High SEPT9_i1 Protein Expression Is Associated with High-Grade Prostate Cancers

Septins are a family of GTP-binding cytoskeleton proteins expressed in many solid tumors. Septin 9 (SEPT9) in particular was found overexpressed in diverse carcinomas. Herein, we studied the expression of SEPT9 isoform 1 protein (SEPT9_i1) in human prostate cancer specimens. We utilized immunohistochemical staining to study the expression of SEPT9_i1 protein. Staining level was analyzed in association with clinical characteristics and the pathological Gleason grade and score. Fifty human prostate cancer specimens (42 primary tumors and 8 metastatic lesions) were stained by SEPT9_i1 antibody and analyzed. SEPT9_i1 protein was expressed in prostate cancer cells but absent in normal epithelial cells. The intensity of staining was correlated proportionally to pretreatment prostate-specific antigen (PSA) blood levels and Gleason score (P < 0.05). SEPT9_i1 was highly expressed in all metastatic lesions. A significant assocation between SEPT9_i1 expression and high Gleason score on multivariate linear regression analysis was found. We conclude that SEPT9_i1 is expressed in high-grade prostate tumors suggesting it has a significant role in prostate tumorigenesis and that it could serve as a molecular marker for prostate tumor progression.     

Circulating Methylated Septin 9 Nucleic Acid in the Plasma of Patients with Gastrointestinal Cancer in the Stomach and Colon

BACKGROUND: Methylated Septin 9 (mSEPT9) in plasma has recently been suggested as a screening marker for colorectal cancer (CRC) with variable sensitivity. We aimed to determine the usefulness of plasma mSEPT9 for screening CRC and gastric cancer (GC) and its diagnostic role in postoperative CRC patients. METHODS: A total of 350 peripheral blood samples from 101 CRC patients, 153 GC patients, and 96 healthy persons were collected. In addition, we obtained 35 follow-up blood samples from 27 CRC patients after curative radical surgery. Plasma mSEPT9, serum carcinoembryonic antigen (CEA), and serum CA19-9 were evaluated with clinicopathologic features. RESULTS: The sensitivity of plasma mSEPT9 was 36.6% for detecting CRC and 17.7% for detecting GC, and the specificity was 90.6%. During follow-up periods, mSEPT9 showed negative conversion in eight of nine CRC patients (88.9%) whose plasma mSEPT9 had been positive before radical surgery. The patients with plasma mSEPT9 had a tendency of presence of distant metastasis and lower disease-free survival in both CRC and GC. In GC patients, plasma mSEPT9 was more frequently observed in intestinal (23.5%) and mixed type (40.0%) than diffuse type (7.3%; P =.009). Combined analysis of mSEPT9, CEA, and CA19-9 increased the sensitivity for diagnosing GC to 32.7% (P = .002). CONCLUSION: Considering the high incidence of plasma mSEPT9 in intestinal or mixed type GCs similar to CRCs, GC should be examined through the plasma mSEPT9 screening test. In addition, plasma mSEPT9 is proposed as a follow-up marker in CRC patients, but further validation is required.     

Prognostic Value of Tumor-Infiltrating Lymphocytes for Patients With Head and Neck Squamous Cell Carcinoma

BACKGROUND: The prognostic value of tumor-infiltrating lymphocytes (TILs) in head and neck squamous cell carcinoma (HNSCC) remains controversial. Additionally, there is no standardized approach or cutoff value for evaluating TIL levels. The aim of this study was to establish a feasible method and criterion to assess TIL levels for future clinical practice and research use and to explore the relationship between TIL levels and prognosis. PATIENTS AND METHODS: This retrospective cohort study reviewed the records and pathological sections of 202 patients with HNSCC who were surgically treated at Beijing Stomatological Hospital, Capital Medical University, from January 1998 to January 2011. The predictor variable was the TIL level. The main outcome assessment parameters were disease-free survival (DFS) and disease-specific survival (DSS). RESULT: The T stage (P = .008), smoking history (P = .042), alcohol history (P = .048), need for radiotherapy (P = .012) and microscopic extracapsular spread (ECS) (P = .012) were associated with the TIL level. A cutoff value equal to 70% could be taken as a threshold for TIL assessment, with a TIL level higher than 70% associated with a better prognosis (DFS rate: 51.9%, P = .018; DSS rate: 59.3%, P = .049). The Cox regression model showed that the TIL level was an independent prognostic factor for DFS (hazard ratio (HR): 0.786, 95% CI: 0.618-0.999, P = .049). CONCLUSION: The TIL level is closely related to the prognosis of patients with HNSCC. A threshold value of 70% is appropriate for TIL assessment, as patients with a TIL level higher than 70% show a better prognosis. Thus, the TIL level might serve as an independent predictor for HNSCC recurrence.     

