TTF-1/p63-positive poorly differentiated NSCLC: A histogenetic hypothesis from the basal reserve cell of the terminal respiratory unit

TTF-1 is expressed in the alveolar epithelium and in the basal cells of distal terminal bronchioles. It is considered the most sensitive and specific marker to define the adenocarcinoma arising from the terminal respiratory unit (TRU). TTF-1, CK7, CK5/6, p63 and p40 are useful for typifying the majority of non-small-cell lung cancers, with TTF and CK7 being typically expressed in adenocarcinomas and the latter three being expressed in squamous cell carcinoma. As tumors with coexpression of both TTF-1 and p63 in the same cells are rare, we describe different cases that coexpress them, suggesting a histogenetic hypothesis of their origin. We report 10 cases of poorly differentiated non-small-cell lung carcinoma (PD-NSCLC). Immunohistochemistry was performed by using TTF-1, p63, p40 (∆Np63), CK5/6 and CK7. EGFR and BRAF gene mutational analysis was performed by using real-time PCR. All the cases showed coexpression of p63 and TTF-1. Six of them showing CK7+ and CK5/6− immunostaining were diagnosed as “TTF-1+ p63+ adenocarcinoma”. The other cases of PD-NSCLC, despite the positivity for CK5/6, were diagnosed as “adenocarcinoma, solid variant”, in keeping with the presence of TTF-1 expression and p40 negativity. A “wild type” genotype of EGFR was evidenced in all cases. TTF1 stained positively the alveolar epithelium and the basal reserve cells of TRU, with the latter also being positive for p63. The coexpression of p63 and TTF-1 could suggest the origin from the basal reserve cells of TRU and represent the capability to differentiate towards different histogenetic lines. More aggressive clinical and morphological features could characterize these “basal-type tumors” like those in the better known “basal-like” cancer of the breast

Immunoistochemical expression of PD-1 and PD-L1 in bone marrow biopsies of patients with acute myeloid leukemia

