Multi‐regional sequencing reveals intratumor heterogeneity and positive selection of somatic mtDNA mutations in hepatocellular carcinoma and colorectal cancer

Recent studies have revealed significant intratumor heterogeneity (ITH) of nuclear genome mutations and highlighted its function in tumor progression and treatment resistance. However, the ITH of somatic mitochondrial DNA (mtDNA) mutations detected in cancers remains unknown. In this study, we performed multiregional mtDNA sequencing of tumor and paratumor tissue samples from 12 hepatocellular carcinoma (HCC) and 13 colorectal cancer (CRC) patients. A substantial level of mtDNA mutations was found in paired non‐HCC inflammatory tissues, suggesting that these tissues might not be mtDNA‐genetically “normal.” Moreover, our data indicated that the ITH of somatic mtDNA mutations was a common feature in HCC and CRC patients. In addition, we found that shared mutations which were observed in at least 2 samples in each patient exhibited a significantly higher heteroplasmic level than mutations that were private to a specific tumor region from both HCC (p = 0.039) and CRC patients (p = 0.001). The heteroplasmic level of shared mutations was positively correlated with intratumoral recurrence of mtDNA mutations. We also found that shared mutations in tumor tissues with a higher degree of pathogenicity risk exhibited a higher heteroplasmic level and intratumoral recurrence in both HCC and CRC patients. These findings suggest that some mtDNA mutations may undergo positive selection during the clonal expansion. Taken together, our analyses identified various levels of ITH of somatic mtDNA mutations in HCC and CRC patients and provided evidence supporting the positive selection working on some somatic mtDNA mutations in tumor tissues.     

Survival of squamous cell carcinoma of the head and neck in relation to human papillomavirus infection: review and meta-analysis

Human papillomavirus (HPV) has been associated with head and neck squamous cell carcinomas (HNSCC), especially of the oropharynx, with highest distribution in the tonsils. HPV infection has been associated with improved outcome, although not all the studies show consistent results. The reason for this is not clear. We reviewed all published articles and conducted a meta-analysis on the overall relationship between HPV infection and overall survival (OS) and disease-free survival (DFS) in HNSCC. Patients with HPV-positive HNSCC had a lower risk of dying (meta HR: 0.85, 95% CI: 0.7-1.0), and a lower risk of recurrence (meta HR: 0.62, 95%CI: 0.5-0.8) than HPV-negative HNSCC patients. Site-specific analyses show that patients with HPV-positive oropharyngeal tumours had a 28% reduced risk of death (meta HR: 0.72, 95%CI: 0.5-1.0) in comparison to patients with HPV-negative oropharyngeal tumours. Similar observations were made for DFS (meta HR: 0.51, 95% CI: 0.4-0.7). There was no difference in OS between HPV-positive and negative non-oropharyngeal patients. The observed improved OS and DFS for HPV-positive HNSCC patients is specific to the oropharynx; these tumours may have a distinct etiology from those tumours in non-oropharyngeal sites.     

Serum matrix metalloproteinase-9 in head and neck squamous cell carcinoma is a prognostic marker

The aim of this study was to determine whether serum matrix metalloproteinase-9 (MMP-9) could predict cause-specific and relapse-free survival in patients with squamous cell carcinoma of head and neck. Furthermore, this study was designed to investigate whether there is an association between MMP-9 immunohistochemical staining and serum MMP-9 levels. Pretreatment serum levels of MMP-9 were quantitatively measured by ELISA assay in 67 patients presenting with a primary head and neck squamous cell carcinoma. The results were compared with the corresponding immunohistochemical staining results, clinical data and the patients' outcome. The follow-up time for all of the patients was at least 5 years. There was a statistically significant correlation between circulating MMP-9 and MMP-9 immunohistochemical staining in the corresponding tumors (p = 0.028). The cause-specific and relapse-free survival rates were clearly lower among patients with high MMP-9 serum levels (> 73 ng/ml). The 5-year cause-specific survival-rate was 40% in a patient group with high serum MMP-9, and 69% for patients with a low MMP-9 level (p = 0.027). In the same follow-up period, the cumulative relapse-free survival rate was 36% in patients presenting with a high serum MMP-9 and 66% in those with a low MMP-9 level. No correlation was found between MMP-9 serum levels and the traditional clinical or histopathologic factors. The results suggest for the first time that pretreatment serum MMP-9 level could serve as a prognostic factor in head and neck squamous cell carcinoma.     

