ABSTRACT
PURPOSE: The objective of this study was to determine the incidence, necessity for neurosurgical intervention, and overall results of the treatment of pediatric peripheral nerve injuries associated with dislocated supracondylar fractures of the distal humerus. METHOD: A retrospective analysis of pediatric patients with supracondylar fractures treated from April 2019 to April 2022 with a minimum follow-up of 3 months was conducted. RESULTS: Of 453 included patients, there were 51 recorded peripheral nerve injuries. The ulnar nerve was the most frequently injured nerve. Nine patients required neurosurgical intervention, with the most common procedure being the release of entrapped nerves. The combination of a supracondylar fracture and arterial injury was identified as a significant risk factor for peripheral nerve injury (p < 0.001). Only one patient experienced an unsatisfactory outcome. CONCLUSION: Although the prognosis for peripheral nerve injuries in children with supracondylar fractures is generally favorable, these injuries must be properly identified. We recommend an active neurosurgical approach in children with persisting neurological deficits to minimize the risk of permanent neurological impairment.
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OBJECTIVE: One of the possible risks of sinonasal malignancy is its possible spread in the orbit. However, there is no clear consensus among the different departments as to whether it is necessary to exenterate the orbit in limited tumorous infiltration of periorbital fat. The purpose of the study was to demonstrate that periorbital infiltration and periorbital fat invasion without involvement of deeper orbital tissues are not the indication of orbital exenteration. MATERIALS AND METHODS: Retrospective analysis was performed over a 17-year period of patients undergoing surgical treatment for sinonasal malignancy with histologically verified periorbital infiltration or deeper invasion into the orbit. A total of 32 patients were included in the study. For each group, the following data were analysed: sex, age, preoperative imaging studies, histological findings, site of origin, stage, surgical reconstruction, oncological treatment, survival, cause of death, number of recurrences in the orbit and functional status of preserved eyes. RESULTS: Based on our criteria for orbital exenteration, orbital preservation was feasible in 18 patients. Orbital exenteration was performed in 14 patients with deeper tumor infiltration. There was a statistically insignificant difference in survival between the two groups. The 5-year overall survival (OS) was 44% for the orbital preservation group (only 2 patients died from local tumor recurrence) and 34% for the orbital exenteration group. The groups did not differ in other observed factors other than the extent of orbital infiltration. In 11 (61.1%) patients, vision was without significant change after radiation therapy. In 2 (11.1%) patients, visual function was impaired due to diplopia. 5 (27.8%) patients had severely impaired vision due to optic nerve atrophy after radiation therapy. CONCLUSIONS: Our results show a relatively high survival rate in the group of patients with orbital preservation with a high chance of vision preservation, which justifies our approach to orbital preservation even in some tumors with periorbital infiltration.
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Plasmacytoid dendritic cells (pDCs) are the most potent type I interferon-producing cells and play an important role in antiviral immunity. Tumor-infiltrating pDCs were shown to be predominantly pro-tumorigenic, with reduced ability to produce interferon alpha (IFNα) and confirmed capacity to prime regulatory T cells (Tregs) by the ICOS/ICOS-L pathway. Because a significant number of HNSCCs are induced by human papillomaviruses and show markedly different immune profiles than non-virally induced tumors, we compared the phenotype and functional capacity of HNSCC-infiltrating pDCs to the HPV status of the tumor. We observed a reduced capacity of pDCs to produce IFNα upon toll-like receptor activation in HPV-negative samples and a rather uncompromised functionality in HPV-associated tumors. Additionally, supernatants from non-virally induced but not HPV-associated tumor cell suspensions significantly inhibited IFNα production by peripheral blood-derived pDCs. We identified IL-10 and TNFα as the soluble pDC-suppressive factors with the highest variability between HPV-negative and HPV-positive tumor-derived supernatants. Additionally, we observed a positive correlation of tumor-infiltrating pDCs with Tregs in HPV-negative samples but not in virally induced tumors. Overall, our study indicates that the immunosuppressive cytokine milieu rich in IL-10 and TNFα in HPV-negative but not in HPV-positive HNSCC significantly affects the functional capacity of tumor-infiltrating pDCs, and such dysfunctional pDCs may further support the immunosuppressive tumor microenvironment by promoting the expansion of Tregs in the tumor tissue.
