Diversity of intratumoral regulatory T cells in B-cell non-Hodgkin lymphoma.

Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3- Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.

TIGIT and PD-1 Mark Intratumoral T Cells with Reduced Effector Function in B-cell Non-Hodgkin Lymphoma.

Checkpoint blockade can reverse T-cell exhaustion and promote antitumor responses. Although blocking the PD-1 pathway has been successful in Hodgkin lymphoma, response rates have been modest in B-cell non-Hodgkin lymphoma (NHL). Coblockade of checkpoint receptors may therefore be necessary to optimize antitumor T-cell responses. Here, characterization of coinhibitory receptor expression in intratumoral T cells from different NHL types identified TIGIT and PD-1 as frequently expressed coinhibitory receptors. Tumors from NHL patients were enriched in CD8+ and CD4+ T effector memory cells that displayed high coexpression of TIGIT and PD-1, and coexpression of these checkpoint receptors identified T cells with reduced production of IFNγ, TNFα, and IL2. The suppressed cytokine production could be improved upon in vitro culture in the absence of ligands. Whereas PD-L1 was expressed by macrophages, the TIGIT ligands CD155 and CD112 were expressed by lymphoma cells in 39% and 50% of DLBCL cases and in some mantle cell lymphoma cases, as well as by endothelium and follicular dendritic cells in all NHLs investigated. Collectively, our results show that TIGIT and PD-1 mark dysfunctional T cells and suggest that TIGIT and PD-1 coblockade should be further explored to elicit potent antitumor responses in patients with NHL.

Evaluation of CHK1 activation in vulvar squamous cell carcinoma and its potential as a therapeutic target in vitro.

CHK1 is an important regulator of the cell cycle and DNA damage response, and its altered expression has been identified in various tumors. Chk1 inhibitors are currently being evaluated as monotherapy and as potentiators of chemotherapy in clinical settings. However, to our knowledge, no previous study has investigated either the activation status or the therapeutic potential of CHK1 targeting in vulvar cancer. Therefore, we examined the expression status of activated CHK1 forms pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 in 294 vulvar squamous cell carcinomas (VSCC) using immunohistochemistry and analyzed their relationships with various clinicopathological variables and clinical outcome. To aid translation of preclinical studies, we also assessed cell sensitivity to the Chk1 inhibition in two vulvar cancer cell lines. Compared to the levels of pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 in normal vulvar squamous epithelium, high nuclear pCHK1Ser345 expression was found in 57% of vulvar carcinomas, whereas low nuclear pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 expressions were observed in 58%, 64%, and 40% of the cases, respectively. Low levels of pCHK1Ser317 and pCHK1Ser280 in the nucleus correlated significantly with advanced tumor behaviors and aggressive features. None of pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 forms were identified as prognostic factors. In vitro inhibition of CHK1 by small molecular inhibitors or siRNA reduced viability by inducing DNA damage and apoptosis of vulvar cancer cell lines. In summary, we conclude that cellular functions regulated by CHK1 are phosphorylation/localization-dependent and deregulation of CHK1 function occurs in VSCC and might contribute to tumorigenesis. Targeting CHK1 might represent as a useful antitumor strategy for the subgroup of VSCC harboring p53 mutations.

Epithelial-mesenchymal transition markers in malignant ovarian germ cell tumors.

The purpose of this study was to determine the expression and potential clinical role of epithelial-to-mesenchymal transition (EMT)-related factors in malignant ovarian germ cell tumors (MOGCT). Protein expression of E-cadherin, N-cadherin, P-cadherin, Zeb1, HMGA2, and vimentin by immunohistochemistry was analyzed in 42 MOGCT from patients treated in Norway during the period 1981-2001. Expression was analyzed for association with clinicopathologic parameters. E-cadherin (p = 0.016) and HMGA2 (p = 0.002) expression was significantly higher in immature teratomas and yolk sac tumors compared with dysgerminomas. Vimentin (p < 0.001) and Zeb1 (p = 0.029) staining was significantly higher in immature teratomas compared with yolk sac tumors and dysgerminomas, whereas no significant differences were observed for N-cadherin and P-cadherin. EMT-associated markers were not significantly related to clinicopathologic parameters including age, tumor diameter, and FIGO stage. In conclusion, based on this limited series, EMT-associated markers are not associated with clinical parameters in MOGCT, in contrast to ovarian carcinoma. EMT-related proteins are differentially expressed among various MOGCT subtypes, suggesting differences in biological characteristics associated with invasion and metastasis.

