Synergetic effect of high dose rate radiations (10× FFF/2400 MU/min/10 MV x-rays) and paclitaxel selectively eliminates melanoma cells.

BACKGROUND: Melanoma is one of the most aggressive cancers, with 1.6% of total cancer deaths in the United States. In recent years treatment options for metastatic melanoma have been improved by the FDA approval of new therapeutic agents. However, these inhibitors-based therapies are non-specific and have severe toxicities, including hyperkeratosis, photosensitivity, hepatitis, arthralgia, and fatigue. AIMS: The aim of this study is to determine the synthetic lethal effect (paclitaxel and radiations) on melanoma cells and reduce the total radiation doses by increasing the dose rates up to 2400 MU/min. METHODS AND RESULTS: We previously reported a radiation treatment (10 MV x-rays, 10X-FFF, dose rate 2400MU/min, low total dose 0.5 Gy) that kills melanoma cells with 80% survival of normal HEM in vitro. In this study, we extended the radiation cycle up to four and included paclitaxel treatment to study the synthetic lethal effect on melanoma and two other normal primary cells, HDF and HEK. Cells were treated with paclitaxel prior to the radiation at a dose rate of 400 and 2400 MU/min with a total radiation dose of only 0.5 Gy. Mitochondrial respiration assay, DNA damage assay, and colony formation assays were performed to study apoptosis and cell death induction. Four days of consequent radiation treatment with paclitaxel significantly reduces the survival of melanoma cells by inducing apoptosis and mitochondrial damage. After treatment, excessive DNA damage in melanoma cells leads to an increase in the expression of pro-apoptotic genes (Caspase-3) and a decrease in the expression of DNA repair gene (PARP1) and anti-apoptotic gene (Bcl-2) to activate the apoptosis pathway. The combination of paclitaxel and radiation reduces the survival of melanoma cells colonies compared to radiation alone. CONCLUSION: Our study indicates that radiations with paclitaxel have a potential synthetic lethal effect on melanoma cells and can be developed as a melanoma therapy without toxicities or harmful effects on normal primary skin cells.

Amplification of the PLAG-family genes-PLAGL1 and PLAGL2-is a key feature of the novel tumor type CNS embryonal tumor with PLAGL amplification.

Pediatric central nervous system (CNS) tumors represent the most common cause of cancer-related death in children aged 0-14 years. They differ from their adult counterparts, showing extensive clinical and molecular heterogeneity as well as a challenging histopathological spectrum that often impairs accurate diagnosis. Here, we use DNA methylation-based CNS tumor classification in combination with copy number, RNA-seq, and ChIP-seq analysis to characterize a newly identified CNS tumor type. In addition, we report histology, patient characteristics, and survival data in this tumor type. We describe a biologically distinct pediatric CNS tumor type (n = 31 cases) that is characterized by focal high-level amplification and resultant overexpression of either PLAGL1 or PLAGL2, and an absence of recurrent genetic alterations characteristic of other pediatric CNS tumor types. Both genes act as transcription factors for a regulatory subset of imprinted genes (IGs), components of the Wnt/ß-Catenin pathway, and the potential drug targets RET and CYP2W1, which are also specifically overexpressed in this tumor type. A derived PLAGL-specific gene expression signature indicates dysregulation of imprinting control and differentiation/development. These tumors occurred throughout the neuroaxis including the cerebral hemispheres, cerebellum, and brainstem, and were predominantly composed of primitive embryonal-like cells lacking robust expression of markers of glial or neuronal differentiation (e.g., GFAP, OLIG2, and synaptophysin). Tumors with PLAGL1 amplification were typically diagnosed during adolescence (median age 10.5 years), whereas those with PLAGL2 amplification were diagnosed during early childhood (median age 2 years). The 10-year overall survival was 66% for PLAGL1-amplified tumors, 25% for PLAGL2-amplified tumors, 18% for male patients, and 82% for female patients. In summary, we describe a new type of biologically distinct CNS tumor characterized by PLAGL1/2 amplification that occurs predominantly in infants and toddlers (PLAGL2) or adolescents (PLAGL1) which we consider best classified as a CNS embryonal tumor and which is associated with intermediate survival. The cell of origin and optimal treatment strategies remain to be defined.

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.

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.

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.

HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas.

High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.