LRP1B mutation is associated with tumor HPV status and promotes poor disease outcomes with a higher mutation count in HPV-related cervical carcinoma and head & neck squamous cell carcinoma

Human papillomavirus (HPV) infection and gene mutations were reputed as key factors in cervical carcinoma (CC) and head and neck squamous cell carcinoma (HNSCC). However, the associations of HPV status and gene mutations remain to be determined. This study aims to identify molecular patterns of LRP1B mutation and HPV status via rewiring tumor samples of HNSCC (n=1478) and CC (n=178) from the TCGA dataset. Here, we found that LRP1B mutation was associated with HPV status in CC (P=0.040) and HNSCC (P=0.044), especially in HPV 16 integrated CC (P=0.036). Cancer survival analysis demonstrated that samples with LRP1B mutation showed poor disease outcomes in CC (P=0.013) and HNSCC (P=0.0124). In addition, the expression status of LPR1B was more favorable for prediction than TP53 or RB1 in CC and HNSCC. Mutation clustering analysis showed that samples with LRP1B mutation showed higher mutation count in CC (P=1.76e-67) and HNSCC (P<10e-10). Further analysis identified 289 co-occurrence genes in these two cancer types, which were enriched in PI3K signaling, cell division process, and chromosome segregation process, et al. The 289-co-occurrence gene signature identified a cluster of patients with a higher portion of copy number variation (CNV) lost in the genome, different tumor HPV status (P<10e-10), higher mutation count (P<10e-10), higher fraction genome altered value (P=2.078e-4), higher aneuploidy score (P=3.362e-4), and earlier started the smoking year (P=2.572e-4), which were associated with shorter overall survival (P=0.0103) in CC and HNSCC samples. Overall, LRP1B mutation was associated with tumor HPV status and was an unfavorable prognostic biomarker for CC and HNSCC.     

Superfluous role of mammalian septins 3 and 5 in neuronal development and synaptic transmission

The septin family of GTPases, first identified for their roles in cell division, are also expressed in postmitotic tissues. SEPT3 (G-septin) and SEPT5 (CDCrel-1) are highly expressed in neurons, enriched in presynaptic terminals, and associated with synaptic vesicles. These characteristics suggest that SEPT3 or SEPT5 might be important for synapse formation, maturation, or synaptic vesicle traffic. Since Sept5^−/− mice do not show any overt neurological phenotypes, we generated Sept3^−/− and Sept3^−/− Sept5^−/− mice and found that SEPT3 and SEPT5 are not essential for development, fertility, or viability. Changes in the expression of septins were noted in the absence of SEPT3, SEPT5, and both septins. SEPT5 association with other septins in brain tissue was unaffected by the removal of SEPT3. No abnormalities were observed in the gross morphology and synapses of the hippocampus. Similarly, axon development and synapse formation were unaffected in vitro. In cultured hippocampal neurons, the size of the recycling synaptic vesicle pool was unaltered in the absence of SEPT3. Furthermore, synaptic transmission at two different central synapses was not significantly affected in Sept3^−/− Sept5^−/− mice. These results indicate that SEPT3 and SEPT5 are dispensable for neuronal development as well as for synaptic vesicle fusion and recycling.     

PRF1 is a prognostic marker and correlated with immune infiltration in head and neck squamous cell carcinoma

PurposeHead and neck squamous cell carcinoma (HNSCC) is a highly invasive malignancy with poor survival. Perforin (PRF1) plays essential roles in host immunity. Our research intended to identify the correlations of PRF1 with clinical prognosis and tumor immune infiltration in HNSCC.MethodsWe explored PRF1 expression and its associations with the clinical features of HNSCC via the Tumor Immune Estimation Resource (TIMER), Oncomine and The Cancer Genome Atlas (TCGA) databases. The prognostic value of PRF1 for HNSCC was further explored by Kaplan–Meier plotter and TIMER. Finally, the relation between PRF1 and immune infiltration in HNSCC was estimated via CIBERSORT and TIMER.ResultsPRF1 expression was remarkably elevated in HNSCC and associated with clinical stage and HPV infection. High PRF1 expression predicted favorable outcomes in HNSCC, especially in HPV+ HNSCC. Moreover, higher infiltration of CD8+ T cells and CD4+ T cells were found in the PRF1^high group of HNSCC. PRF1 expression in HNSCC was strongly correlated with infiltrating CD8+ T cells and dendritic cells (DCs), with higher relevance in HPV+ HNSCC.ConclusionOur findings suggested that PRF1 could be a novel prognostic biomarker in HNSCC and that its expression was related to immune cell infiltration, which was impacted by HPV status.Key words: PRF1, prognosis, head and neck squamous cell carcinoma, tumor immune infiltration, HPV     