Progesterone Receptor Expression Is an Independent Prognosticator in FIGO Stage I Uterine Leiomyosarcoma.

OBJECTIVES: To analyze the clinical role of hormone receptors in a large uterine sarcomas series with long-term follow-up. METHODS: Protein expression of estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemistry was studied in tissue microarrays from 294 patients diagnosed with uterine sarcoma in Norway from 1970 to 2000 and analyzed for an association with clinicopathologic parameters and outcome. RESULTS: ER and PR were detected in 136 of 291 and 184 of 291 tumors (three noninformative cases each), respectively. Expression was unrelated to histology, patient age, tumor diameter, the degree of atypia, the presence of necrosis or vascular invasion, or mitotic counts. ER and PR expression was unrelated to survival in the analysis of the entire cohort. When survival analysis was confined to stage I leiomyosarcoma (n = 147), higher PR score was significantly related to longer overall survival (OS) (P = .042). Clinicopathologic prognosticators in this group were age (P = .041), tumor diameter (P = .001), and mitotic count (P = .007), with a trend for atypia (P = .087). In Cox multivariate analysis, PR score (P = .019), tumor diameter (P = .013), and mitotic count (P = .002) were independent prognosticators of OS. CONCLUSIONS: Hormone receptor expression is not informative of outcome in the analysis of uterine sarcomas of all stages and histologic types. PR expression identifies patients with longer survival in stage I leiomyosarcoma.

Expression of CDK1(Tyr15), pCDK1(Thr161), Cyclin B1 (total) and pCyclin B1(Ser126) in vulvar squamous cell carcinoma and their relations with clinicopatological features and prognosis.

Cyclin B1-CDK1 complex plays an important role in the regulation of cell cycle. Activation of Cyclin B1 and CDK1 and the formation of the complex in G2/M are under multiple regulations involving many regulators such as isoforms of 14-3-3 and CDC25 and Wee1. Abnormal expression of Cyclin B1 and CDK1 has been detected in various tumors. However, to our knowledge no previous study has investigated Cyclin B1 and CDK1 in vulvar cancer. Therefore, we evaluated the statuses of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 in 297 cases of vulvar squamous cell carcinomas by immunohistochemistry. Statistical analyses were performed to explore their clinicopathological and prognostic values. In at least 25% of tumor cases high expression of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 was observed, compared to the low levels in normal vulvar squamous epithelium. Elevated levels of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 were correlated with advanced tumor behaviors and aggressive features. Although CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 could not be identified as prognostic factors, combinations of (pCDK1Thr161 C+N + 14-3-3σN), (pCDK1Thr161 C+N + 14-3-3ηC), (pCDK1Thr161 C+N + Wee1C) and (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C) were correlated with disease-specific survival (p = 0.036, p = 0.029, p = 0.042 and p = 0.007, respectively) in univariate analysis. The independent prognostic significance of (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C) was confirmed by multivariate analysis. In conclusion, CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 may be involved in progression of vulvar squamous cell carcinoma. The combination of pCDK1Thr161, 14-3-3σ, 14-3-3η and Wee1 was a statistically independent prognostic factor.

Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an important oncogene contributing to cancer progression partially by regulating cMYC and AKT. We examined CIP2A expression in cutaneous melanomas, its association with clinicopathological parameters and mapped molecular mechanisms regulated by CIP2A in vitro. CIP2A expression was analyzed by immunohistochemistry in 17 nevi, 132 primary melanomas and 49 metastases. Effects of siRNA-mediated down-regulation on proliferation, apoptosis and signaling pathways were assessed in melanoma cell lines. In superficial spreading melanomas (SSM), high nuclear CIP2A expression was associated with poor overall survival (OS) (P = 0.0018). Surprisingly, high cytoplasmic expression was related to improved relapse-free (P = 0.031) and OS (P = 0.014) in nodular melanomas (NM). In vitro experiments revealed that CIP2A can regulate proliferation and/or apoptosis partially through the PI3K/AKT pathway but also independently. In summary, CIP2A could represent a potential therapeutic target in SSM. However, in NM cytoplasmic CIP2A is associated with improved prognosis indicating that CIP2A has distinct, complex functions dependent on the molecular context and histological subtype. As seen in other cancer types, CIP2A can influence cMYC and AKT, but our data also suggest that in melanoma it has additional targets which need to be identified.

