Switching anti-EGFR antibody re-sensitizes head and neck cancer patient following acquired resistance to cetuximab.

Cetuximab induces responses in about 13% of head and neck squamous cell carcinomas (HNSCC). We describe the molecular mechanism of acquired resistance to cetuximab, which could be overcome by switching to a different anti-EGFR antibody. Biopsies were collected at three different time points: before the start of cetuximab (PRE-cetux), at acquired resistance to cetuximab (AR-cetux), and at acquired resistance to duligotuzumab (AR-duligo). Biopsies were analyzed using tumor and normal whole-exome sequencing, RNASeq, and targeted panel sequencing with ultra-deep coverage to generate differential mutation and expression profiles. WES and targeted sequencing analysis identified an EGFR p.G465R extracellular domain mutation in AR-cetux biopsy. Furthermore, RNASeq confirmed the expression of this mutation in the tumor tissue. This mutation prevented the binding of cetuximab to EGFR and was not present in PRE-cetux and AR-duligo biopsies, suggesting a potential mechanism of acquired resistance to cetuximab. Molecular dynamic simulations confirmed that duligotuzumab effectively binds EGFR with a p.G465R mutation. Interestingly, the p.G465R mutation improved the stability of the duligotuzumab-EGFR complex as compared to the wild-type EGFR. This is the first report of an EGFR ECD mutation associated with acquired resistance to cetuximab, posing a need for further validation. We suggest appropriate serial mutational profiling to identify ECD mutations should be considered for select patients with initial cetuximab benefit.

Sialyltransferases and Neuraminidases: Potential Targets for Cancer Treatment.

Cancers are the leading cause of death, causing around 10 million deaths annually by 2020. The most common cancers are those affecting the breast, lungs, colon, and rectum. However, it has been noted that cancer metastasis is more lethal than just cancer incidence and accounts for more than 90% of cancer deaths. Thus, early detection and prevention of cancer metastasis have the capability to save millions of lives. Finding novel biomarkers and targets for screening, determination of prognosis, targeted therapies, etc., are ways of doing so. In this review, we propose various sialyltransferases and neuraminidases as potential therapeutic targets for the treatment of the most common cancers, along with a few rare ones, on the basis of existing experimental and in silico data. This compilation of available cancer studies aiming at sialyltransferases and neuraminidases will serve as a guide for scientists and researchers working on possible targets for various cancers and will also provide data about the existing drugs which inhibit the action of these enzymes.

Low expression of TMPRSS2-a SARS-CoV-2 internalization protease-associates with basal subtype of head and neck squamous cell carcinoma.

SARS-CoV-2 is a single-stranded RNA virus that has caused the ongoing COVID-19 pandemic. ACE2 and other genes utilized by SARS-CoV-2 to enter human cells have been shown to express in Head and Neck Squamous Cell Carcinoma (HNSCC) patients. However, their expression pattern in different subtypes has not been investigated. Hence, in the current study, we have analyzed the expression of ACE2, TMPRSS2 and FURIN in 649 HNSCC patients from two independent cohorts. Our analysis showed significantly lower expression of TMPRSS2 while significantly increased expression of ACE2 and FURIN in HPV-negative HNSCC. Comparison of expression of these genes in the three subtypes of HNSCC patients (basal, classical and inflamed/mesenchymal) showed no significant difference in the expression of ACE2 among the three subtypes; however, the basal subtype showed significantly reduced expression of TMPRSS2 but significantly increased expression of FURIN. Comparison of expression of these genes between the HPV-negative patients of basal subtype vs all others confirmed significantly lower expression of TMPRSS2 in HPV-negative patients of basal subtype as compared to all others. Our study shows that the different subtypes of HNSCC patients have different expression patterns of genes utilized by the SARS-CoV-2 to enter human cells, and hence, their susceptibility to SARS-CoV-2 may also be different. As the expression of TMPRSS2 is significantly lower in the HNSCC patients of the basal subtype, we predict that these patients would be less susceptible to SARS-CoV-2 infection than the patients of other subtypes. However, these findings need to be further validated.