Clinical significance and biological functions of chemokine CXCL3 in head and neck squamous cell carcinoma

CXCL3 plays extensive roles in tumorigenesis in various types of human cancers through its roles in tumor cell differentiation, invasion, and migration. However, the mechanisms of CXCL3 in head and neck squamous cell carcinoma (HNSCC) remain unclear. In our study, multiple databases were used to explore the expression level, prognostic value, and related mechanisms of CXCL3 in human HNSCC through bioinformatic methods. We also performed further experiments in vivo and in vitro to evaluate the expression of CXCL3 in a human head and neck tissue microarray and the underlying effect mechanisms of CXCL3 on the tumor biology of HNSCC tumor cells. The result showed that the expression level of CXCL3 in patients with HNSCC was significantly higher as compared with that in normal tissues (P<0.05). Kaplan–Meier survival analysis demonstrated that patients with high CXCL3 expression had a lower overall survival rate (P=0.038). CXCL3 was further identified as an independent prognostic factor for HNSCC patients by Cox regression analysis, and GSEA exhibited that several signaling pathways including Apoptosis, Toll-like receptor, Nod-like receptor, Jak-STAT, and MAPK signaling pathways may be involved in the tumorigenesis of HNSCC. CAL27 cells overexpressing or HNSCC cells treated with exogenous CXCL3 exhibited enhanced cell malignant behaviors, whereas down-regulating CXCL3 expression resulted in decreased malignant behaviors in HSC4 cells. In addition, CXCL3 may affect the expression of several genes, including ERK1/2, Bcl-2, Bax, STAT3, and NF-κB. In summary, our bioinformatics and experiment findings effectively suggest the information of CXCL3 expression, roles, and the potential regulatory network in HNSCC.     

Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80% (with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high κ concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.     

DNA repair gene expression level in peripheral blood and tumour tissue from non-small cell lung cancer and head and neck squamous cell cancer patients

BackgroundThe nucleotide excision repair pathway is crucial for cellular DNA integrity and the ERCC1 helicase is also potentially involved in resistance to platinum-based chemotherapy, and high levels of ERCC1 mRNA in tumours have been associated with cisplatin resistance in different human cancers. The aim of this work was to investigate the correlation between DNA repair gene expression levels in tumour tissue, normal tissue and peripheral blood samples from patients with two common human cancers, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (HNSCC), to test if blood gene expression could be a proxy for tumour tissue gene expression to predict response to platinum-based chemotherapy.MethodsUsing RT-qPCR we determined ERCC1, ERCC2, ERCC4, XPA, XPC, XRCC1, XRCC3, APEX, OGG1, MGMT mRNA levels in fresh NSCLC, normal lung and HNSCC tissue, as well as blood, from NSCLC and HNSCC patients who were treated surgically.ResultsTarget gene expression in NSCLC and HNSCC tissue was higher than in blood. A statistically significant correlation (p < 0.05) was found between target gene mRNA expression in tumour tissue and blood, in particular ERCC1, MGMT, XPC, XRCC1 and XRCC3 in NSCLC and APEX, ERCC1, ERCC2, ERCC4, XRCC1 and XRCC3 in HNSCC.ConclusionsThe existence of a significant correlation between blood and tumour tissue expression of some genes of clinical interest, such as ERCC1 in NSCLC and HNSCC, could allow the introduction in clinical practice of a simple test that would measure mRNA levels of DNA repair genes in peripheral blood samples instead of tissue samples to determine prognostic and predictive factors in NSCLC and HNSCC patients.     