Gene expression signatures of primary and metastatic uterine leiomyosarcoma.

Leiomyosarcoma (LMS) is the most common uterine sarcoma. Although the disease is relatively rare, it is responsible for considerable mortality due to frequent metastasis and chemoresistance. The molecular events related to LMS metastasis are unknown to date. The present study compared the global gene expression patterns of primary uterine LMSs and LMS metastases. Gene expression profiles of 13 primary and 15 metastatic uterine LMSs were analyzed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. To identify differently expressed genes between primary and metastatic tumors, we performed one-way analysis of variance with Benjamini-Hochberg correction. This led to identification of 203 unique probes that were significantly differentially expressed in the 2 tumor groups by greater than 1.58-fold with P < .01, of which 94 and 109 were overexpressed in primary and metastatic LMSs, respectively. Genes overexpressed in primary uterine LMSs included OSTN, NLGN4X, NLGN1, SLITRK4, MASP1, XRN2, ASS1, RORB, HRASLS, and TSPAN7. Genes overexpressed in LMS metastases included TNNT1, FOLR3, TDO2, CRYM, GJA1, TSPAN10, THBS1, SGK1, SHMT1, EGR2, and AGT. Quantitative real-time PCR confirmed significant anatomical site-related differences in FOLR3, OSTN, and NLGN4X levels; and immunohistochemistry showed significant differences in TDO2 expression. Gene expression profiling differentiates primary uterine LMSs from LMS metastases. The molecular signatures unique to primary and metastatic LMSs may aid in understanding tumor progression in this cancer and in providing a molecular basis for prognostic studies and therapeutic target discovery.

Primary tumor vascularity, HIF-1α and VEGF expression in vulvar squamous cell carcinomas: their relationships with clinicopathological characteristics and prognostic impact.

BACKGROUND: Increased vascularity is a crucial event in the tumor progression and has prognostic significance in various cancers. However, the ultimate role of angiogenesis in the pathogenesis and clinical outcome of vulvar carcinoma patients is still not settled. METHODS: Tumor vascularity using CD34 stained slides measured by Chalkley counting method as well as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) immunoexpression was examined in 158 vulvar squamous cell carcinomas. Associations between vascular Chalkley count, HIF-1α and VEGF expression and clinicopathological factors and clinical outcome were evaluated. RESULTS: High CD34 Chalkley count was found to correlate with larger tumor diameter (P = 0.002), deep invasion (P < 0.001) and HIF-1α (P = 0.04), whereas high VEGF expression correlate significantly with poor tumor differentiation (P = 0.007). No significant association between CD34 Chalkley counts and VEGF expression and disease-specific survival was observed. High HIF-1α expression showed better disease specific survival in both univariate and multivariate analyses (P = 0.001). CONCLUSIONS: A significant association between high tumor vascularity and larger tumor size as well as deeper tumor invasion suggests an important role of angiogenesis in the growth and progression of vulvar carcinomas. HIF-1α expression in vulvar carcinomas was a statistically independent prognostic factor.

Expression of p15INK4b and p57KIP² and relationship with clinicopathological features and prognosis in patients with vulvar squamous cell carcinoma.

