidermal growth factor receptor testing standardisation on LBCs.

Sanger sequencing in routine KRAS testing: a review of 1720 cases from a pathologist's perspective.

BACKGROUND: Sanger sequencing (SS) of PCR products is still the most frequent method to test colorectal cancer for KRAS mutations in routine practice. METHODS: An audit of SS on 1720 routine cases was carried out, taking into account age, gender, specimen type (resection vs biopsies), tumour site (primary vs metastasis), tumour stage, neoplastic cells abundance (>30% vs <30%) and fixation type (buffered formalin vs simple formalin). In a subset of 50 wild-type (WT) patients correlations between SS findings and response rate (RR), progression-free survival (PFS) and overall survival (OS) were also evaluated. RESULTS: The tests were informative in 1691 cases (98.3%). Mutations were detected in 671 cases (39.6%). No significant differences in mutation rates were observed with respect to age (p=0.2), gender (p=0.2), specimen type (p=0.3) and formalin fixation (p=0.08). Conversely, KRAS mutant rate was higher in metastatic tissue (50% vs 39%, p=0.02), in samples with over 30% of neoplastic cells (43.4% vs 26.6%, p=0.02) and in tumours tested in stage IV (p=0.05). The RR of SS KRAS WT patients was 26% (one complete and 12 partial responses). The disease control rate (objective responses plus stable disease) was 56%. Median PFS was 4.4 months and median OS was 10.4 months. CONCLUSIONS: Pathological criteria that make SS a more robust method for KRAS testing and treatment response prediction are neoplastic cell abundance, metastatic tissue sample and stage IV primary tumour.

BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma.

PURPOSE: The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. PATIENTS AND METHODS: In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. RESULTS: BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. CONCLUSION: In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.