Subject(s)
Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Papillomavirus Infections/complications , Papillomavirus Infections/virology , Squamous Cell Carcinoma of Head and Neck/etiology , Squamous Cell Carcinoma of Head and Neck/metabolism , Tumor Microenvironment , Biomarkers , Case-Control Studies , Cell Transformation, Viral , Dendritic Cells/pathology , Disease Susceptibility , Gene Expression , Humans , Interferon-alpha/immunology , Interferon-alpha/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Tumor Microenvironment/immunologyABSTRACT
Cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components of the tumor stroma. CAFs can impact many important steps of cancerogenesis and may also influence treatment resistance. Some of these effects need the direct contact of CAFs and cancer cells, while some involve paracrine signals. In this study, we investigated the ability of head and neck squamous cell carcinomas (HNSCC) patient-derived CAFs to promote or inhibit the colony-forming ability of HNSCC cells. The effect of cisplatin on this promoting or inhibiting influence was also studied. The subsequent analysis focused on changes in the expression of genes associated with cancer progression. We found that cisplatin response in model HNSCC cancer cells was modified by coculture with CAFs, was CAF-specific, and different patient-derived CAFs had a different "sensitizing ratio". Increased expression of VEGFA, PGE2S, COX2, EGFR, and NANOG in cancer cells was characteristic for the increase of resistance. On the other hand, CCL2 expression was associated with sensitizing effect. Significantly higher amounts of cisplatin were found in CAFs derived from patients who subsequently experienced a recurrence. In conclusion, our results showed that CAFs could promote and/or inhibit colony-forming capability and cisplatin resistance in HNSCC cells via paracrine effects and subsequent changes in gene expression of cancer-associated genes in cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/metabolism , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Head and Neck Neoplasms/metabolism , Squamous Cell Carcinoma of Head and Neck/metabolism , Aged , Aged, 80 and over , Cell Line, Tumor , Coculture Techniques , Female , Gene Expression Regulation, Neoplastic/drug effects , Head and Neck Neoplasms/pathology , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/pathology , Paracrine Communication/drug effects , Squamous Cell Carcinoma of Head and Neck/pathology , Tumor Stem Cell AssayABSTRACT
Symplastic haemangioma is a rare vascular tumor presented with regressive and degenerative atypia in stromal cells. Its morphology represents a challenge in classification of vascular tumors, regarding their biological behaviour in particular. We present a case report of a 47-years-old female with a history of left-sided breast adenocarcinoma treated by resection followed by adjuvant chemotherapy and radiotherapy. Three years after the primary diagnosis a tumorous mass appeared in the region of upper margin of left scapula, in subcutaneous tissues and the trapezius muscle. Histologically, the tumor was formed by multiple blood vessels of varied diameter and wall thickness. Endothelial lining was bland, without atypia; thromboses were observed in vascular spaces. In the interstitium, a population of spindle and pleomorphic cells with distinctive atypia and bizarre nuclei was found. These cells showed positivity in immunohistochemical expression of smooth muscle actin, further extensive immunohistochemistry including cytokeratines was negative. Mitoses were absent, proliferating activity was minimal. Signs of infiltrative growth pattern were not found and the tumor lacked hallmarks of malignant behaviour. A diagnosis of symplastic haemangioma was established. Above mentioned atypical stromal cells show myofibroblastic and sporadically smooth muscle differentiation. Their atypical appearence is associated with degenerative alterations similar to changes in leiomyomas with bizarre nuclei or ancient schwannomas. Etiopathogenesis of these changes is not clear, there are hypotheses considering long-lasting persistence of the lesion, regression of ischaemic or postinflammatory origin, or, like in our case, postirradiative degeneration. Differential diagnosis of symplastic haemangioma is widespred and contains many histological entities of variant histogenesis and biological potential. For proper classification, an extensive investigation including immunohistochemistry, clinical and anamnestic data and imaging methods is necessary.