BACKGROUND: The cyclin-dependent kinase inhibitors p15(INK4b) and p57(KIP2) are important regulators of the cell cycle, and their abnormal expression has been detected in various tumors. However, little is known about the role of p15(INK4b) and p57(KIP2) in the pathogenesis of vulvar carcinoma, and the prognostic impact is still unknown. In our current study, we examined the expression of p15(INK4b) and p57(KIP2) in a large series of vulvar squamous cell carcinomas to elucidate the prognostic impact. METHODS: Expression of p15(INK4b) and p57(KIP2) were examined in 297 vulvar squamous cell carcinomas using immunohistochemistry. Both uni- and multivariate analysis of prognostic factors were performed, and correlations with clinicopathologic parameters were examined. RESULTS: Compared to the high levels of p15(INK4b) and p57(KIP2) in normal vulvar squamous epithelium, low levels of p15(INK4b) and p57(KIP2) were found in 82% and 44% of vulvar carcinomas, respectively. Low levels of p15(INK4b) and p57(KIP2) correlated significantly with malignant features, including large tumor diameter (p = 0.03 and p = 0.001, respectively) and increased invasiveness (p = 0.003 and p = 0.04, respectively). Although p15(INK4b) and p57(KIP2) levels could not be identified as prognostic markers, combined analysis of p14(ARF)/p15(INK4b)/p16(INK4a) showed that patients whose tumors expressed low levels of two or three of these INK4 proteins had a worse prognosis than those with only low levels of one or no protein (univariate analysis p = 0.02). The independent prognostic significance of these INK4 proteins was confirmed by multivariate analysis (p = 0.008). CONCLUSIONS: We show for the first time that p15(INK4b) and p57(KIP2) may be involved in the progression of vulvar carcinomas and the combined p14(ARF)/p15(INK4b)/p16(INK4a) status was a statistically independent prognostic factor.

CD105 (Endoglin) expression in breast carcinoma effusions is a marker of poor survival.

We analyzed the expression and clinical role of endoglin (CD105) in breast carcinoma effusions. Endoglin levels were measured in 36 effusion supernatants by ELISA and studied for association with the cancer-associated markers calprotectin, VEGF, and the VEGF receptor sFlt1. Endoglin expression was further studied in 46 effusions and 22 primary carcinomas using immunohistochemistry. The four secreted molecules were detected in all specimens and their levels significantly correlated (p < 0.001). In effusions, endoglin was localized to carcinoma cells and reactive mesothelium using immunohistochemistry. Tumor cell expression was higher in effusions compared to primary carcinomas (p = 0.025), and in post-chemotherapy compared to pre-chemotherapy effusions (p = 0.017). Higher tumor endoglin expression was associated with poor overall (p = 0.021) and disease-free (p = 0.032) survival in univariate analysis, and was an independent predictor in Cox multivariate analysis (p = 0.001 and p = 0.038, respectively). Our data suggest that endoglin may be an important therapeutic target in metastatic breast cancer.

Exploring the peritoneal surface malignancy phenotype--a pilot immunohistochemical study of human pseudomyxoma peritonei and derived animal models.

Peritoneal surface malignancies are characterized by the propensity for tumor growth on peritoneal surfaces without development of extraperitoneal metastases, but the molecular basis for this phenomenon is incompletely understood. Five human tumors and corresponding orthotopic animal models of human pseudomyxoma peritonei and peritoneal mucinous carcinomatosis from colorectal carcinoma were extensively characterized by immunohistochemical analysis of molecular markers of tissue differentiation (carcinoembryonal antigen, CK20, CK7, and vimentin), proliferation and metastasis (Ki-67, vascular endothelial growth factor, and S100A4), mucins (MUC1, MUC2, MUC4, MUC5AC), and adhesion molecules (E-cadherin, N-cadherin, P-cadherin, claudin 1, claudin 3, and claudin 4). Macro- and microscopic growth patterns of implanted human tissues were preserved through passages in the animals, as were with few exception immunohistochemical staining profiles, supporting the relevance of the models as tools for studying the human disease. Tissue differentiation marker expression was in accordance with previously published results and high Ki-67 score confirmed high proliferative capacity, whereas absence of metastatic capacity was supported by low expression levels of the studied metastasis markers. These mucinous tumors expressed high levels of MUC2 and MUC4, whereas MUC1 was not expressed and MUC5AC expression was variable. Similarly, specific adhesion molecules from the cadherin and claudin families were shown to be of relevance in the investigated samples. The results indicate that mucinous peritoneal surface malignancies of intestinal origin are characterized by the presence of specific molecular markers and represent a step toward understanding the complexity of this intriguing phenotypic entity.