Therapeutic approaches for the treatment of head and neck squamous cell carcinoma-An update on clinical trials.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common non-skin cancer with a tobacco consumption and infection with high-risk human papillomavirus (HPV) being major risk factors. Despite advances in numerous therapy modalities, survival rates for HNSCC have not improved considerably; a vast number of clinical outcomes have demonstrated that a combination strategy (the most well-known docetaxel, cisplatin, and 5-fluorouracil) is the most effective treatment choice. Immunotherapy that targets immunological checkpoints is being tested in a number of clinical trials, either alone or in conjunction with chemotherapeutic or targeted therapeutic drugs. Various monoclonal antibodies, such as cetuximab and bevacizumab, which target the EGFR and VEGFR, respectively, as well as other signaling pathway inhibitors, such as temsirolimus and rapamycin, are also being studied for the treatment of HNSCC. We have reviewed the primary targets in active clinical studies in this study, with a particular focus on the medications and drug targets used.

Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is continuously evolving, and this poses a major threat to antibody therapies and currently authorized Coronavirus Disease 2019 (COVID-19) vaccines. It is therefore of utmost importance to investigate and predict the putative mutations on the spike protein that confer immune evasion. Antibodies are key components of the human immune system's response to SARS-CoV-2, and the spike protein is a prime target of neutralizing antibodies (nAbs) as it plays critical roles in host cell recognition, fusion, and virus entry. The potency of therapeutic antibodies and vaccines partly depends on how readily the virus can escape neutralization. Recent structural and functional studies have mapped the epitope landscape of nAbs on the spike protein, which illustrates the footprints of several nAbs and the site of escape mutations. In this review, we discuss (1) the emerging SARS-CoV-2 variants; (2) the structural basis for antibody-mediated neutralization of SARS-CoV-2 and nAb classification; and (3) identification of the RBD escape mutations for several antibodies that resist antibody binding and neutralization. These escape maps are a valuable tool to predict SARS-CoV-2 fitness, and in conjunction with the structures of the spike-nAb complex, they can be utilized to facilitate the rational design of escape-resistant antibody therapeutics and vaccines.

Immune-related adverse events are associated with improved response, progression-free survival, and overall survival for patients with head and neck cancer receiving immune checkpoint inhibitors.

BACKGROUND: The authors hypothesized that patients developing immune-related adverse events (irAEs) while receiving immune checkpoint inhibition (ICI) for recurrent/metastatic head and neck cancer (HNC) would have improved oncologic outcomes. METHODS: Patients with recurrent/metastatic HNC received ICI at 2 centers. Univariate and multivariate logistic regression, Kaplan-Meier methods, and Cox proportional hazards regression were used to associate the irAE status with the overall response rate (ORR), progression-free survival (PFS), and overall survival (OS) in cohort 1 (n = 108). These outcomes were also analyzed in an independent cohort of patients receiving ICI (cohort 2; 47 evaluable for irAEs). RESULTS: The median follow-up was 8.4 months for patients treated in cohort 1. Sixty irAEs occurred in 49 of 108 patients with 5 grade 3 or higher irAEs (10.2%). ORR was higher for irAE+ patients (30.6%) in comparison with irAE- patients (12.3%; P = .02). The median PFS was 6.9 months for irAE+ patients and 2.1 months for irAE- patients (P = .0004), and the median OS was 12.5 and 6.8 months, respectively (P = .007). Experiencing 1 or more irAEs remained associated with ORR (P = .03), PFS (P = .003), and OS (P = .004) in multivariate analyses. The association between development of irAEs and prolonged OS persisted in a 22-week landmark analysis (P = .049). The association between development of irAEs and favorable outcomes was verified in cohort 2. CONCLUSIONS: The development of irAEs was strongly associated with an ICI benefit, including overall response, PFS, and OS, in 2 separate cohorts of patients with recurrent/metastatic HNC.