Background. Haematological and non-haematological malignancies are able to escape the host immune by the capacity to hijack the immune check-points. Several immune check-point molecules are known, such as T cell immunoglobulin mucin-3 (TIM-3), cytotoxic T-cell antigen-4 (CTLA-4), programmed death-1 (PD-1) with its ligand PD-L1 and others.1 The function of these immune check-points is to prevent the damage resulting from an excessive activation of the immune response in the setting of chronic antigenic stimulation, thus leading to autoimmune phenomena, as proved in knock-out mice models. PD-1 is normally present on activated T lymphocytes membrane, acting as a negative costimulatory receptor. PD-L1 is constitutively expressed at low levels by resting lymphocytes, antigen presenting cells and certain immunologically privileged tissues like placenta and testis. PD-L1 expression can be induced as well. In an inflammatory/infective context when T cells recognize antigens expressed by MHC-complex they start to produce inflammatory cytokines. The resulting inflammation leads to the expression of PD-L1 by hematopoietic, epithelial and endothelial cells, activating PD-1 on the surface of T-cells and therefore blocking the immune response. Previous studies have found out that PD-1 is highly expressed on T-reg cells and their binding with PD-L1 enhances suppressor T-reg functions2,3; the activation of PD-1/PD-L1 pathway reduces the lytic capacity of NK cells and B cell antibody production. In solid neoplasms PD-L1 expression by cancer cells and persistent up-regulation of PD-1 by tumourinfiltrating lymphocytes is common45. All these findings brought to the development of check-point inhibitors in the contest of solid tumors and lymphoproliferative neoplasms such as lymphoma and myeloma where the immune checkpoint blockade treatment have shown efficacy in refractory/relapsed neoplasms6. Few investigations only have been conducted on the role of PD1/PD-L1 in myeloid neoplasms, such as acute myeloid leukemia, a haematological cancer characterised by high-risk of relapse and poor prognosis. In leukemia, the bone marrow serves as a sanctuary for neoplastic cells, these cells interact with the tumour microenvironment (TME), constituted by stromal cells, endothelial cells and immune cells. The marked activation of the PD-1/PD-L1 pathway contributes to the maintenance of an immunosuppressive microenvironment. In fact, blasts are able, through the production of immunoinhibitory factors, to suppress the function of immunosurveillance and immuno-elimination of the tumor by the effector T cells. The effector T cells are "exhausted" in their capacity to secrete granzyme B, perforine and interferon gamma, and there is an upregulation of the T-reg functions, in addition to the presence of myeloid-derived suppressor cells7. PD-L1 expression and his link with PD-1 on activated lymphocytes results in an impaired antitumoral activity in murine models8. Zhang et al. investigated the role of PD-1/PD-L1 engagement in murine AML showing that PD-1-/-mice generated augmented antitumoral response in comparison with wild type mice. Similar results were obtained using anti-PD-L1 antibodies.9 A study from Zhou et al. reported how the function of adoptively transferred AML-reactive CTLs was reduced by AML-associated Tregs and how Treg depletion followed by PD-1/PD-L1 blockade showed efficacy for AML eradication in murine models.10 Objective of the study. To assess the presence of PD-1 and PD-L1 positive cells, by immunohistochemistry, in bone marrow biopsies of patients with AML. Material and methods. Four micron thickness sections were obtained from the formalin-fixed paraffin-embedded specimens. Haematoxylin-eosin staining was performed to assess the morphologic features of bone marrow. Immunohistochemical stainings were performed using a Ventana Benchmark Ultra automated staining instrument according to the manufacturer’s recommendations, using anti-PD-1 (clone NAT105, Ventana) and anti-PD-L1 (clone 22C3, Dako) antibodies. Results. We obtained 34 bone marrow trephine biopsies from newly diagnosed AML patients, 17 males and 17 females with a 67.3 mean age. We used 10 healthy bone marrow specimens as normal controls. None out of 10 control bone marrows resulted positive for either PD-1 or PD-L1 expressing cells as expected. Eleven out of 34 AML bone marrows (32,4%) showed at least 1% of PD-L1 positive cells (fig.2 b,d,f), while 6 AML bone marrow samples (17,6%) were positive for at least 1% of PD-1+ cells (fig. 1 b). Discussion. Despite the presence of relevant preclinical data regarding the role of immune check-points, few studies to evaluate the PD1/PDL1 axis have been conducted in myeloid neoplasm. Jia et al. performed flow cytometry analysis on PBMCs and BMMCs of 22 newly diagnosed AML patients and observed a significantly increased frequency of PD-1 expressing CD8 T cells in bone marrow compared to peripheral blood, suggesting a more exhausted status of these cells in relation to the suppressivePaper environment11. Dail et al. measured PD-L1 expression in 7 AML patients using immunohistochemistry and flow cytometry and found that PD-L1 was detectable (>2% cells) in all patients.12 Yang et al. assessed 45 bone marrow biopsies of MDS, CMML and AML patients and found that leukemic blasts of 9 patients (20%) were PD-L1+, while 3 (7%) were positive for PD-1. All 4 controls tested were negative for both PD-1 and PD-L1.13 Daver et al. measured PD-1 expression on bone marrow aspirates of 74 AML patients using flow cytometry. The results showed higher PD-1 expression compared to healthy controls (n=8).14Acceped To our this is the largest study evaluating by immunohistochemistry the expression of PD-1 and PDL1 in AML bone marrows and shows a significative positivity of the activation of the PD-1/PD-L1 pathway which gives a rationale to further studies regarding the charateristics of the cells involved in the PD-1/PD-L1 pathway and the immunosuppressive microenvironment. An implementation of the PD-1/PD-L1 pathway evaluation in clinical setting could have prognostic significance since the expression of PD-L1 by AML blasts has been associated with poor-risk and intermediate-risk AML15, furthermore immune checkpoint inhibitors have shown promising results in maintenance treatment of high-risk AML16, underlining not only a prognostic but also therapeutic value of the PD1/PD-L1 evaluation

Solitary Fibrous Tumor of the Lower Leg: A Rare and Difficult Diagnosis

Solitary fibrous tumor (SFT) is a rare neoplasm that commonly originates in the pleura. Extrapleural locations are rare and for this reason sometimes difficult to diagnose. Malignant forms with local recurrence or distant metastases have been reported, also as a consequence of inappropriate treatment. In this article, we report the case of an SFT of the lower leg in a 37-year-old man. Leg SFT is a rare occurrence, and differential diagnosis may be difficult because they can mimic a variety of benign and malignant mesenchymal tumors; immunohistochemical analysis for CD34, CD99, vimentin, and Bcl-2 is necessary. Misdiagnosis carries a significant risk of inadequate removal with subsequent increased risk of recurrence and distant metastases