Subject(s)
Hemangioma , Leiomyoma , Skin Neoplasms , Diagnosis, Differential , Female , Hemangioma/radiotherapy , Humans , Immunohistochemistry , Middle AgedABSTRACT
Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods--the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.
Subject(s)
Carcinoma, Squamous Cell/virology , Head and Neck Neoplasms/virology , Papillomavirus Infections/virology , Tonsillar Neoplasms/virology , Virus Integration/genetics , Blotting, Southern , Female , Head and Neck Neoplasms/mortality , Humans , Male , Papillomavirus Infections/complications , Prognosis , Proportional Hazards Models , Squamous Cell Carcinoma of Head and NeckABSTRACT
BACKGROUND: Better insights into the molecular changes involved in virus-associated and -independent head and neck cancer may advance our knowledge of HNC carcinogenesis and identify critical disease biomarkers. Here we aimed to characterize the expression profiles in a matched set of well-characterized HPV-dependent and HPV-independent tonsillar tumors and equivalent immortalized keratinocyte clones to define potential and clinically relevant biomarkers of HNC of different etiology. METHODS: Fresh frozen tonsillar cancer tissues were analyzed together with non-malignant tonsillar tissues and compared with cervical tumors and normal cervical tissues. Furthermore, relative miRNAs abundance levels of primary and immortalized human keratinocyte clones were evaluated. The global quantitation of miRNA gene abundance was performed using a TaqMan Low Density Array system. The confirmation of differentially expressed miRNAs was performed on a set of formalin-fixed paraffin-embedded tumor samples enriched for the tumor cell fraction by macrodissection. RESULTS: We defined 46 upregulated and 31 downregulated miRNAs characteristic for the HPV-positive tonsillar tumors and 42 upregulated miRNAs and 42 downregulated miRNAs characteristic for HPV-independent tumors. In comparison with the expression profiles in cervical tumors, we defined miR-141-3p, miR-15b-5p, miR-200a-3p, miR-302c-3p, and miR-9-5p as specific for HPV induced malignancies. MiR-335-5p, miR-579-3p, and miR-126-5p were shared by the expression profiles of HPV-positive tonsillar tumors and of the HPV immortalized keratinocyte clones, whereas miR-328-3p, miR-34c-3p, and miR-885-5p were shared by the miRNA profiles of HPV-negative tonsillar tumors and the HPV-negative keratinocytes. CONCLUSIONS: We identified the miRNAs characteristic for HPV-induced tumors and tonsillar tumors of different etiology, and the results were compared with those of the model system. Our report presents the basis for further investigations leading to the identification of clinically relevant diagnostic and/or therapeutic biomarkers for tumors of viral and non-viral etiology.
Subject(s)
Keratinocytes/cytology , MicroRNAs/genetics , Papillomavirus Infections/genetics , Tonsillar Neoplasms/genetics , Uterine Cervical Neoplasms/genetics , Cells, Cultured , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Keratinocytes/pathology , Oligonucleotide Array Sequence Analysis , Principal Component Analysis , Tonsillar Neoplasms/virology , Uterine Cervical Neoplasms/virologyABSTRACT
Soft tissue tumors (SSTs) constitute a broad spectrum of neoplasms with diverse biological properties. Rare or unusual types are often difficult to classify. Recent studies show, that a significant subset of SSTs including many types of sarcomas are associated with specific genetic changes such as chromosomal translocations producing chimeric genes, which play a role in the pathogenesis of SSTs. Because SSTs represent a diagnostically challenging group of tumors, molecular-genetic techniques (FISH or PCR) are useful as supplementary and/or confirmatory diagnostic tools. In the present paper we demonstrate the usefulness of a combined diagnostic approach using the tools of classical histopathology and immunohistochemistry together with the molecular diagnostic approach in selected nosologic entites.