Identification and Evaluation of Apoptosis-Inducing Activity of Ipomone from Ipomoea nil: A Novel, Unusual Bicyclo-[3.2.1] Octanone Containing Gibberic Acid Diterpenoid.

Ipomone (1), a novel diterpenoid along with seven known compounds (2-8), was isolated for the first time from the acidified hydroalcoholic extract of Ipomoea nil seeds. The structures of the isolated compounds were elucidated via comprehensive NMR spectroscopic data. The absolute configuration of 1 was ascertained through NOESY, NMR, and ECD analyses. Compound 1 was found to contain an unusual bicyclo-[3.2.1] octanone, which appeared first time in any natural product that might be an artifact resulting from the acid-catalyzed 1,2 alkyl shift/rearrangement. The novel compound was screened for cytotoxic activity against a panel of 12 human cancer cell lines and exhibited weak cytotoxicity with IC50 values in the range of 34-86 µM (except for HEK-293 cells). Microscopic studies revealed that compound 1 induced apoptosis and autophagy in A549 cells. To further explore the signaling pathway involved, immunoblot analysis was performed that confirmed inhibition of apoptotic proteins PARP-1 and caspase-3 expression and upregulation of LC3B expression by compound 1. The compound was further subjected to molecular docking studies to evaluate its binding affinity with p110α, PARP-1, and caspase-3 proteins.

Pan-Cancer Analysis Shows Enrichment of Macrophages, Overexpression of Checkpoint Molecules, Inhibitory Cytokines, and Immune Exhaustion Signatures in EMT-High Tumors.

Epithelial-mesenchymal transition (EMT) is a highly dynamic process that occurs under normal circumstances; however, EMT is also known to play a central role in tumor progression and metastasis. Furthermore, role of tumor immune microenvironment (TIME) in shaping anticancer immunity and inducing the EMT is also well recognized. Understanding the key features of EMT is critical for the development of effective therapeutic interventions. Given the central role of EMT in immune escape and cancer progression and treatment, we have carried out a pan-cancer TIME analysis of The Cancer Genome Atlas (TCGA) dataset in context to EMT. We have analyzed infiltration of various immune cells, expression of multiple checkpoint molecules and cytokines, and inflammatory and immune exhaustion gene signatures in 22 cancer types from TCGA dataset. A total of 16 cancer types showed a significantly increased (p < 0.001) infiltration of macrophages in EMT-high tumors (mesenchymal samples). Furthermore, out of the 17 checkpoint molecules we analyzed, 11 showed a significant overexpression (p < 0.001) in EMT-high samples of at least 10 cancer types. Analysis of cytokines showed significant enrichment of immunosuppressive cytokines-TGFB1 and IL10-in the EMT-high group of almost all cancer types. Analysis of various gene signatures showed enrichment of inflammation, exhausted CD8+ T cells, and activated stroma signatures in EMT-high tumors. In summary, our pan-cancer EMT analysis of TCGA dataset shows that the TIME of EMT-high tumors is highly immunosuppressive compared to the EMT-low (epithelial) tumors. The distinctive features of EMT-high tumors are as follows: (i) the enrichment of tumor-associated macrophages, (ii) overexpression of immune checkpoint molecules, (iii) upregulation of immune inhibitory cytokines TGFB1 and IL10, and (iv) enrichment of inflammatory and exhausted CD8+ T-cell signatures. Our study shows that TIMEs of different EMT groups differ significantly, and this would pave the way for future studies analyzing and targeting the TIME regulators for anticancer immunotherapy.

Immune profiles in primary squamous cell carcinoma of the head and neck.

