Immune infiltration at the primary tumor is associated with clinical outcome of patients with extranodal extension of lymph node metastasis in oral cancer.

BACKGROUND: Extranodal extension (ENE) of lymph node metastasis is one of the most reliable prognostic indicators for patients with locally advanced oral cancer. Although multiple reports have found a close relationship between immune infiltration of tumors and patient clinical outcomes, its association with ENE is unknown. METHODS: We identified 234 human papillomavirus-negative (HPV-) oral cavity squamous cell carcinoma (OSCC) patients in The Cancer Genome Atlas and investigated the immune infiltration profiles of primary tumors and their association with survival. RESULTS: Hierarchical clustering analysis clearly classified the overall immune infiltration status in OSCC into high immune or low immune groups. The combination of ENE positivity and low immune infiltration was strongly associated with poor overall survival (OS) compared to the combination of ENE positivity and high immune infiltration [hazard ratio 2.04 (95 %CI, 1.08-3.83); p = 0.024]. The immune infiltration status was not associated with OS rates in patients with ENE-negative or node negative tumors. CONCLUSION: Overall Immune infiltration at the primary site was significantly associated with clinical outcome of OSCC patients with ENE.

TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV-associated oral squamous cell carcinoma.

BACKGROUND: TP53, the most mutated gene in solid cancers, has a profound impact on most hallmarks of cancer. Somatic TP53 mutations occur in high frequencies in head and neck cancers, including oral squamous cell carcinoma (OSCC). Our study aims to understand the role of TP53 gain-of-function mutation in modulating the tumor immune microenvironment (TIME) in OSCC. METHODS: Short hairpin RNA knockdown of mutant p53R172H in syngeneic oral tumors demonstrated changes in tumor growth between immunocompetent and immunodeficient mice. HTG EdgeSeq targeted messenger RNA sequencing was used to analyze cytokine and immune cell markers in tumors with inactivated mutant p53R172H. Flow cytometry and multiplex immunofluorescence (mIF) confirmed the role of mutant p53R172H in the TIME. The gene expression of patients with OSCC was analyzed by CIBERSORT and mIF was used to validate the immune landscape at the protein level. RESULTS: Mutant p53R172H contributes to a cytokine transcriptome network that inhibits the infiltration of cytotoxic CD8+ T cells and promotes intratumoral recruitment of regulatory T cells and M2 macrophages. Moreover, p53R172H also regulates the spatial distribution of immunocyte populations, and their distribution between central and peripheral intratumoral locations. Interestingly, p53R172H-mutated tumors are infiltrated with CD8+ and CD4+ T cells expressing programmed cell death protein 1, and these tumors responded to immune checkpoint inhibitor and stimulator of interferon gene 1 agonist therapy. CIBERSORT analysis of human OSCC samples revealed associations between immune cell populations and the TP53R175H mutation, which paralleled the findings from our syngeneic mouse tumor model. CONCLUSIONS: These findings demonstrate that syngeneic tumors bearing the TP53R172H gain-of-function mutation modulate the TIME to evade tumor immunity, leading to tumor progression and decreased survival.

Dysregulation and Epigenetic Reprogramming of NRF2 Signaling Axis Promote Acquisition of Cisplatin Resistance and Metastasis in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. EXPERIMENTAL DESIGN: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. RESULTS: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. CONCLUSIONS: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1-NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Evolutionary Action Score of TP53 Analysis in Pathologically High-Risk Human Papillomavirus-Negative Head and Neck Cancer From a Phase 2 Clinical Trial: NRG Oncology Radiation Therapy Oncology Group 0234.