Subject(s)
Pathology, Molecular/methods , Soft Tissue Neoplasms/diagnosis , Soft Tissue Neoplasms/genetics , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Polymerase Chain Reaction , Sarcoma/diagnosis , Sarcoma/geneticsABSTRACT
BACKGROUND: A proportion of head and neck carcinomas (HNSCCs) are induced by high-risk human papillomaviruses (HPVs) and are associated with better patient outcomes compared to patients with HNSCCs related to tobacco and alcohol abuse. In the microenvironment of solid tumors, including HNSCCs, oxygen levels are often reduced, and a hypoxic state is induced. This can lead to a poor treatment response and a worse patient prognosis. One of the hypoxia-responsive genes is aspartate-ß-hydroxylase (ASPH), whose activity promotes the growth, invasiveness, and metastasis of many types of solid tumors. METHODS: In our study, HNSCC samples were analyzed for the expression of ASPH and selected endogenous hypoxia markers by real-time PCR and/or multiplex fluorescence immunohistochemistry. RESULTS: Except for the EPAS1 gene, which had higher mRNA expression in the HPV-negative group of HNSCC (p < 0.05), we found no other differences in the expression of the tested genes that were related to HPV status. On the contrary, a statistically significantly higher number of cells producing ASPH (p < 0.0001), HIF1A (p < 0.0001), GLUT1 (p < 0.0001), and MMP13 (p < 0.05) proteins were detected in the HPV-positive tumor group than in the HPV-negative sample group. All the evaluated markers, except for MMP9/13, were more abundant in the tumor parenchyma than in the tumor stroma. The Cox proportional hazard models showed that increased numbers of cells with GLUT1 and HIF1A protein expression were positive prognostic markers for overall and disease-specific survival in patients independent of HPV tumor status. CONCLUSION: The study examined HNSCC samples and found that elevated ASPH and hypoxia marker proteins, typically associated with poor prognosis, may actually indicate active HPV infection, the strongest prognostic factor in HNSCC patients. In cases where HPV status is uncertain, increased expression of HIF1A and GLUT1 can serve as positive prognostic factors.
ABSTRACT
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.
ABSTRACT
BACKGROUNDS: Oncological outcomes of the robotic low anterior rectal resection with total mesorectal excision (TME) are still under discussion. Few studies have proven that robotic TME (rTME) is a safe and equivalent method for treatment of rectal carcinoma. But there is almost no comparison between the rTME and conventional TME in terms of the number of lymph nodes obtained and the quality of the TME. METHODS: A single institution retrospective study was designed in a cohort of 261 patients. Cohort was divided into two groups depending on the type of surgery (rTME versus TME) and within these two groups, patients were divided according to whether they underwent neoadjuvant chemoradiation (nCHRT) or did not. The primary objective of the study was to compare obtained number of the lymph nodes in specimen. Secondary objectives were comparison of the quality of the TME and the number of positive circumferential resection margins. RESULTS: Results of the study have shown no significant difference in number of the lymph nodes obtained by the rTME and TME. There was no difference in the quality of the TME, neither in the group with the previous nCHRT nor in the group without a nCHRT. CONCLUSION: With results from the study we consider the rTME to be non-inferior to the conventional TME. Therefore, at least identical oncological results can be expected in patients treated by the rTME.