OBJECTIVES: In this study we describe the tumor microenvironment, the signaling pathways and genetic alterations associated with the presence or absence of CD8+ T-cell infiltration in primary squamous cell carcinoma of the head and neck (SCCHN) tumors. MATERIALS AND METHODS: Two SCCHN multi-analyte cohorts were utilized, the Cancer Genome Atlas (TCGA) and the Chicago Head and Neck Genomics (CHGC) cohort. A well-established chemokine signature classified SCCHN tumors into high and low CD8+ T-cell inflamed phenotypes (TCIP-H, TCIP-L respectively). Gene set enrichment and iPANDA analyses were conducted to dissect differences in signaling pathways, somatic mutations and copy number aberrations for TCIP-H versus TCIP-L tumors, stratified by HPV status. RESULTS: TCIP-H SCCHN tumors were enriched in multiple immune checkpoints irrespective of HPV-status. HPV-positive tumors were enriched in markers of T-regulatory cells (Tregs) and HPV-negative tumors in protumorigenic M2 macrophages. TCIP-L SCCHN tumors were enriched for the ß-catenin/WNT and Hedgehog signaling pathways, had frequent mutations in NSD1, amplifications in EGFR and YAP1, as well as CDKN2A deletions. TCIP-H SCCHN tumors were associated with the MAPK/ERK, JAK/STAT and mTOR/AKT signaling pathways, and were enriched in CASP8, EP300, EPHA2, HRAS mutations, CD274, PDCD1LG2, JAK2 amplifications. CONCLUSIONS: Our findings support that combinatorial immune checkpoint blockade and depletion strategies targeting Tregs in HPV-positive and M2 macrophages in HPV-negative tumors may lead to improved antitumor immune responses in patients with TCIP-H SCCHN. We highlight novel pathways and genetic events that may serve as candidate biomarkers and novel targeted therapies to enhance the efficacy of immunotherapy in SCCHN patients.

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer.

PURPOSE: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy. EXPERIMENTAL DESIGN: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n = 275) and validated using two independent cohorts (n = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining. RESULTS: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxialow/immunehigh, hypoxiahigh/immunelow, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxialow/immunehigh and hypoxiahigh/immunelow tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3+ T cells (r = -0.5464; P = 0.0377), further corroborating the transcription-based classification. CONCLUSIONS: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Characterization of the T-Cell Receptor Repertoire and Immune Microenvironment in Patients with Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck.

Purpose: Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer with a suboptimal 5-year overall survival of approximately 50% with surgery and/or definitive chemoradiotherapy. Novel treatments are thus urgently awaited. Immunotherapy with checkpoint blockade has emerged as a promising option for patients with recurrent/metastatic SCCHN; however, it has not been investigated in the curative-intent setting yet. The purpose of this study was to investigate the T-cell receptor repertoire and the tumor microenvironment in tumor tissues of SCCHN patients with locoregionally advanced disease.Experimental Design: We performed T-cell receptor sequencing of tumor tissues from 44 patients with locoregionally advanced SCCHN prior to treatment with definitive chemoradiotherapy and correlated the T-cell clonality and the mRNA expression levels of immune-related genes with clinicopathologic parameters.Results: Clonal expansion of T cells was significantly higher in human papilloma virus (HPV)-negative compared with HPV-positive tumors, signifying more robust antigen presentation in HPV-negative tumors. The latter was supported by the higher percentage of HPV-negative tumors expressing HLA-A protein compared with HPV-positive tumors (P = 0.049). Higher GRZB levels correlated significantly with longer recurrence-free survival (log-rank, P = 0.003) independent of tumor size, nodal stage, and HPV status.Conclusions: Our findings support clonal expansion of T cells in SCCHN patients with locoregionally advanced disease and imply differences in the antigen presentation capacity between HPV-negative and HPV-positive tumors. Elevated GRZB mRNA levels may also serve as a favorable and independent predictor of outcome in SCCHN patients treated with chemoradiotherapy. These data provide rationale for the introduction of immunotherapeutic approaches in the curative-intent setting. Clin Cancer Res; 23(16); 4897-907. ©2017 AACR.