Plasma Membrane Proteomics of Tumor Spheres Identify CD166 as a Novel Marker for Cancer Stem-like Cells in Head and Neck Squamous Cell Carcinoma

Patients with advanced head and neck squamous cell carcinoma (HNSCC) have a poor prognosis with the currently available therapy, and tumor recurrence is frequently observed. The discovery of specific membrane-associated cancer stem cell (CSC) markers is crucial for the development of novel therapeutic strategies to target these CSCs. To address this issue, we established sphere cultures to enrich CSCs and used them for plasma membrane proteomics to identify specific membrane signatures of the HNSCC spheres. Of a dataset that included a total of 376 identified proteins, 200 were bona fide membrane proteins. Among them, 123 proteins were at least 1.5-fold up- or down-regulated in the spheres relative to the adherent cultures. These proteins included cell adhesion molecules, receptors, and transporter proteins. Some of them play key roles in wnt, integrin, and TGFβ signaling pathways. When we compared our dataset with two published hESC membrane protein signatures, we found 18 proteins common to all three of the databases. CD166 and CD44 were two such proteins. Interestingly, the expression of CD166, rather than that of the well-established HNSCC CSC marker CD44, was significantly related to the malignant behavior of HNSCC. Relative to CD166^low HNSCC cells, CD166^high HNSCC cells had a greater sphere-formation ability in vitro and tumor formation ability in vivo. Patients whose tumors expressed high levels of CD166 had a significantly poorer clinical outcome than those whose tumors expressed low levels of CD166 (cohort 1: 96 cases, p = 0.040), whereas the level of CD44 expression had only a marginal influence on the clinical outcome of patients with HNSCC (p = 0.078). The level of CD166 expression in HNSCC tumors was also associated with the tumor recurrence rate (cohort 2: 104 cases, p = 0.016). This study demonstrates that CD166 is a valuable cell surface marker for the enrichment of HNSCC stem cells and that plasma membrane proteomics is a promising biological tool for investigating the membrane proteins of CSCs.Head and neck squamous cell carcinoma (HNSCC)¹ is the sixth most common cancer worldwide. Despite ongoing improvement in traditional treatments, the long-term survival rate of patients with HNSCC has not significantly improved over the past several decades. More than 60% of patients with advanced tumors or localized lymph node metastases die within five years of their diagnosis (1). Tumor recurrence and resistance to therapy are the major causes of death. Recently, newly recognized cancer stem cells (CSCs) or tumor-initiating cells have been associated in a cause-and-effect manner with tumor recurrence and resistance to therapy. The concept of CSCs was established because of the heterogeneous nature of cancer and suggests that CSCs are a subpopulation of cancer cells with stem-cell-like traits and the source of all cells in the cancer. Conventional cancer therapies such as chemotherapy and radiotherapy may destroy only those cells that form the bulk of the tumor, leaving the CSCs intact and able to give rise to tumor recurrence. Based on this theory, researchers are searching for therapies that would destroy CSCs in the hope of finally curing cancer (2). In order to develop strategies that target CSCs, experimental assays are required to determine how to distinguish CSCs from their progeny. Different methods have been used to isolate CSCs from a range of hematopoietic and solid tumors, and some CSC-specific cell surface markers have been found. These markers are primarily selected from the corresponding normal stem-cell markers based on their heterogeneous expression in the pertinent cancers. Despite some controversy, the CD34+CD38- marker signature was chosen to define the CSCs of leukemia (3), the CD44+CD24- signature was chosen to define breast cancer CSCs (4), and the CD44 marker was chosen to define the CSCs of HNSCC (5). Though membrane proteins represent only one-third of the proteins encoded by the human genome, they represent more than two-thirds of the known protein targets of drugs. These cell surface markers are not only useful for enriching CSCs from different tumors, but also of significant interest for drug discovery.However, as more cell surface markers for different cancers have been identified, conflicting results have been reported regarding the usefulness of some of the markers and the reproducibility of some of the marker profiles (6). Quintana et al. examined the expression of 22 common CSC markers in melanoma and found that none of them were exclusively enriched in tumorigenic cells relative to non-tumorigenic cells derived from melanoma (7). CD133 is a widely accepted cell surface marker for glioblastoma CSCs, but Beier et al. found that some glioblastoma CSCs were CD133- (8). CD44 is a CSC marker that is commonly expressed by different malignancies of hematopoietic and epithelial origin, including HNSCC (5). However, increasing data have demonstrated a high level of expression of CD44 in the great majority of cells in head and neck tissues, including normal mucosa and carcinomas, and its subsequent expression could not be used to distinguish normal from benign or malignant epithelia of the head and neck. These observations suggest the need for a comprehensive investigation and greater understanding of the cell surface molecules of CSCs.Many different “omic” technologies have shown promise as means to identify markers for cancer stem cells and tumors (9). Among them, membrane proteomics can directly detect changes in the cell surface content and provide insights into the post-translational regulation of cell surface functions. Therefore, in this study, we chose to use membrane proteomics both to investigate the cell surface molecules of CSCs that were enriched from the HNSCC cell populations based on their ability to form spheres and to relate their expression to that of stem cell traits. Our results may contribute to further clinical applications of CSCs by providing tools for purifying and identifying CSCs.     

