Thomas Krausz

Intratumor depletion of CD4+ cells unmasks tumor immunogenicity leading to the rejection of late-stage tumors

Tumor environment can be critical for preventing the immunological destruction of antigenic tumors. We have observed a selective accumulation of CD4+CD25+ T cells inside tumors. In a murine fibrosarcoma Ld-expressing Ag104, these cells made up the majority of tumor-infiltrating lymphocytes at the late stage of tumor progression, and their depletion during the effector phase, rather than priming phase, successfully enhanced antitumor immunity. We show here that CD4+CD25+ T cells suppressed the proliferation and interferon-γ production of CD8+ T cells in vivo at the local tumor site. Blockade of the effects of IL-10 and TGF-β partially reversed the suppression imposed by the CD4+ cells. Furthermore, local depletion of CD4+ cells inside the tumor resulted in a change of cytokine milieu and led to the eradication of well-established highly aggressive tumors and the development of long-term antitumor memory. Therefore, CD4+CD25+ T cells maintained an environment in the tumor that concealed the immunogenicity of tumor cells to permit progressive growth of antigenic tumors. Our study illustrates that the suppression of antitumor immunity by regulatory T cells occurs predominantly at the tumor site, and that local reversal of suppression, even at a late stage of tumor development, can be an effective treatment for well-established cancers.

HPV testing in primary screening of older women.

SummaryCertain types of the human papilloma virus (HPV) are well established as the primary cause of cervical cancer. Several studies have shown that HPV testing can improve the detection rate of high-grade cervical intraepithelial neoplasia (CIN), but these have been carried out primarily in younger women. In this study we evaluated the role of HPV testing as an adjunct to cytology in women aged 35 or over. An additional aim was to evaluate commercially available kits for HPV testing. A total of 2988 eligible women aged 34 or more attending for a routine smear in 40 general practitioner practices received HPV testing in addition to routine cytology, after having given written informed consent. Samples were assayed by polymerase chain reaction (PCR) and two versions of the Hybrid Capture test for HPV, and women were invited for colposcopy if there was any cytological abnormality (including borderline smears) or the PCR test was positive. Any apparent abnormality was biopsied and loop-excision was performed as necessary. CIN was judged by histology; 42 women had high-grade CIN, of which six were cytology negative (86% sensitivity for borderline or worse) and three had a borderline smear (79% sensitivity for mild dyskaryosis or worse). The positive predictive value of a borderline smear was only 3.1%. Eleven high-grade lesions were negative by the PCR HPV test (sensitivity 74%). The first generation Hybrid Capture II test had a similar sensitivity but an unacceptably high false positive rate (18.3%), while the newer Hybrid Capture II microtitre kit had a 95% sensitivity and a 2.3% positivity rate in normal women when used at a 2 pg ml–1 cut-off (positive predictive value 27%). Cytology performed very well in this older cohort of women. The newer Hybrid Capture II microtitre test may be a useful adjunct, especially if the results reported here are reproducible in other studies. A combined screening test offers the possibility of greater protection and/or longer screening intervals, which could reduce the overall cost of the screening programme.

Guidelines for pathologic diagnosis of Malignant Mesothelioma: 2017 Update of the consensus statement from the International Mesothelioma Interest Group

CONTEXT - Malignant mesothelioma (MM) is an uncommon tumor that can be difficult to diagnose. OBJECTIVE - To provide updated, practical guidelines for the pathologic diagnosis of MM. DATA SOURCES - Pathologists involved in the International Mesothelioma Interest Group and others with an interest and expertise in the field contributed to this update. Reference material included up-to-date, peer-reviewed publications and textbooks. CONCLUSIONS - There was discussion and consensus opinion regarding guidelines for (1) distinguishing benign from malignant mesothelial proliferations (both epithelioid and spindle cell lesions), (2) cytologic diagnosis of MM, (3) recognition of the key histologic features of pleural and peritoneal MM, (4) use of histochemical and immunohistochemical stains in the diagnosis and differential diagnosis of MM, (5) differentiating epithelioid MM from various carcinomas (lung, breast, ovarian, and colonic adenocarcinomas, and squamous cell and renal cell carcinomas), (6) diagnosis of sarcomatoid MM, (7) use of molecular markers in the diagnosis of MM, (8) electron microscopy in the diagnosis of MM, and (9) some caveats and pitfalls in the diagnosis of MM. Immunohistochemical panels are integral to the diagnosis of MM, but the exact makeup of panels employed is dependent on the differential diagnosis and on the antibodies available in a given laboratory. Depending on the morphology, immunohistochemical panels should contain both positive and negative markers for mesothelial differentiation and for lesions considered in the differential diagnosis. Immunohistochemical markers should have either sensitivity or specificity greater than 80% for the lesions in question. Interpretation of positivity generally should take into account the localization of the stain (eg, nuclear versus cytoplasmic) and the percentage of cells staining (>10% is suggested for cytoplasmic and membranous markers). Selected molecular markers are now being used to distinguish benign from malignant mesothelial proliferations. These guidelines are meant to be a practical diagnostic reference for the pathologist; however, some new pathologic predictors of prognosis and response to therapy are also included.