Growth factor expression mediates resistance to EGFR inhibitors in head and neck squamous cell carcinomas.

OBJECTIVES: Epidermal growth factor receptor (EGFR)-targeted therapy is frequently used in the treatment of advanced head and neck squamous cell carcinoma (HNSCC). However, constitutive or acquired resistance is common and underlying resistance mechanisms remain poorly understood. We investigated the expression levels of growth factors (GF) in tumor-associated stroma and tumor from HNSCC patients and determined the influence of GFs on EGFR inhibitor efficacy in vitro. MATERIALS AND METHODS: The Chicago HNC Genomic Cohort (CHGC) was queried for GF and receptor tyrosine kinase (RTK) expression. Viability assays were used to evaluate the effect of EGFR inhibition (gefitinib), GF treatment, or both in HNSCC cell lines. Caspase-based assays were used to measure apoptotic activity. Expression of RTKs was determined and correlated with GF treatment effects. RESULTS: Amphiregulin (AREG), transforming growth factor (TGFß1), insulin like growth factor (IGF1), fibroblast growth factors (FGF1/FGF2) and the corresponding RTKs were highly expressed in 30-50% of HNSCC, and expression was usually concurrent. While EGFR inhibition was markedly efficacious in HNC cell lines (HN5/HN13/H400/SCC61), co-treatment with most GFs increased viability up to 100%. Only TGFß1 treatment was additive to EGFR inhibition. GFs also reduced apoptotic effects of EGFR inhibition. RTK expression showed strong positive correlation with respective GF treatment effect for IGF1-IGF1R, less strong for HGF-MET/AREG-EGFR and a moderate negative correlation for TGFß1-TGFBR1/2. CONCLUSION: High expression of GFs/RTKs occurs in HNSCC. Co-expression is common. GF expression contributes to EGFR inhibition resistance in our model system, and may be a common mechanism of constitutive or acquired resistance to EGFR inhibition in HNSCC.

Genetic profiling of advanced radioactive iodine-resistant differentiated thyroid cancer and correlation with axitinib efficacy.

Biomarkers predicting which patients with advanced radioiodine-resistant differentiated thyroid cancer (DTC) may benefit from multi-kinase inhibitors are unavailable. We aimed to describe molecular markers in DTC that correlate with clinical outcome to axitinib. Pretreatment thyroid cancer blocks from 18 patients treated with axitinib were collected and genomic DNA was isolated. The OncoCarta™ Mutation Panel was used to test for 238 oncogenic mutations. Copy number of VEGFR1-3 and PIK3CA was determined using qPCR on enriched tumor samples. Genomic DNA was analyzed for all coding regions of VEGFR1-3 with custom primers. Protein expressions of VEGFR1-3, c-Met, and PIK3CA were evaluated with immunohistochemistry. Clinical response to axitinib, including best response (BR) and progression free survival (PFS), was ascertained from corresponding patients. Fisher's exact test and logistic regression models were used to correlate BR with molecular findings. Cox proportional hazards regression was used to correlate PFS with molecular defects. A total of 22 pathology samples (10 primary, 12 metastatic) were identified. In patients with 2 samples (n = 4), genetic results were concordant and only included once for analysis. Tumors from 4 patients (22%) harbored BRAF V600E mutations, 2 (11%) had KRAS mutations (G12A, G13D) and 2 (11%) had HRAS mutations (Q61R, Q61K). One metastatic sample with mutated KRAS also harbored a PIK3CA (H1047R) mutation. qPCR showed increased copy numbers of PIK3CA in 6 (33%) tumors, VEGFR1 in 0 (0%) tumors, VEGFR2 in 4 (22%) tumors, and VEGFR3 in 6 (33%) tumors. VEGFR sequencing was significant for a possibly damaging non-synonymous SNP in VEGFR2 (G539R) in 2 samples (11%), a possibly damaging SNP in VEGFR3 (E350V) in 1 sample (6%), and a potentially novel mutation in VEGFR2 (T439I) in 2 samples (11%). Immunohistochemistry (VEGFR1, -2, -3; c-MET; PIK3CA) revealed positive staining in the majority of samples. No significant relationship was seen between BR or PFS and the presence of molecular alterations. Molecular evaluation of DTC specimens did not predict clinical response to axitinib but our data were limited by sample size. We did identify molecular changes in VEGFR that should be further explored. While DTC is genetically heterogeneous, primary and metastatic lesions showed identical oncogenic alterations in four cases.