Purpose: An evolutionary action scoring algorithm (EAp53) based on phylogenetic sequence variations stratifies patients with head and neck squamous cell carcinoma (HNSCC) bearing TP53 missense mutations as high-risk, associated with poor outcomes, or low-risk, with similar outcomes as TP53 wild-type, and has been validated as a reliable prognostic marker. We performed this study to further validate prior findings demonstrating that EAp53 is a prognostic marker for patients with locally advanced HNSCC and explored its predictive value for treatment outcomes to adjuvant bio-chemoradiotherapy. Methods and Materials: Eighty-one resection samples from patients treated surgically for stage III or IV human papillomavirus-negative HNSCC with high-risk pathologic features, who received either radiation therapy + cetuximab + cisplatin (cisplatin) or radiation therapy + cetuximab + docetaxel (docetaxel) as adjuvant treatment in a phase 2 study were subjected to TP53 targeted sequencing and EAp53 scoring to correlate with clinical outcomes. Due to the limited sample size, patients were combined into 2 EAp53 groups: (1) wild-type or low-risk; and (2) high-risk or other. Results: At a median follow-up of 9.8 years, there was a significant interaction between EAp53 group and treatment for overall survival (P = .008), disease-free survival (P = .05), and distant metastasis (DM; P = .004). In wild-type or low-risk group, the docetaxel arm showed significantly better overall survival (hazard ratio [HR] 0.11, [0.03-0.36]), disease-free survival (HR 0.24, [0.09-0.61]), and less DM (HR 0.04, [0.01-0.31]) than the cisplatin arm. In high-risk or other group, differences between treatments were not statistically significant. Conclusions: The docetaxel arm was associated with better survival than the cisplatin arm for patients with wild-type or low-risk EAp53. These benefits appear to be largely driven by a reduction in DM.

Fusobacterium is enriched in oral cancer and promotes induction of programmed death-ligand 1 (PD-L1).

Recently, increased number of studies have demonstrated a relationship between the oral microbiome and development of head and neck cancer, however, there are few studies to investigate the role of oral bacteria in the context of the tumor microenvironment in a single head and neck subsite. Here, paired tumor and adjacent normal tissues from thirty-seven oral tongue squamous cell carcinoma (SCC) patients were subjected to 16S rRNA gene sequencing and whole exome sequencing (WES), in addition to RNA sequencing for tumor samples. We observed that Fusobacterium was significantly enriched in oral tongue cancer and that Rothia and Streptococcus were enriched in adjacent normal tissues. A decrease in alpha diversity was found in tumor when compared to adjacent normal tissues. While increased Fusobacterium in tumor samples was not associated with changes in immune cell infiltration, it was associated with increased PD-L1 mRNA expression. Therefore, we examined the effects of Fusobacterium on PD-L1 expression in head and neck SCC cell lines. We demonstrated that infection with Fusobacterium species can increase both PD-L1 mRNA and surface PD-L1 protein expression on head and neck cancer cell lines. The correlation between Fusobacterium and PD-L1 expression in oral tongue SCC, in conjunction with the ability of the bacterium to induce PD-L1 expression in vitro suggests a potential role for Fusobacterium on modulation of the tumor immune microenvironment in head and neck cancer.

High-Risk TP53 Mutations Are Associated with Extranodal Extension in Oral Cavity Squamous Cell Carcinoma.

Purpose: Development of extranodal extension (ENE) has been associated with poor survival in patients with oral cavity squamous cell carcinoma (OSCC). Here, we sought to confirm the role of ENE as a poor prognostic factor, and identify genomic and epigenetic markers of ENE in order to develop a predictive model and improve treatment selection.Experimental Design: An institutional cohort (The University of Texas MD Anderson Cancer Center) was utilized to confirm the impact of ENE on clinical outcomes and evaluate the genomic signature of primary and ENE containing tissue. OSCC data from The Cancer Genome Atlas (TCGA) were analyzed for the presence of molecular events associated with nodal and ENE status.Results: ENE was associated with decreased overall and disease-free survival. Mutation of the TP53 gene was the most common event in ENE+ OSCC. The frequency of TP53 mutation in ENE+ tumors was higher compared with ENE- tumors and wild-type (WT) TP53 was highly represented in pN0 tumors. pN+ENE+ patients had the highest proportion of high-risk TP53 mutations. Both primary tumors (PT) and lymph nodes with ENE (LN) exhibited a high rate of TP53 mutations (58.8% and 58.8%, respectively) with no significant change in allele frequency between the two tissue sites.Conclusions: ENE is one of the most significant markers of OSCC OS and DFS. There is a shift toward a more aggressive biological phenotype associated with high-risk mutations of the TP53 gene. Prospective clinical trials are required to determine whether TP53 mutational status can be used for personalized treatment decisions. Clin Cancer Res; 24(7); 1727-33. ©2018 AACR.

Characterizing Genetic Transitions of Copy Number Alterations and Allelic Imbalances in Oral Tongue Carcinoma Metastasis.