Cathepsin D in tissue and serum of patients with squamous cell carcinoma of the head and neck

Aspartic proteinase cathepsin D (CD) is believed to be associated with proteolytic processes leading to local invasion and seeding of tumour cells. To estimate a potential prognostic value of cathepsin D in squamous cell carcinoma of the head and neck, its total concentration was measured immunoradiometrically (ELSA-CATH-D kit, CIS bio international) in cytosols of tumour and adjacent normal tissue samples from 111 patients; in 42/111 patients, the CD concentration was determined in serum samples obtained at diagnosis (serum no. 1) and after the therapy (serum no. 2) from each of these patients. Sera of 15 healthy volunteers served as controls. A significantly elevated concentration of CD was measured in tumour cytosols as compared to normal tissue cytosols (31.1 versus 12.6 pmol/mgp, P<0.0001) and in cytosols of normal laryngeal tissue than of the oral cavity or pharynx (13.3 versus 11.2 pmol/mgp, P=0.03). The higher CD tumour concentration correlated with the age of the patients (≤60 versus >60 years, 28.8 versus 32.8 pmol/mgp, P=0.045) and histopathological tumour grade (G₁₊₂ versus G₃, 32.6 versus 24.4 pmol/mgp, P=0.02). In serum samples, a lower concentration of CD was measured in the control group than in the patients (3.6 versus 4.1 pmol/mls, P=0.045) and in serum no. 1 than in serum no. 2 (4.1 versus 5.1 pmol/mls, P=0.05). The CD concentration in sera obtained at diagnosis was stage-dependent (S_I–III versus S_IV, 3.9 versus 4.7 pmol/mls, P=0.09); there was a trend towards lower CD concentrations with an increasing time delay in serum no. 2 sampling (R_S=−0.20, P=0.21). No correlation was observed between cytosolic and serum concentrations of CD. We conclude that our results confirm a specific role of CD in the process of invasion and metastasis of squamous cell carcinoma of the head and neck, which might also be of prognostic value in this particular cancer type.     

Current applications and challenges of circulating tumor DNA (ctDNA) in squamous cell carcinoma of the head and neck (SCCHN)

Liquid biopsies (LB) are emerging in the oncology field, with promising data as new diagnostic, prognostic and treatment-monitoring tools. Squamous cell carcinoma of the head and neck (SCCHN) is a heterogenous disease and many challenges remain to improve patient outcomes. Liquid biopsy could be of interest at different stages of SCCHN disease, including better screening to diagnose more patients at an early stage, early detection of relapse after curative treatment, and the implementation of precision medicine. As LB is very attractive by the ease of sampling, this field is moving fast. Therefore, it is important to be aware of the potential applications but also the limitations of these new tools in regards to technical aspects and interpretation of the data. In this review, we will first give an overview of potential clinical applications and technical challenges of circulating tumor DNA (ctDNA) and then focus on current available data of ctDNA in SCCHN. Although the literature on ctDNA analysis for SCCHN is scarce compared to other tumors, preliminary results seem to hold promise for the future, including the detection of minimal residual disease or the detection of potentially targetable events through liquid biopsy. Prospective liquid-biopsy driven clinical trials are needed to validate its clinical relevance.     

MicroRNA-137 promoter methylation is associated with poorer overall survival in patients with squamous cell carcinoma of the head and neck

BACKGROUND:

The overall 5‐year survival rate of approximately 60% for head and neck cancer patients has remained essentially unchanged over the past 30 years. MicroRNA‐137 (miR‐137) plays an essential role in cell‐cycle control at the G1/S‐phase checkpoint. However, the aberrant miR‐137 promoter methylation observed in squamous cell carcinoma of the head and neck (SCCHN) suggests a tumor‐specific molecular defect that may contribute to disease progression.

METHODS:

The goal of this study was to assess, in formalin‐fixed, paraffin‐embedded tumor tissue, the association between miR‐137 promoter methylation and survival (both overall and disease free) and with prognostic factors including stage, tumor size, lymph node positivity, tumor grade, and surgical tumor margin positivity.

RESULTS:

The promoter methylation status of miR‐137 was ascertained by methylation‐specific polymerase chain reaction and detected in 11 of 67 SCCHN patients (16.4%), with no significant differences according to site (oral cavity, pharynx, larynx). Methylation of the miR‐137 promoter was significantly associated with overall survival (hazard ratio, 3.68; 95% confidence interval, 1.01‐13.38) but not with disease‐free survival or any of the prognostic factors evaluated.