Subject(s)
Laparoscopy , Rectal Neoplasms , Robotic Surgical Procedures , Humans , Robotic Surgical Procedures/methods , Retrospective Studies , Laparoscopy/methods , Treatment Outcome , Rectal Neoplasms/surgery , Rectal Neoplasms/pathology , Lymph Nodes/surgery , Lymph Nodes/pathologyABSTRACT
Head and neck squamous cell carcinomas (HNSCCs) belong to a group of diverse tumors, which can be induced by infection with human papillomavirus (HPV) or tobacco and alcohol consumption. The viral etiology of HNSCC relates to better clinical outcomes reflecting a different immune system response. Here, we retrospectively analyzed 97 tissue samples from oral and oropharyngeal carcinomas associated and non-associated with HPV infection using multispectral fluorescent immunohistochemistry. To evaluate the immune cell infiltration in tumor and stroma compartments, we designed four panels of four to five antibodies. We detected more T lymphocytes in the stroma, compared to the tumor parenchyma. In HPV positive (HPV+) in comparison to HPV negative (HPV-) tumors, higher counts of CD3+CD4+, CD3+CD8+, PD1+CD4+, PD1+CD8+ T cells, and ICOS- Treg cells were detected while more ICOS+ Treg cells and CTLA4+CD4+ T cells were observed in HPV- than in HPV+ tumors. The results of the univariate and multivariate analyses confirmed the predominant impact of HPV status on prognosis. More importantly, the number of CD8+PD-1+ T cells was identified as an independent factor, influencing the overall and/or disease-specific survival of patients with oral cavity or oropharyngeal carcinomas.
ABSTRACT
Head and neck squamous cell carcinomas (HNSCC) belong among severe and highly complex malignant diseases showing a high level of heterogeneity and consequently also a variance in therapeutic response, regardless of clinical stage. Our study implies that the progression of HNSCC may be supported by cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) and the heterogeneity of this disease may lie in the level of cooperation between CAFs and epithelial cancer cells, as communication between CAFs and epithelial cancer cells seems to be a key factor for the sustained growth of the tumour mass. In this study, we investigated how CAFs derived from tumours of different mRNA subtypes influence the proliferation of cancer cells and their metabolic and biomechanical reprogramming. We also investigated the clinicopathological significance of the expression of these metabolism-related genes in tissue samples of HNSCC patients to identify a possible gene signature typical for HNSCC progression. We found that the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes can support the growth of primary cancer cells or metastases. Specifically, during coculture, cancer cell colony supporting effect and effect of CAFs on cell stiffness of cancer cells are driven by the mRNA subtype of the tumour from which the CAFs are derived. The degree of colony-forming support is reflected in cancer cell glycolysis levels and lactate shuttle-related transporters.
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BACKGROUND: Paediatric tumours are often characterised by the presence of recurrent DNA copy number alterations (CNAs). These DNA copy number profiles, obtained from a tissue biopsy, can aid in the correct prognostic classification and therapeutic stratification of several paediatric cancer entities (e.g. MYCN amplification in neuroblastoma) and are part of the routine diagnostic practice. Liquid biopsies (LQBs) offer a potentially safer alternative for such invasive tumour tissue biopsies and can provide deeper insight into tumour heterogeneity. PROCEDURE: The robustness and reliability of LQB CNA analyses was evaluated. We performed retrospective CNA profiling using shallow whole-genome sequencing (sWGS) on paired plasma circulating cell-free DNA (cfDNA) and tissue DNA samples from routinely collected samples from paediatric patients (n = 128) representing different tumour entities, including osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, Wilms tumour, brain tumours and neuroblastoma. RESULTS: Overall, we observed a good concordance between CNAs in tissue DNA and cfDNA. The main cause of CNA discordance was found to be low cfDNA sample quality (i.e. the ratio of cfDNA (<700 bp) and high molecular weight DNA (>700 bp)). Furthermore, CNAs were observed that were present in cfDNA and not in tissue DNA, or vice-versa. In neuroblastoma samples, no false-positives or false-negatives were identified for the detection of the prognostic marker MYCN amplification. CONCLUSION: In future prospective studies, CNA analysis on LQBs that are of sufficient quality can serve as a complementary assay for CNA analysis on tissue biopsies, as either cfDNA or tissue DNA can contain CNAs that cannot be identified in the other biomaterial.
Subject(s)
Biomarkers, Tumor/genetics , Cell-Free Nucleic Acids/genetics , DNA Copy Number Variations/genetics , Liquid Biopsy/methods , Adolescent , Child , Child, Preschool , Feasibility Studies , Female , Humans , Male , Prospective Studies , Retrospective StudiesABSTRACT
Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV-) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV- tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers-cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)-was detected in HPV- patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.