IRX-2, a novel immunotherapeutic, enhances and protects NK-cell functions in cancer patients

Background

IRX-2 is a primary biologic which has been used for the therapy of head and neck squamous cell cancer (HNSCC) with promising clinical results. Since NK-cell function is compromised in HNSCC patients, we tested the effects of IRX-2 on the restoration of human NK-cell functions in vitro.

Methods

Peripheral blood mononuclear cells (PBMC) were isolated from 23 HNSCC patients and 10 normal controls (NC). The NK-cell phenotype and functions were compared before and after culture?±?IRX-2 or?±?50?IU/ml rhIL-2. Flow cytometry was used to study the NK-cell phenotype, cytotoxic activity and cytokine expression.

Results

Impaired NK-cell cytotoxicity in HNSCC patients was related to lower expression of NKG2D, NKp30 and NKp46 receptors (P?P?P?P?P?Conclusions IRX-2 was more effective than IL-2 in enhancing NK-cell cytotoxicity and protecting NK-cell function of HNSCC patients in vitro, emphasizing the potential advantage of IRX-2 as a component of future therapies for HNSCC.     

The expression and clinical significance of connective tissue growth factor in advanced head and neck squamous cell cancer

Connective tissue growth factor (CTGF) has been reported to play critical roles in the tumorigenesis of several human malignancies. This study was performed to evaluate CTGF protein expression in head and neck squamous cell carcinoma (HNSCC). Surgical specimens from 76 primary HNSCC were obtained with written informed consents and the expression level of CTGF was immunohistochemically evaluated. The cytoplasmic immunoreactivity of CTGF in cancer cells was semiquantitatively classified into low and high expression. Among all 76 cases with or without neoadjuvant therapy, low CTGF showed significantly longer (P = 0.0282) overall survival (OS), but not disease-free survival (DFS) than high CTGF. Although low CTGF in patients with stage I, II and III did not result in any significant difference of the OS and DFS, stage IV HNSCC patients with low CTGF showed significantly longer OS (P = 0.032) and DFS (P = 0.0107) than those with high CTGF. These differences in stage IV cases were also confirmed using multivariate analyses. These results suggest that low CTGF in stage IV HNSCC is an independent prognostic factor, despite with or without neoadjuvant therapy.     

Induction of CD44 Variant 9-Expressing Cancer Stem Cells Might Attenuate the Efficacy of Chemoradioselection and Worsens the Prognosis of Patients with Advanced Head and Neck Cancer

Background

At our institute, a chemoradioselection strategy has been used to select patients for organ preservation on the basis of response to an initial 30–40 Gy concurrent chemoradiotherapy (CCRT). Patients with a favorable response (i.e., chemoradioselected; CRS) have demonstrated better outcomes than those with an unfavorable response (i.e., nonchemoradioselected; N-CRS). Successful targeting of molecules that attenuate the efficacy of chmoradioselection may improve results. Thus, the aim of this study was to evaluate the association of a novel cancer stem cell (CSC) marker, CD44 variant 9 (CD44v9), with cellular refractoriness to chemoradioselection in advanced head and neck squamous cell carcinoma (HNSCC).

Materials and Methods

Through a medical chart search, 102 patients with advanced HNSCC treated with chemoradioselection from 1997 to 2008 were enrolled. According to our algorithm, 30 patients were CRC following induction CCRT and 72 patients were N-CRS. Using the conventional immunohistochemical technique, biopsy specimens and surgically removed tumor specimens were immunostained with the anti-CD44v9 specific antibodies.

Results

The intrinsic expression levels of CD44v9 in the biopsy specimens did not correlate with the chemoradioselection and patient survival. However, in N-CRS patients, the CD44v9-positive group demonstrated significantly (P = 0.008) worse prognosis, than the CD44v9-negative group. Multivariate analyses demonstrated that among four candidate factors (T, N, response to CCRT, and CD44v9), CD44v9 positivity (HR: 3.145, 95% CI: 1.235–8.008, P = 0.0163) was significantly correlated with the poor prognosis, along with advanced N stage (HR: 3.525, 95% CI: 1.054–9.060, P = 0.0228). Furthermore, the survival rate of the CD44v9-induced group was significantly (P = 0.04) worse than the CD44v9-non-induced group.

Conclusions

CCRT-induced CD44v9-expressing CSCs appear to be a major hurdle to chemoradioselection. CD44v9-targeting seems to be a promising strategy to enhance the efficacy of chemoradioselection and consequent organ preservation and survival.