Rare occurrence of EGFRvIII deletion in head and neck squamous cell carcinoma.

BACKGROUND: The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor and is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) cases. The EGFR truncation mutation, EGFR variant III (EGFRvIII), harbors an in-frame deletion of exons 2-7 (801 bp) that leads to the constitutive activation of downstream signaling. EGFRvIII has been reported in ∼40% of glioblastomas (GBM), but its presence in HNSCC remains controversial. METHODS: EGFRvIII deletion in 638 HNSCC samples was analyzed using: (i) quantitative Real-Time polymerase chain reaction (qRT-PCR) on 108 HNSCC samples with direct detection of the EGFRvIII breakpoint, (ii) RNA-Seq analysis on 7 HNSCC tumor tissues and 425 The Cancer Genome Atlas (TCGA) HNSCC samples, and (iii) immunohistochemistry (IHC) for EGFRvIII using an established antibody (L8A4) on a tissue microarray of 105 HNSCC samples. RESULTS: qRT-PCR did not show the presence of EGFRvIII in any of the samples analyzed. Furthermore, we could not detect any EGFRvIII transcripts in the RNA-Seq data of the seven HNSCC samples. However, 2 samples out of 425 TCGA HNSCC samples had EGFRvIII specific reads. EGFRvIII IHC results were assessed as negative for all samples. CONCLUSION: Our results firmly establish that EGFRvIII is very rare in HNSCC as only 2 out of 638 (0.31%) samples we analyzed overall, or 2 out of 540 (0.37%) using mRNA based approaches, were positive for EGFRvIII. EGFRvIII is extremely rare in HNSCC and the clinical significance remains unclear. We propose not to include EGFRvIII testing in regular diagnostic tests for HNSCC.

Integrative analysis of head and neck cancer identifies two biologically distinct HPV and three non-HPV subtypes.

PURPOSE: Current classification of head and neck squamous cell carcinomas (HNSCC) based on anatomic site and stage fails to capture biologic heterogeneity or adequately inform treatment. EXPERIMENTAL DESIGN: Here, we use gene expression-based consensus clustering, copy number profiling, and human papillomavirus (HPV) status on a clinically homogenous cohort of 134 locoregionally advanced HNSCCs with 44% HPV(+) tumors together with additional cohorts, which in total comprise 938 tumors, to identify HNSCC subtypes and discover several subtype-specific, translationally relevant characteristics. RESULTS: We identified five subtypes of HNSCC, including two biologically distinct HPV subtypes. One HPV(+) and one HPV(-) subtype show a prominent immune and mesenchymal phenotype. Prominent tumor infiltration with CD8(+) lymphocytes characterizes this inflamed/mesenchymal subtype, independent of HPV status. Compared with other subtypes, the two HPV subtypes show low expression and no copy number events for EGFR/HER ligands. In contrast, the basal subtype is uniquely characterized by a prominent EGFR/HER signaling phenotype, negative HPV-status, as well as strong hypoxic differentiation not seen in other subtypes. CONCLUSION: Our five-subtype classification provides a comprehensive overview of HPV(+) as well as HPV(-) HNSCC biology with significant translational implications for biomarker development and personalized care for patients with HNSCC.

Integrative and comparative genomic analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas.