CONCLUSIONS:

The results of this study indicate that miR‐137 is methylated in tumor tissue from pharyngeal and laryngeal squamous cancers, in addition to oral squamous cell carcinoma, and that miR‐137 promoter methylation has potential utility as a prognostic marker for SCCHN. Cancer 2011. © 2010 American Cancer Society.     

Global gene expression profiles of human head and neck squamous carcinoma cell lines

For genomewide monitoring and identification of biomarkers of head and neck squamous cell carcinoma (HNSCC), we have conducted a systematic characterization of gene expression profiles, using human cDNA microarrays containing 9K clones, in 25 HNSCC cell lines and 1 immortalized human oral keratinocyte cell line. We used normal human oral keratinocytes (NHOKs) as a reference. Our study showed that genes primarily involved in cell cycle regulation, oncogenesis, cell proliferation, differentiation, apoptosis and cell adhesion were widely altered in the 26 cell lines. Upregulated genes included known oncogenes, protein kinases, DNA-binding proteins and cell cycle regulators, while those commonly downregulated included differentiation markers, cell adhesion proteins, extracellular matrix proteins, structural proteins (keratins) and protease inhibitor proteins. Compared to NHOK, we observed a striking reduction in the expression of genes involved in terminal differentiation, suggesting that a loss in this process is an important signature of HNSCC. In addition, hierarchical clustering analysis as well as principal component analysis revealed 2 distinctive subtypes of gene expression patterns among the 26 cell lines, reflecting a degree of heterogeneity in HNSCC. By applying significance analysis of microarrays, 128 genes were selected for being distinctively expressed between the 2 groups. Genes differentially expressed in the 2 subgroups include cell proliferation-related genes, IGFBP6, EGFR and VEGFC; tumor suppression and apoptosis-related genes such as Tp53, Tp63; as well as cell cycle regulators such as CCND1 and CCND2 (cyclins D1 and D2), suggesting that the 2 subgroups might have undergone different pathways of carcinogenesis.     

Loss of MTBP expression is associated with reduced survival in a biomarker-defined subset of patients with squamous cell carcinoma of the head and neck

BACKGROUND:

Recent genetic studies have implicated p53 mutation as a significant risk factor for therapeutic failure in squamous cell carcinoma of the head and neck (SCCHN). However, in a recent meta‐analysis in the literature of p53 from major anatomical subsites (larynx, oral cavity, oropharynx/hypopharynx), associations between patient survival and p53 status were ambiguous.

METHODS:

The authors examined a cohort of SCCHNs using a previously developed biomarker combination that likely predicts p53 status based on p53/MDM2 expression levels determined by immunohistochemistry (IHC). In addition, the authors generated and validated an antibody to MTBP (an MDM2 binding protein that alters p53/MDM2 homeostasis and may contribute to metastatic suppression) and have incorporated data for MTBP expression into the current analyses.

RESULTS:

Analysis of expression data for p53 and MDM2 in 198 SCCHN patient samples revealed that the biomarker combination p53 + ve/MDM2‐low (likely indicative of p53 mutation) was significantly associated with reduced overall survival (log‐rank P = .035) and was an independent prognostic factor (P = .013; HR, 1.705; 95% CI, 1.12‐2.60); thus, these data were compatible with earlier genetic analyses. By using IHC for p53 and MDM2 to dichotomize patients, the authors found that loss of MTBP expression was significantly associated with reduced survival (log‐rank P = .004) and was an independent prognostic factor (P = .004; HR, 2.78; 95% CI, 1.39‐5.54) in p53 + ve/MDM2‐low patients.

CONCLUSIONS:

These results represent the first examination of MTBP expression in human tissues and provide evidence for a p53 status‐dependent role for MTBP in suppressing disease progression in SCCHN patients as well as confirming a role for p53 pathway function in delaying disease progression. Cancer 2011. © 2011 American Cancer Society.     

Clinical significance of FAT1 gene mutation and mRNA expression in patients with head and neck squamous cell carcinoma

The FAT1 gene functions as a tumor suppressor or promoter and remains incompletely understood. We examined the clinical significance of FAT1 in head and neck squamous cell carcinoma (HNSCC) using four publicly available HNSCC cohorts and one HNSCC cohort enrolled at a tertiary medical center. We developed FAT1 signatures reflecting FAT1 mutations and mRNA expression using one cohort. Patients with HNSCC were classified into FAT1‐associated low risk (FAT1‐LR; n = 195) and FAT1‐associated high risk (FAT1‐HR; n = 371) subgroups. The five‐year overall survival and recurrence‐free survival rates were significantly lower in the FAT1‐HR subgroup than in the FAT1‐LR subgroup (P = 0.01 and 0.003, respectively). The clinical significance of FAT1 was validated using four independent cohorts. Cox proportional hazards models showed that the FAT1 signature was an independent prognostic factor for HNSCC patients. In addition, FAT1 signature was associated with the response to radiotherapy, advanced stage, and human papilloma virus (HPV) status in HNSCC patients. In conclusion, the FAT1 gene signature was associated with prognosis of HNSCC and may help to provide personalized treatments for HNSCC patients.     