Primary tumor (PT) heterogeneity can significantly affect the genetic profile of clones at metastatic sites. To understand the mechanisms underlying metastasis, we compared the genetic profile of paired PT and metastatic lymph node (MLN) samples obtained from patients with oral tongue squamous cell carcinoma (OTSCC). Large-scale genetic profiling was performed on paired PT-MLN samples obtained from 10 OTSCC patients using high-density single-nucleotide polymorphism microarrays. We compared the genetic profile of PT and MLN OTSCC samples to identify common and specific copy number alterations and copy-neutral loss-of-heterozygosity (CN-LOH). Unsupervised hierarchical clustering analysis indicated that 8 of the 10 PT-MLN sample pairs formed clusters, indicating that the primary and metastatic tumors were composed of predominantly genetically similar tumor cells. In 6 of the 10 pairs, 8q11.21, 8q12.2-3, and 8q21.3 gains, and 22q11.23 loss were detected in both the PT and MLN. In addition, 16p11.2 CN-LOH was identified in 9 of the 10 pairs. Conversely, 20q11.2 gain was only observed in the MLNs of 5 of the 10 sample pairs, indicating that genes in this chromosomal region may play a significant role in OTSCC lymph node metastasis. To confirm this, we investigated the expression of two candidate 20q11.2 genes in a separate patient cohort. The expression of one of these genes, E2F1, was significantly increased during the process of metastasis. This study indicates that additional genetic changes, such as 20q11.2 gain, which encodes the E2F1 gene, can be acquired through clonal evolution, and may be required for the metastatic process. © 2016 Wiley Periodicals, Inc.

The high-temperature requirement factor A3 (HtrA3) is associated with acquisition of the invasive phenotype in oral squamous cell carcinoma cells.

OBJECTIVES: Previous studies have identified several genes involved in the carcinogenesis of oral cancer; however, the detailed mechanisms underlying this process have not been elucidated. Previously, we established a database of the transcriptional progression profile of oral carcinogenesis and identified 15 candidate genes with continuously increasing or decreasing expression (Sumino et al., 2013). MATERIALS AND METHODS: In the present study, using this database, we attempted to identify genes that may specifically contribute to progression from oral dysplastic lesions to invasive tumours. RESULTS: We identified 4 candidate genes. Using a literature survey, we narrowed down the candidates and focused on the high-temperature requirement factor A3 (HtrA3). Quantitative real-time reverse transcription polymerase chain reaction and immunohistochemical analysis confirmed that HtrA3 expression significantly increased during this process. In addition, high HtrA3 expression was significantly associated with decreased disease-free survival (P=0.045) and overall survival (P=0.003). Multivariate Cox proportional hazards analysis found that high HtrA3 expression significantly correlated with overall survival (P=0.018). CONCLUSION: The findings of this study demonstrated that the HtrA3 is likely to be associated with the acquisition of the invasive phenotype in oral squamous cell carcinoma cells and may be a potential prognostic marker for oral cancer.

Gene expression changes in initiation and progression of oral squamous cell carcinomas revealed by laser microdissection and oligonucleotide microarray analysis.

Oral carcinogenesis is a complex process involving multiple genes. However, the genetic changes involved in this process are not apparent in identical oral squamous cell carcinomas (OSCCs). According to pathological characteristics, samples of normal tissue, oral dysplastic lesions (ODLs), and invasive cancers were obtained from identical OSCCs using laser microdissection (LMD). Large-scale gene expression profiling was carried out on 33 samples derived from 11 OSCCs. We analyzed genes differentially expressed in normal tissues vs. ODLs and in ODLs vs. invasive tumors and identified 15 candidate genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these genes, ISG15, was chosen for further characterization. Real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that ISG15 expression consistently increased during oral tumorigenesis. An ISG15 high-expression level was significantly associated with poor prognosis (p = 0.027). In addition, patients with high-expression tumors had a poorer 5-year survival rate than patients with low expression levels (p = 0.019). In conclusion, we identified 15 genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these, ISG15, is likely to be associated with both dysgenesis and tumorigenesis and may be a potential prognostic marker for oral cancer.

Loss of NKX3-1 as a potential marker for an increased risk of occult lymph node metastasis and poor prognosis in oral squamous cell carcinoma.

