Tissue contexture determines the pattern and density of tumor-infiltrating immune cells in HPV-associated squamous cell carcinomas of oropharynx and uterine cervix.

The profile of the antitumor immune response is an important factor determining patient clinical outcome. However, the influence of the tissue contexture on the composition of the tumor microenvironments of virally induced tumors is not clearly understood. Therefore, we analyzed the immune landscape of two HPV-associated malignancies: oropharyngeal squamous cell carcinoma (OPSCC) and squamous cell carcinoma of uterine cervix (CESC). We employed multiplex immunohistochemistry and immunofluorescence to evaluate the density and spatial distribution of immune cells in retrospective cohorts of OPSCC and CESC patients. This approach was complemented by transcriptomic analysis of purified primary tumor cells and in silico analysis of publicly available RNA sequencing data. Transcriptomic analysis showed similar immune profiles in OPSCC and CESC samples. Interestingly, immunostaining of OPSCC tissues revealed high densities of immune cells in both tumor stroma and tumor epithelium, whereas CESC samples were mainly characterized by the lack of immune cells in the tumor epithelium. However, in contrast to other immune cell populations, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were abundant in both segments of CESC samples and CESC-derived tumor cells expressed markedly higher levels of the PMN-MDSC chemoattractants CXCL1, CXCL5, and CXCL6 than OPSCC tumor cells. Taken together, despite their having the same etiologic agent, the immune infiltration pattern significantly differs between OPSCC and CESC, with a noticeable shift toward prominent MDSC infiltration in the latter. Our data thus present a rationale for a diverse approach to targeted therapy in patients with HPV-associated tumors of different tissue origins.

Non-conventional mucosal lesions (serrated epithelial change, villous hypermucinous change) are frequent in patients with inflammatory bowel disease-results of molecular and immunohistochemical single institutional study.

Chronically inflamed mucosa in inflammatory bowel disease (IBD) is associated with an increased risk of cancer. Besides IBD-associated dysplasia, there are non-conventional mucosal changes that may act as potential precursors. The aim of the study was to retrospectively review samples from IBD patients focusing on detection of such lesions with evaluation of their immunohistochemical and molecular properties. Surgical specimens and/or endoscopical biopsy samples of IBD patients examined during a 10-year period were reviewed. Detected mucosal lesions were divided into three groups-group 1 (non-conventional or putative precursor lesions - PPLs) with serrated or villous hypermucinous morphology, group 2 (true serrated polyps fulfilling valid criteria), and group 3 (IBD-associated neoplasia). Lesions from all groups were analyzed with antibodies against MLH1, p53, and MGMT and by molecular testing of KRAS, NRAS, and BRAF mutation. Samples from 309 IBD patients were reviewed. A total of 88 mucosal lesions were found in 51 patients. Most common were lesions from group 1 with superficial serrated epithelial change seen in 41 samples (46.6%) and villous hypermucinous change in 6 (6.8%). Group 2 consisted of 15 true serrated polyps. Six conventional IBD-dysplasia cases and 11 carcinomas were seen in group 3. Six lesions from group 1 were associated with invasive carcinoma whereas two shared the same mutation in KRAS or BRAF. Lesions from group 1 were characterized by loss of MGMT expression in 44.6%, aberrant p53 expression, and by mutations in KRAS gene in 42.9% of cases. This study proves the existence of mucosal changes other than conventional IBD-dysplasia and extends the knowledge about their immunohistochemical and molecular properties and relation to carcinoma further supporting their potential role in IBD-related carcinogenesis.

Tumor-infiltrating B cells affect the progression of oropharyngeal squamous cell carcinoma via cell-to-cell interactions with CD8+ T cells.

BACKGROUND: Standard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with high morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients. Therefore, a better stratification of patients and the development of novel therapeutic protocols are crucially needed. The importance of tumor-infiltrating B cells (TIL-Bs) in shaping antitumor immunity remains unclear; therefore, we analyzed frequency, phenotype, prognostic value and possible roles of TIL-Bs in OPSCC. METHODS: We utilized transcriptomic analysis of immune response-related genes in 18 OPSCC samples with respect to human papillomavirus (HPV) status. The density and localization of CD20+, CD8+ and DC-LAMP+ cells were subsequently analyzed in 72 tissue sections of primary OPSCC samples in relation to patients' prognosis. The immunohistochemical approach was supplemented by flow cytometry-based analysis of phenotype and functionality of TIL-Bs in freshly resected primary OPSCC tissues. RESULTS: We observed significantly higher expression of B cell-related genes and higher densities of CD20+ B cells in HPV-associated OPSCC samples. Interestingly, CD20+ TIL-Bs and CD8+ T cells formed non-organized aggregates with interacting cells within the tumor tissue. The densities of both intraepithelial CD20+ B cells and B cell/CD8+ T cell interactions showed prognostic significance, which surpassed HPV positivity and CD8+ TIL density in stratification of OPSCC patients. High density of TIL-Bs was associated with an activated B cell phenotype, high CXCL9 production and high levels of tumor-infiltrating CD8+ T cells. Importantly, the abundance of direct B cell/CD8+ T cell interactions positively correlated with the frequency of HPV16-specific CD8+ T cells, whereas the absence of B cells in tumor-derived cell cultures markedly reduced CD8+ T cell survival. CONCLUSIONS: Our results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8+ T cell interactions can predict patients with excellent prognosis, who would benefit from less invasive treatment. We propose that in extensively infiltrated tumors, TIL-Bs might recruit CD8+ T cells via CXCL9 and due to a highly activated phenotype contribute by secondary costimulation to the maintenance of CD8+ T cells in the tumor microenvironment.