Human papilloma virus circulating tumor DNA assay predicts treatment response in recurrent/metastatic head and neck squamous cell carcinoma

Despite the rising incidence of human papillomavirus related (HPV+) oropharyngeal squamous cell carcinoma (OPSCC), treatment of metastatic disease remains palliative. Even with new treatments such as immunotherapy, response rates are low and can be delayed, while even mild tumor progression in the face of an ineffective therapy can lead to rapid death. Real-time biomarkers of response to therapy could improve outcomes by guiding early change of therapy in the metastatic setting. Herein, we developed and analytically validated a new droplet digital PCR (ddPCR)-based assay for HPV16 circulating tumor DNA (ctDNA) and evaluated plasma HPV16 ctDNA for predicting treatment response in metastatic HPV+ OPSCC. We found that longitudinal changes HPV16 ctDNA correlate with treatment response and that ctDNA responses are observed earlier than conventional imaging (average 70 days, range: 35–166). With additional validation in multi-site studies, this assay may enable early identification of treatment failure, allowing patients to be directed promptly toward clinical trials or alternative therapies.     

Role of miRNA in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Evidence suggests that miRNAs play an important role in progression, recurrence, metastasis and postoperative survival of HNSCC. Studies have investigated the utility of miRNAs as diagnostic/prognostic tools and as potential therapeutic targets and biomarkers that may improve the management and outcomes of HNSCC. The aim of this article is to review the current literature on aberrant expression profiles of miRNAs in biopsy samples of HNSCC and their role in cancer development, metastasis, prognosis and survival of these patients. This review gives an overview that miRNAs deregulation play major role in the development of HNSCC. They offer the potential to be used as biomarkers or novel therapeutic targets. Future research is required to test their use in both of these fields.     

Adjuvant radiotherapy improves overall survival for patients with lymph node-positive head and neck squamous cell carcinoma

BACKGROUND: Although adjuvant radiotherapy (RT) is often recommended for locally advanced squamous cell carcinoma of the head and neck (HNSCC), its effect on overall or cancer-specific survival has not been clearly demonstrated. In the current study, the frequency and effect of adjuvant RT on overall survival was investigated in patients with resected lymph node-positive head and neck cancer. METHODS: Within the Surveillance, Epidemiology, and End Results (SEER) database, patients were selected with lymph node-positive HNSCC (American Joint Committee on Cancer and SEER stage 3/4) who were treated either with surgery alone or surgery and RT and were diagnosed between 1988 and 2001. A total of 8795 patients who met the inclusion criteria for analysis comprised the study population, with a median follow-up of 4.3 years for patients still alive at the time of last follow-up. RESULTS: Adjuvant RT was utilized in 84% of patients. Adjuvant RT improved the 5-year overall survival (43.2% [95% confidence interval (95% CI), 41.9-44.4%] for surgery + RT vs 33.4% [95% CI, 30.7-36.0%] for surgery alone; P < .001) and cancer-specific survival (50.9% for surgery + RT vs 42.1% for surgery) on univariate analysis. On multivariate analysis, adjuvant RT (hazards ratio [HR] of 0.78; 95% CI, 0.71-0.86 [P < .001]) remained a significant predictor of improved survival. The significant benefit of radiation on overall survival was noted for lymph node-positive patients with both primary tumors localized to the involved organ (HR of 0.81; 95% CI, 0.71-0.94 [P = .007]) and more locally invasive primary tumors (HR of 0.77; 95% CI, 0.68-0.87 [P < .001]). CONCLUSIONS: In what to the authors' knowledge is the largest reported analysis of adjuvant RT in patients with locally advanced HNSCC published to date, adjuvant RT resulted in an approximately 10% absolute increase in 5-year cancer-specific survival and overall survival for patients with lymph node-positive HNSCC compared with surgery alone. Despite combined surgery and adjuvant RT, outcomes in this high-risk population remain suboptimal, emphasizing the need for continued investigation of innovative treatment approaches.     