PURPOSE: The genetic differences between human papilloma virus (HPV)-positive and -negative head and neck squamous cell carcinomas (HNSCC) remain largely unknown. To identify differential biology and novel therapeutic targets for both entities, we determined mutations and copy-number aberrations in a large cohort of locoregionally advanced HNSCC. EXPERIMENTAL DESIGN: We performed massively parallel sequencing of 617 cancer-associated genes in 120 matched tumor/normal samples (42.5% HPV-positive). Mutations and copy-number aberrations were determined and results validated with a secondary method. RESULTS: The overall mutational burden in HPV-negative and HPV-positive HNSCC was similar with an average of 15.2 versus 14.4 somatic exonic mutations in the targeted cancer-associated genes. HPV-negative tumors showed a mutational spectrum concordant with published lung squamous cell carcinoma analyses with enrichment for mutations in TP53, CDKN2A, MLL2, CUL3, NSD1, PIK3CA, and NOTCH genes. HPV-positive tumors showed unique mutations in DDX3X, FGFR2/3 and aberrations in PIK3CA, KRAS, MLL2/3, and NOTCH1 were enriched in HPV-positive tumors. Currently targetable genomic alterations were identified in FGFR1, DDR2, EGFR, FGFR2/3, EPHA2, and PIK3CA. EGFR, CCND1, and FGFR1 amplifications occurred in HPV-negative tumors, whereas 17.6% of HPV-positive tumors harbored mutations in fibroblast growth factor receptor genes (FGFR2/3), including six recurrent FGFR3 S249C mutations. HPV-positive tumors showed a 5.8% incidence of KRAS mutations, and DNA-repair gene aberrations, including 7.8% BRCA1/2 mutations, were identified. CONCLUSIONS: The mutational makeup of HPV-positive and HPV-negative HNSCC differs significantly, including targetable genes. HNSCC harbors multiple therapeutically important genetic aberrations, including frequent aberrations in the FGFR and PI3K pathway genes. See related commentary by Krigsfeld and Chung, p. 495.

A phase II study of lapatinib in recurrent/metastatic squamous cell carcinoma of the head and neck.

PURPOSE: This study sought to determine the efficacy and safety profile of lapatinib in patients with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN). EXPERIMENTAL DESIGN: This phase II multiinstitutional study enrolled patients with recurrent/metastatic SCCHN into two cohorts: those without (arm A) and those with (arm B) before exposure to an epidermal growth factor receptor (EGFR) inhibitor. All subjects were treated with lapatinib 1,500 mg daily. Primary endpoints were response rate (arm A) and progression-free survival (PFS; arm B). The biologic effects of lapatinib on tumor growth and survival pathways were assessed in paired tumor biopsies obtained before and after therapy. RESULTS: Forty-five patients were enrolled, 27 in arm A and 18 in arm B. Diarrhea was the most frequent toxicity occurring in 49% of patients. Seven patients experienced related grade 3 toxicity (3 fatigue, 2 hyponatremia, 1 vomiting, and 1 diarrhea). In an intent-to-treat analysis, no complete or partial responses were observed, and stable disease was the best response observed in 41% of arm A (median duration, 50 days, range, 34-159) and 17% of arm B subjects (median, 163 days, range, 135-195). Median PFS was 52 days in both arms. Median OS was 288 (95% CI, 62-374) and 155 (95% CI, 75-242) days for arms A and B, respectively. Correlative analyses revealed an absence of EGFR inhibition in tumor tissue. CONCLUSION: Lapatinib as a single agent in recurrent/metastatic SCCHN, although well tolerated, appears to be inactive in either EGFR inhibitor naive or refractory subjects.

APOB gene signal peptide deletion polymorphism is not associated with infertility in Indian men.