Morphological, immunohistochemical and molecular features of inflammatory bowel disease associated colorectal carcinoma and associated mucosal lesions - Single institution experience.

BACKGROUND: Patients with inflammatory bowel disease (IBD) - ulcerative colitis (UC) and Crohn's disease (CD) have an elevated risk of developing colorectal carcinoma (CRC). Major risk factor in IBD patients is the continuous chronic inflammation leading to development of dysplasia and carcinoma. Nevertheless, other types of non-conventional but suspicious mucosal changes serrated change/dysplasia, NOS and villous hypermucinous change, have also been reported in IBD patients. Preneoplastic potential of these lesions is still not well elucidated. AIMS: The aim of this study was identification of IBD-associated CRCs focusing on finding related precursor lesions in the surgical specimen or in archival biopsy samples followed by a detailed morphological, immunohistochemical and molecular evaluation. For the purpose of the study the mucosal lesions were divided into conventional IBD-associated dysplasia and non-conventional lesions that were merged under a provisory term of putative preneoplastic lesions (PPL). METHODS: A total of 309 consecutive IBD colectomy specimens diagnosed during a 10-year period were reviewed. Detailed morphological evaluation, immunohistochemical analysis of mismatch repair (MMR) proteins, p53 and O6-methylguanine DNA methyltransferase (MGMT) expression and molecular analysis for KRAS, NRAS and BRAF gene mutation were performed in the retrieved CRC cases as well as in the detected dysplasia and PPLs of these patients. RESULTS: We identified 11 cases of morphologically heterogenous IBD-associated CRCs, occurring in 5 males and 6 females, aged 26-79 years (mean 44 years). A total of 22 mucosal lesions were revealed in 8 CRC patients comprising conventional IBD-associated dysplasia (4 lesions), PPLs as serrated change/dysplasia NOS (11 lesions), villous hypermucinous change (5 lesions), and two true serrated lesions (one sessile serrated adenoma and one traditional serrated adenoma). More than one type of lesion was found in 6 patients. Seven CRC cases harbored mutation of KRAS/NRAS and one case of BRAF. Two patients with KRAS-mutated CRC showed the same mutation in PPL in the same specimen (one serrated change NOS and one TSA with high-grade dysplasia). Similarly, one BRAF-mutated carcinoma case presented the same mutation in serrated change/dysplasia, NOS in the same specimen. Of the CRCs, two showed deficient MMR system profile, six presented with loss of MGMT expression, and six showed aberrant p53 expression. PPLs showed deficient MGMT expression (14 cases) and aberrant p53 (10 cases) as well. CONCLUSION: IBD-associated CRCs are very heterogeneous entities. Besides conventional IBD-related dysplasia, other types of mucosal lesions may be associated with long lasting IBD and CRC e.g. villous hypermucinous change and serrated change/dysplasia, NOS. Since these lesions share certain genetic or immunohistochemical changes with the related CRC, a suspicion is raised that these lesions may also have preneoplastic potential. Awareness of these changes is necessary to prevent their missing and under-reporting, and further studies of these lesions should be carried out.

Cervical human papillomavirus infection in women with preterm prelabor rupture of membranes.

OBJECTIVE: To evaluate the association between cervical human papillomavirus (HPV) infection at the time of admission and the presence of microbial invasion of the amniotic cavity (MIAC) and intra-amniotic inflammation (IAI) in women with preterm prelabor rupture of membranes (PPROM) and to determine the association between cervical HPV infection and short-term neonatal morbidity. METHODS: One hundred women with singleton pregnancies complicated by PPROM between the gestational ages of 24+0 and 36+6 weeks were included in the study. The presence of HPV DNA was evaluated in scraped cervical cells using polymerase chain reaction (PCR). Amniotic fluid samples were obtained by transabdominal amniocentesis. RESULTS: The rate of cervical HPV infection in women with PPROM was 24%. The rates of MIAC and IAI were not different between women with cervical HPV infection and those without cervical HPV infection [MIAC: with HPV: 21% (5/24) vs. without HPV: 22% (17/76), p = 1.00; IAI: with HPV: 21% (5/24) vs. without HPV: 18% (14/76), p = 0.77]. There were no differences in the selected aspects of short-term neonatal morbidity between women with and without cervical HPV infection. CONCLUSIONS: In women with PPROM, the presence of cervical HPV infection at the time of admission is not related to a higher risk of intra-amniotic infection-related and inflammatory complications or worse short-term neonatal outcomes.