Frequency and clinical aspects of neurological and psychiatric symptoms in patients with non-celiac wheat sensitivity

Background: Non-Celiac Wheat Sensitivity (NCWS) is characterized by both intestinal and extra-intestinal symptoms. The study aims to investigate the frequency of neuropsychiatric manifestations in NCWS patients and identify their clinical and demographic characteristics. Methods: 278 clinical records of NCWS patients, diagnosed by a double-blind placebo-controlled wheat challenge between 2006 and 2020, were retrospectively revised. Fifty-two patients with Celiac Disease (CD) and 54 patients with Irritable Bowel Syndrome (IBS) served as controls. Results: 87% of the NCWS patients had an IBS-like clinical presentation. The NCWS group showed a longer duration of symptoms, a higher frequency of positive serum anti-nuclear antibodies than CD and IBS patients, and a higher frequency of DQ2/DQ8 haplotypes and duodenal mucosa lymphocytosis than IBS controls. In addition, 50% of NCWS patients showed neuropsychiatric manifestations, while lower percentages were observed in CD (25%) and IBS (28%) controls. Neuropsychiatric symptoms in NCWS were more frequently associated with the male sex, longer duration of symptoms, and IBS-diarrhea-like clinical presentation. Conclusions: Our data suggest that in patients with IBS-like symptoms and neuropsychiatric manifestations of unknown cause, it could be useful to investigate a correlation of these symptoms with wheat ingestion to identify NCWS patients with this ‘atypical’ manifestation

Persistence of Nonceliac Wheat Sensitivity, Based on Long-term Follow-up

We investigated how many patients with a diagnosis of nonceliac wheat sensitivity (NCWS) still experienced wheat sensitivity after a median follow-up time of 99 months. We collected data from 200 participants from a previous study of NCWS, performed between July and December 2016 in Italy; 148 of these individuals were still on a strict wheat- free diet. In total, 175 patients (88%) improved (had fewer symptoms) after a diagnosis of NCWS; 145 of 148 patients who adhered strictly to a gluten-free diet (98%) had reduced symptoms, compared with 30 of 52 patients who did not adhere to a gluten-free diet (58%) (P < .0001). Of the 22 patients who repeated the double-blind, placebo- controlled challenge, 20 reacted to wheat. We conclude that NCWS is a persistent condition. Clinicaltrials.gov registration number: NCT02823522

Anti-Transglutaminase Antibody Assay of the Culture Medium of Intestinal Biopsy Specimen Can Improve the Accuracy of Celiac Disease Diagnosis.

BACKGROUND: We measured anti-transglutaminase (anti-tTG) antibody in the culture medium of intestinal biopsy specimens from patients with suspected celiac disease (CD) and evaluated the relationship between antibody production and severity of intestinal mucosal damage. METHODS: We performed diagnostic testing for CD on 273 consecutive patients. In addition to routine histologic evaluation of duodenal biopsy specimens, we assayed anti-tTG antibodies in serum and in the culture medium of duodenal biopsy specimens. RESULTS: CD was diagnosed in 191 of the 273 patients. Sensitivity and specificity of the serum anti-endomysium (EmA) and anti-tTG assays were 83% and 85% and 99% and 95%, respectively, and both had 88% diagnostic accuracy. EmA and anti-tTG assayed in the culture medium had 98% sensitivity, 100% specificity, and 98% diagnostic accuracy (vs serum assays; P <0.0001). Twenty-nine CD patient specimens (16%) were negative for serum anti-tTG and EmA; for 24 of these patients, anti-tTG assay of the culture medium was positive. The CD patients whose biopsy specimens were positive for serum antibodies showed the following intestinal histologies: total villous atrophy, 35%; severe villous atrophy, 25%; mild atrophy, 25%; villi with no atrophy but with increased intraepithelial lymphocytes, 15%. None of the CD patients whose specimens were negative for serum antibodies showed total or severe villous atrophy; 77% had mild villous atrophy, and 23% had no villous atrophy but had increased intraepithelial lymphocyte counts. Mild villous atrophy was also seen in specimens from approximately 15% of patients without CD. CONCLUSION: Anti-tTG assay of the culture medium of biopsy specimens can improve the accuracy of CD diagnosis in patients negative for serum antibodies