Conditional survival in head and neck squamous cell carcinoma: results from the SEER dataset 1973-1998

BACKGROUND: Survival statistics for patients with head and neck squamous cell carcinomas (HNSCC) are commonly calculated from the time of diagnosis. The less commonly employed conditional survival (CS) analyzes survival for patients who have survived a period of time after diagnosis. Useful prognostic information for cancer survivors is provided by CS analysis. Estimated baseline CS parameters for HNSCC were sought using large-scale cancer registry data. METHODS: HNSCC cases identified from the Surveillance, Epidemiology, and End Results (SEER) Program were accessed to identify those diagnosed between 1973 and 1998. Five-year observed, relative, and cumulative CS calculations were performed, with secondary stratification by site, extent of disease, and age. RESULTS: The overall 5-year observed survival for all sites increased from 47.8% for 76,181 included patients from the time of diagnosis to 64.4% for those 43,985 patients alive at 3 years, and thereafter plateaus. The greatest increase in CS was for oropharyngeal cancers, which more than doubled over the first decade of surveillance (26.5%-60%). Distant disease showed a 10-year increase in CS (17.4%-60.4%), whereas localized disease CS was essentially static, ranging from 66.1% to 68.5%; for those over 65 at diagnosis it ranged from 39.9-52.9%, whereas patients <65 years at diagnosis ranged from 53.8-73.5%. CONCLUSIONS: Benchmark CS estimates for domestic HNSCC cohorts were developed from the SEER database. CS is a useful tool to assist clinicians in predicting the probability of demise from HNSCC for patients surviving 1 or more year after diagnosis.     

Hypermethylation of E-cadherin is an independent predictor of improved survival in head and neck squamous cell carcinoma

BACKGROUND.

The loss of E‐cadherin (ECAD) protein expression has been linked to aggressive head and neck squamous cell carcinoma (HNSCC). Promoter hypermethylation of the cadherin 1, type 1 (CDH1) gene (encoding ECAD) is 1 mechanism by which this protein can be inactivated, although this epigenetic alteration of the gene has not been linked conclusively to poorer patient outcome and, in fact, may be associated with better patient prognosis.

METHODS.

The authors investigated the prevalence of CDH1 promoter hypermethylation in a population‐based case series of 340 primary HNSCC tumors using methylation‐specific polymerase chain reaction. They also studied the association between CDH1 hypermethylation and patient demographic characteristics using multivariate analysis and examined the impact of CDH1 hypermethylation on patient survival using both univariate and multivariate methods.

RESULTS.

Hypermethylation of CDH1 was significantly more prevalent (P < .03) among individuals with a low smoking history independent of whether they were seropositive for human papillomavirus type 16 (HPV‐16). Patients who had tumors with CDH1 hypermethylation had significantly better overall survival compared with patients who had tumors without hypermethylation (P < .02; log‐rank test). This effect was independent of HPV‐16 status and demonstrated a significant hazard ratio of 0.5 (95% confidence interval, 0.3‐0.9) in a model that controlled for HPV‐16 serology, age, sex, and tumor stage.

CONCLUSIONS.

The current results suggested that hypermethylation of CDH1 occurs more commonly in patients with HNSCC who are low smokers, suggesting that an additional factor may be driving this epigenetic alteration. Clinically, CDH1 hypermethylation may hold powerful prognostic potential in addition to that observed with HPV serology, and the authors concluded that it should be pursued in additional studies. Cancer 2008. © 2008 American Cancer Society.     

Circulating small non coding RNA signature in head and neck squamous cell carcinoma

The Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common human cancer, causing 350,000 individuals die worldwide each year. The overall prognosis in HNSCC patients has not significantly changed for the last decade. Complete understanding of the molecular mechanisms in HNSCC carcinogenesis could allow an earlier diagnosis and the use of more specific and effective therapies. In the present study we used deep sequencing to characterize small non-coding RNAs (sncRNAs) in serum from HNSCC patients and healthy donors. We identified, for the first time, a multi-marker signature of 3 major classes of circulating sncRNAs in HNSCC, revealing the presence of circulating novel and known miRNAs, and tRNA- and YRNA-derived small RNAs that were significantly deregulated in the sera of HNSCC patients compared to healthy controls. By implementing a triple-filtering approach we identified a subset of highly biologically relevant miRNA-mRNA interactions and we demonstrated that the same genes/pathways affected by somatic mutations in cancer are affected by changes in the abundance of miRNAs. Therefore, one important conclusion from our work is that during cancer development, there seems to be a convergence of oncogenic processes driven by somatic mutations and/or miRNA regulation affecting key cellular pathways.