Apolipoprotein B (APOB) plays a key role in lipoprotein metabolism and plasma lipid transport. It has been shown that about two-thirds of male mice heterozygous for ApoB were infertile. Moreover, a 3-codon deletion polymorphism (rs11279109) in the signal peptide region of the APOB gene has been shown to be a risk factor for infertility in Slovenian men, but its association with infertility in Indian men has not been evaluated to date. Hence, in the present study, we have genotyped this polymorphism in 545 Indian men, including 294 infertile and 251 fertile men. Our results show that the distribution of this deletion polymorphism was consistent with the Hardy-Weinberg equilibrium in both infertile and fertile men. No statistically significant difference was observed in the distribution of the APOB signal peptide deletion polymorphism between infertile and fertile men (chi(2) = 0.156, P = .925 for genotypes; chi(2) = 0.015, P = .903 for alleles). Moreover, no significant difference was observed when infertile and fertile men were categorized on the basis of presence (D/D and D/W genotypes) or absence (W/W genotypes) of deletion (odds ratio, 0.955; 95% confidence interval, 0.644-01.418; P = .820). Our study concludes that the APOB gene deletion polymorphism is not a risk factor for the development of infertility in Indian men.

Novel variants in UBE2B gene and idiopathic male infertility.

The UBE2B gene encodes ubiquitin-conjugating enzyme, which is involved in DNA repair. Ube2b knockout mice were found to be infertile because of structural abnormality of sperm. However, there is no genetic study on the role of the UBE2B gene in human fertility; therefore, the present investigation was designed to study genetic variations in the UBE2B gene and its role in human male infertility. Sequence analyses of the UBE2B gene in 530 infertile (350 azoospermic, 105 oligoasthenoteratozoospermic, and 75 oligoasthenozoospermic) and 300 fertile control men revealed the presence of 5 substitution single-nucleotide polymorphisms (SNPs) in 221 individuals (199 infertile [37.5%] and 22 fertile [7.3%] men). Of these, 2 (g.5197:T>G; g.9157:A>G) of the 5 substitutions were novel and observed only in infertile men. Distribution of haplotypes TA, TG, GA, and GG are not uniform between the patient and the control group of this study. Interestingly, our study suggests that the haplotype TG conferred significantly increased risk for male infertility (odds ratio = 5.07, 95% CI = 1.29-23.29, p = .007). In silico analysis of SNPs that were specific to infertile men predicted that these SNPs lead to defective splicing by destroying or creating the potential binding site of splicing factors or causing alteration in predicted regulatory sequences. In the light of the above, our study suggests that the UBE2B gene is associated with male infertility in Indian men, hence, providing evidence for additional genetic factors for male infertility.

Type 1 fimbriae of insecticidal bacterium Xenorhabdus nematophila is necessary for growth and colonization of its symbiotic host nematode Steinernema carpocapsiae.

Xenorhabdus nematophila produces type 1 fimbriae on the surface of Phase I cells. Fimbriae mediate recognition and adhesion of the bacteria to its target cell. To investigate the role of fimbriae in the biology of X. nematophila, we have produced a fimbrial mutant strain by insertional inactivation of the mrxA gene, encoding the structural subunit of type 1 fimbriae. Phenotypic characterization of the mutant revealed loss of fimbriae on the cell surface. Cell surface characteristics like dye absorption, biofilm formation, red blood cell agglutination remained unaltered. The mrxA mutant was defective in swarming on soft agar, although swimming motility was not affected. Flagellar expression was suppressed in the mrxA strain under swarming conditions, but not swimming conditions. Agglutination and cytotoxicity of the mutant to larval haemocytes was also reduced. When the mutant cells were injected in the haemocoel of the fourth instar larvae of Helicoverpa armigera, an increase in the LT(50) of 9-12 h was observed relative to the wild-type strain. The nematode growth was slow on the lawn of the fimbrial mutant. The mrxA negative strain was unable to colonize the nematode gut efficiently. This study demonstrates importance of type 1 fimbriae in establishment of bacteria-nematode symbiosis, a key to successful pest management program.