The prognosis of oral squamous cell carcinoma (OSCC) is significantly dependent on the existence of cervical lymph node metastasis (LNM), with the overall survival rate being much lower in patients with LNM. Primary causes and molecular mechanisms of LNM are still largely unclear. We hypothesized that factors related with cancer progress and/or prognosis in OSCC are revealed by genome-wide investigation of DNA copy number aberrations (CNAs). In order to find biomarkers for occult LNM of OSCC, we comprehensively investigated genomic DNAs from 60 OSCC patients using Affymetrix mapping arrays and statistically analyzed correlations between CNAs of genes and the presence of occult LNM in the patients. The genome-wide CNA study indicated significant correlations between the presence of occult LNM and CNAs of certain genes. Through a literature survey, we narrowed down the candidates and focused on loss of NKX3-1, which is a homeodomain-containing transcription factor. NKX3-1 is known as a tumor suppressor gene in prostate cancer but has never been reported in OSCC. Quantitative RT-PCR and immunohistochemistry (IHC) analyses also showed significantly lower expression of NKX3-1 in the cases with occult LNM, which was further validated by IHC analysis in independent cases. The survival analyses indicated that NKX3-1 loss is a significant risk factor to decrease the disease-free survival (DFS) and the overall survival (OS) rates. This is the first time that the significant association of NKX3-1 loss and occult LNM was indicated in OSCC. The present results suggest that loss of NKX3-1 may be a potential biomarker for occult LNM of OSCC.

Clinical significance of lymphatic and blood vessel invasion in oral tongue squamous cell carcinomas.

Although vascular invasion (VI) is recognized as an important predictor of lymph node metastasis and a significant prognostic factor in head and neck squamous cell carcinoma (HNSCC), there is currently no common definition for the pathological evaluation of VI status. We reviewed the medical records of 63 consecutive resected primary oral tongue SCCs (OTSCCs) without preoperative treatment between June 1999 and April 2008, and evaluated VI status by investigating lymphatic vessel invasion (LVI) and blood vessel invasion (BVI) by using immunohistochemistry (IHC) with monoclonal antibody D2-40 (D2-40) and Elastica van Gieson (EVG) staining, respectively. Subsequently, we analyzed their correlations with cervical lymph node metastasis and prognosis. LVI was found in 16 of the 63 tumors (25.4%) and BVI was in 32 tumors (50.8%). Univariate analysis revealed that the presence of LVI is statistically correlated with lymph node metastasis. Moreover, multivariate logistic regression analysis revealed that LVI is an independent risk factor of nodal metastasis (odds ratio=4.262, 95% confidence interval=1.262-14.397, p=0.020). In contrast, Kaplan-Meier survival analysis revealed that patients with BVI had a significantly shorter disease-free survival (DFS) and overall survival (OS) rates than those without BVI (68.6% versus 90.3%, p=0.028 and 68.6% versus 93.5%, p=0.013, respectively). The present study clearly demonstrated that LVI at primary OTSCC had significant correlation with lymph node metastasis, and that BVI was significantly associated with recurrence and poor prognosis. Evaluation of VI status, as LVI and BVI status separately, using IHC with D2-40 and EVG staining may be useful in predicting lymph node metastasis and poor prognosis in OTSCCs.

EGFR gene copy number alteration is a better prognostic indicator than protein overexpression in oral tongue squamous cell carcinomas.

Although epidermal growth factor receptor (EGFR) is particularly important in the pathogenesis of head and neck squamous cell carcinomas (HNSCCs), conflicting data have been reported on the correlation between EGFR copy number and survival and the association between EGFR copy number and protein expression. Anatomical site of the tumour in HNSCCs may likely contribute to the discordance of the above points as EGFR expression may differ between the sub-sites of HNSCCs. Thus, in this study, we focused on oral tongue squamous cell carcinomas (OTSCCs). To investigate the association between EGFR copy number alteration and overexpression and to determine which is the more reliable prognostic indicator, Fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were performed at a single institution on samples from 89 patients with OTSCCs undergoing surgery as the primary treatment modality. Thirty-two (36%) of 89 cases demonstrated an EGFR copy number alteration. EGFR protein expression was found in all 89 cases, of which 82.0% showed overexpression. No significant correlation was found between gene copy number and protein overexpression. Gene copy number alteration was significantly associated with reduced disease-free survival (P=0.048) and overall survival (P=0.001). Multivariate Cox proportional hazards analysis demonstrated that EGFR copy number increase was significantly correlated with overall survival (P=0.001). EGFR copy number status is a more reliable indicator than protein overexpression of the survival rate in OTSCCs. FISH analysis of the EGFR status is useful in predicting poor prognosis in OTSCCs.