Glucose Utilization In Vivo by Human Pulmonary Neoplasms

Neoplastic tissue in general shows a high rate of glucose consumption under both anaerobic and aerobic conditions. Using positron emission tomography (PET) we measured the rate of uptake of the glucose analogue 18fluoro‐2‐deoxy‐D‐glucose (18FDG) in 12 patients with carcinoma of the lung. The tumor types were six squamous cell, two large cell, two oat cell, one adenocarcinoma, and one undifferentiated carcinoma. In each patient a transaxial plane was selected that contained the bulk of the tumor tissue. Regional density and blood volume were measured. Following the intravenous injection of 18FDG, the rates of uptake in the tumor and normal lung tissue were assessed from sequential scans over 1 hour. In each patient the rate of uptake of 18FDG in the tumor tissue was significantly increased relative to normal lung tissue. For the group the rate of uptake by the tumor was 211.4 ± 69.4 ml/100 g/hr (mean ± SD) compared to 31.9 ± 13.2 in the contralataral lung (P < 0.05). The tumor‐to‐normal tissue ratio of 6.6 (range, 2.7 to 14.6) was higher than previously reported ratios for brain and liver tumors. In contrast to brain tumors there was little correlation between tumor type and rate of 18FDG uptake. Measurements of glucose metabolism taken in vivo in human pulmonary tumors may lead to advances in screening, staging, and therapy.

Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues

Staphylococcus aureus infections are associated with abscess formation and bacterial persistence; however, the genes that enable this lifestyle are not known. We show here that following intravenous infection of mice, S. aureus disseminates rapidly into organ tissues and elicits abscess lesions that develop over weeks but cannot be cleared by the host. Staphylococci grow as communities at the center of abscess lesions and are enclosed by pseudocapsules, separating the pathogen from immune cells. By testing insertional variants in genes for cell wall‐anchored surface proteins, we are able to infer the stage at which these molecules function. Fibrinogen‐binding proteins ClfA and ClfB are required during the early phase of staphylococcal dissemination. The heme scavenging factors IsdA and IsdB, as well as SdrD and protein A, are necessary for abscess formation. Envelope‐associated proteins, Emp and Eap, are either required for abscess formation or contribute to persistence. Fluorescence microscopy revealed Eap deposition within the pseudocapsule, whereas Emp was localized within staphylococcal abscess communities. Antibodies directed against envelope‐associated proteins generated vaccine protection against staphylococcal abscess formation. Thus, staphylococci employ envelope proteins at discrete stages of a developmental program that enables abscess formation and bacterial persistence in host tissues.—Cheng, A. G., Kim, H. K., Burts, M. L., Krausz, T., Schneewind, O., Missiakas, D. M. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. FASEB J. 23, 3393–3404 (2009). www.fasebj.org

Guidelines for pathologic diagnosis of malignant mesothelioma: A consensus statement from the International Mesothelioma Interest Group

CONTEXT Malignant mesothelioma (MM) is an uncommon tumor that can be difficult to diagnose. OBJECTIVE To develop practical guidelines for the pathologic diagnosis of MM. DATA SOURCES A pathology panel was convened at the International Mesothelioma Interest Group biennial meeting (October 2006). Pathologists with an interest in the field also contributed after the meeting. CONCLUSIONS There was consensus opinion regarding (1) distinguishing benign from malignant mesothelial proliferations (both epithelioid and spindle cell lesions), (2) cytologic diagnosis of MM, (3) key histologic features of pleural and peritoneal MM, (4) use of histochemical and immunohistochemical stains in the diagnosis and differential diagnosis of MM, (5) differentiating epithelioid MM from various carcinomas (lung, breast, ovarian, and colonic adenocarcinomas and squamous cell and renal cell carcinomas), (6) diagnosis of sarcomatoid mesothelioma, (7) use of molecular markers in the differential diagnosis of MM, (8) electron microscopy in the diagnosis of MM, and (9) some caveats and pitfalls in the diagnosis of MM. Immunohistochemical panels are integral to the diagnosis of MM, but the exact makeup of panels used is dependent on the differential diagnosis and on the antibodies available in a given laboratory. Immunohistochemical panels should contain both positive and negative markers. The International Mesothelioma Interest Group recommends that markers have either sensitivity or specificity greater than 80% for the lesions in question. Interpretation of positivity generally should take into account the localization of the stain (eg, nuclear versus cytoplasmic) and the percentage of cells staining (>10% is suggested for cytoplasmic membranous markers). These guidelines are meant to be a practical reference for the pathologist.