Malignant epithelial/exocrine tumors of the pancreas

Pancreatic malignant exocrine tumors represent the most important cause of cancerrelated death for pancreatic neoplasms. The most common tumor type in this category is represented by pancreatic ductal adenocarcinoma (PDAC), an ill defined, stroma-rich, scirrhous neoplasm with glandular differentiation. Here we present the relevant characteristics of the most important PDAC variants, namely adenosquamous carcinoma, colloid carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, signet ring carcinoma, medullary carcinoma and hepatoid carcinoma. The other categories of malignant exocrine tumors, characterized by fleshy, stroma-poor, circumscribed neoplasms, include acinar cell carcinoma (pure and mixed), pancreatoblastoma, and solid pseudopapillary neoplasms. The most important macroscopic, histologic, immunohistochemical and molecular hallmarks of all these tumors, highlighting their key diagnostic/pathological features are presented. Lastly, standardized indications regarding gross sampling and how to compile a formal pathology report for pancreatic malignant exocrine tumors will be provided

Reproducibility of the WHO histological criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms.

This study, performed on behalf of the Italian Registry of Thrombocythaemias (Registro Italiano Trombocitemie), aimed to test the inter-observer reproducibility of the histological parameters proposed by the WHO classification for the diagnosis of the Philadelphia chromosome-negative myeloproliferative neoplasms. A series of 103 bone marrow biopsy samples of Philadelphia chromosome-negative myeloproliferative neoplasms consecutively collected in 2004 were classified according to the WHO criteria as follows: essential thrombocythaemia (n=34), primary myelofibrosis (n=44) and polycythaemia vera (n=25). Two independent groups of pathologists reviewed the bone marrow biopsies. The first group was asked to reach a collegial 'consensus' diagnosis. The second group reviewed individually all the cases to recognize the main morphological parameters indicated by the WHO classification and report their results in a database. They were subsequently instructed to individually build a 'personal' diagnosis of myeloproliferative neoplasms subtype just assembling the parameters collected in the database. Our results indicate that high levels of agreement ( 6570%) have been reached for about all of the morphological features. Moreover, among the 18 evaluated histological features, 11 resulted statistically more useful for the differential diagnosis among the different Philadelphia chromosome-negative myeloproliferative neoplasms. Finally, we found a high percentage of agreement (76%) between the 'personal' and 'consensus' diagnosis (Cohen's kappa statistic >0.40). In conclusion, our results support the use of the histological criteria proposed by the WHO classification for the Philadelphia chromosome-negative myeloproliferative neoplasms to ensure a more precise and early diagnosis for these patients

The chaperone system in glioblastoma multiforme and derived cell lines: diagnostic and mechanistic implications

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Novel treatments are needed to counteract the molecular mechanisms of GBM growth and drug resistance. The chaperone system (CS) members are typically cytoprotective but some, termed Hsp, can become pathogenic and participate in carcinogenesis, along with the vascular endothelial growth factor (VEGF), and we investigated them in GBM biopsies and derived cell lines. The objectives were to identify diagnostic-prognostic biomarkers and gather information for developing chaperonotherapy. METHODS: Cell lines from GBMs were established, characterized (morphology, growth characteristics, and specific markers), and stored. Chaperones and angiogenic factors [Hsp10, Hsp27, Hsp60, Hsp70, Hsp90, FLT-1 (VEGFR-1), FLK1 (KDR, VEGFR-2), and FLT-4 (VEGFR-3)] were observed in cells by immunofluorescence while the chaperones were measured in tumor tissue by immunohistochemistry. RESULTS: Four cell lines were derived from four different GBMs; the cells were spindle shaped or polygonal and grew at high rates as adherent monolayers or clusters without evidence of contact inhibition. The astrocyte-specific glial fibrillary acidic protein (GFAP); and the neuronal NSE, malignancy VIM, and proliferation PCNA, markers were determined. The cells expressed GFAP but no NSE, indicating that they were primary glioblastoma cell lines, with high levels of Hsp10, Hsp27, Hsp60, Hsp90, and Flk1; and low levels of Hsp70, Flt1, and Flt4. CONCLUSIONS: Four cell lines were established derived from four out of ten GBM tumors studied. The cell lines showed intense positivity for chaperones studied and factors connected to malignancy and the tumors showed increased levels of chaperones, making them potential diagnostic-prognostic biomarkers and targets for anti-cancer compounds