ABSTRACT
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.
Subject(s)
Cell Communication/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Oropharyngeal Neoplasms/immunology , Papillomavirus Infections/immunology , Squamous Cell Carcinoma of Head and Neck/immunology , Adult , Aged , B-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chemoradiotherapy, Adjuvant , Cohort Studies , Disease Progression , Female , Humans , Kaplan-Meier Estimate , Lymphocyte Activation , Male , Middle Aged , Neck Dissection , Oropharyngeal Neoplasms/mortality , Oropharyngeal Neoplasms/therapy , Oropharyngeal Neoplasms/virology , Oropharynx/pathology , Oropharynx/surgery , Papillomaviridae/immunology , Papillomaviridae/isolation & purification , Papillomavirus Infections/mortality , Papillomavirus Infections/therapy , Papillomavirus Infections/virology , Patient Selection , Prognosis , Squamous Cell Carcinoma of Head and Neck/mortality , Squamous Cell Carcinoma of Head and Neck/therapy , Squamous Cell Carcinoma of Head and Neck/virology , Treatment Outcome , Tumor Microenvironment/immunologyABSTRACT
The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients' samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients' samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.
ABSTRACT
BACKGROUND: Human papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-associated carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed, HPV-specific T cell immunity may play the key role in improving the prognosis of these patients. METHODS: Tumor-derived T cells were expanded in high levels of IL-2 and stimulated with HPV16 E6/E7 peptides in the presence or absence of anti-PD-1 monoclonal antibody nivolumab and soluble Tim-3. RESULTS: HPV16-specific tumor-infiltrating T cells were present in 73.1% of HPV-associated oropharyngeal tumors. HPV16 specific CD8+ TILs were able to produce IFNγ upon specific stimulation and predominantly expressed PD-1 but not Tim-3. Specific IFNγ production was further enhanced after a blockade of both PD-1 and Tim-3 pathways but not after a PD-1 blockade alone. Additionally, the specific stimulation of anti-HPV16 CD8+ T cells suppressed Tim-3 upregulation after the PD-1 blockade. CONCLUSION: Our data provide the rationale for combination cancer immunotherapy approaches, including the dual blockade of PD-1 and Tim-3 and, potentially, the use of HPV16-directed therapeutic vaccines.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Hepatitis A Virus Cellular Receptor 2/metabolism , Human papillomavirus 16/isolation & purification , Oropharyngeal Neoplasms/immunology , Oropharyngeal Neoplasms/virology , Programmed Cell Death 1 Receptor/metabolism , Adult , Aged , Antineoplastic Agents, Immunological/therapeutic use , Cytokines/biosynthesis , Female , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Male , Middle Aged , Nivolumab/therapeutic use , Oropharyngeal Neoplasms/drug therapy , Oropharyngeal Neoplasms/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor EscapeABSTRACT
MicroRNAs are considered as promising prognostic and diagnostic biomarkers of human cancer since their profiles differ between tumor types. Most of the tumor profiling studies were performed on rarely available fresh frozen (FF) samples. Alternatively, archived formalin-fixed paraffin-embedded (FFPE) tissue samples are also well applicable to larger-scale retrospective miRNA profiling studies. The aim of this study was to perform systematic comparison of the miRNA expression profiles between FF and macrodissected FFPE tonsillar tumors using the TaqMan Low Density Array system, with the data processed by different software programs and two types of normalization methods. We observed a marked correlation between the miRNA expression profiles of paired FF and FFPE samples; however, only 27-38% of the differentially deregulated miRNAs overlapped between the two source systems. The comparison of the results with regard to the distinct modes of data normalization revealed an overlap in 58-67% of differentially expressed miRNAs, with no influence of the choice of software platform. Our study highlights the fact that for an accurate comparison of the miRNA expression profiles from published studies, it is important to use the same type of clinical material and to test and select the best-performing normalization method for data analysis.