BAP1 and cancer.

BAP1 is a deubiquitylase that is found associated with multiprotein complexes that regulate key cellular pathways, including the cell cycle, cellular differentiation, cell death, gluconeogenesis and the DNA damage response (DDR). Recent findings indicate that germline BAP1 mutations cause a novel cancer syndrome that is characterized, at least in the affected families that have been studied so far, by the onset at an early age of benign melanocytic skin tumours with mutated BAP1, and later in life by a high incidence of mesothelioma, uveal melanoma, cutaneous melanoma and possibly additional cancers.

In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumours

Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS/RAF/MEK/ERK/MAP‐kinase pathway. Activating mutations in BRAF have recently been identified in melanomas, colorectal cancers, and thyroid and ovarian tumours. In the present study, an extensive characterization of BRAF and KRAS mutations has been performed in 264 epithelial and non‐epithelial ovarian neoplasms. The epithelial tumours ranged from adenomas and borderline neoplasms to invasive carcinomas including serous, mucinous, clear cell, and endometrioid lesions. It is shown that BRAF mutations in ovarian tumours occur exclusively in low‐grade serous neoplasms (33 of 91, 36%); these included serous borderline tumours (typical and micropapillary variants), an invasive micropapillary carcinoma and a psammocarcinoma. KRAS mutations were identified in 26 of 91 (29.5%) low‐grade serous tumours, 7 of 49 (12%) high‐grade serous carcinomas, 2 of 6 mucinous adenomas, 22 of 28 mucinous borderline tumours, and 10 of 18 mucinous carcinomas. Of note, two serous borderline tumours were found to harbour both BRAF and KRAS mutations. The finding that at least 60% of serous borderline tumours harbour mutations in two members of the ERK‐MAP‐kinase pathway (BRAF 36%, KRAS 30%) compared with 12% of high‐grade serous carcinomas (BRAF 0%, KRAS 12%) indicates that the majority of serous borderline tumours do not progress to serous carcinomas. Furthermore, no BRAF mutations were detected in the other 173 ovarian tumours in this study. Copyright

Expression and mutational analysis of MET in human solid cancers

MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) regulate a variety of cellular functions, many of which can be dysregulated in human cancers. Activated MET signaling can lead to cell motility and scattering, angiogenesis, proliferation, branching morphogenesis, invasion, and eventual metastasis. We performed systematic analysis of the expression of the MET receptor and its ligand HGF in tumor tissue microarrays (TMA) from human solid cancers. Standard immunohistochemistry (IHC) and a computerized automated scoring system were used. DNA sequencing for MET mutations in both nonkinase and kinase domains was also performed. MET was differentially overexpressed in human solid cancers. The ligand HGF was widely expressed in both tumors, primarily intratumoral, and nonmalignant tissues. The MET/HGF likely is functional and may be activated in autocrine fashion in vivo. MET and stem cell factor (SCF) were found to be positively stained in the bronchioalevolar junctions of lung tumors. A number of novel mutations of MET were identified, particularly in the extracellular semaphorin domain and the juxtamembrane domain. MET‐HGF pathway can be assayed in TMAs and is often overexpressed in a wide variety of human solid cancers. MET can be activated through overexpression, mutation, or autocrine signaling in malignant cells. Mutations in the nonkinase regions of MET might play an important role in tumorigenesis and tumor progression. MET would be an important therapeutic antitumor target to be inhibited, and in lung cancer, MET may represent a cancer early progenitor cell marker.

Formation of endothelin by cultured airway epithelial cells.

Immunoreactivity to endothelin was detected in conditioned culture medium from both canine and porcine tracheal epithelial cells. Gel permeation chromatography and fast protein liquid chromatography were used to confirm the identity of the endothelin. The two peaks demonstrated on fast protein liquid chromatography co‐eluted with endothelin 1 and endothelin 3 respectively.