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Purpose: The Piedmont study is a prospectively designed retrospective evaluation of a new 48-gene antifolate response signature (AF-PRS) in patients with locally advanced/metastatic NS-NSCLC treated with pemetrexed-containing platinum doublet chemotherapy (PMX-PDC). The study tested the hypothesis that AF-PRS selects for patients with NS-NSCLC that preferentially respond to PMX-PDC, with a goal of providing clinical support for AF-PRS as potential diagnostic test. Experimental Design: Residual pre-treatment FFPE tumor samples and clinical data were analyzed from 105 patients treated with 1st-line (1L) PMX-PDC. 95 patients had sufficient RNA sequencing (RNAseq) data quality and clinical annotation for inclusion in the analysis. Associations between AF-PRS status and associate genes, and outcome measures including progression-free survival (PFS) and clinical response were evaluated. Results: Overall, 53% of patients were AF-PRS(+), which was associated with extended PFS, but not OS, vs. AF-PRS(-) (16.6 vs. 6.6 mo; p = 0.025). In patients who were Stage I-III patients at time of treatment, PFS was further extended in AF-PRS(+) vs. AF-PRS(-) (36.2 vs. 9.3 mo; p = 0.03). Complete response (CR) to therapy was noted in 14 of 95 patients. AF-PRS(+) preferentially selected a majority (79%) of CRs, which were evenly split between patients Stage I-III (6 of 7) and Stage IV (5 of 7) at time of treatment. Conclusions: AF-PRS identified a significant population of patients with extended PFS and/or clinical response following PMX-PDC treatment. AF-PRS may be a useful diagnostic test for patients indicated for systemic chemotherapy, especially when determining the optimal PDC regimen for locally advanced disease.

Pancreatic ductal adenocarcinoma (PDA) tumors have a highly immunosuppressive desmoplastic tumor microenvironment (TME) where immune checkpoint inhibition (ICI) therapy has been exceptionally ineffective. Transforming growth factor-β (TGF-β) receptor activation leads to cancer and immune cell proliferation and phenotype, and cytokine production leading to tumor progression and worse overall survival in PDA patients. We hypothesized that TGF-β receptor inhibition may alter PDA progression and antitumor immunity in the TME. Here, we used a syngeneic preclinical murine model of PDA to explore the impact of TGF-β pathway inhibitor galunisertib (GAL), dual checkpoint immunotherapy (anti-PD-L1 and CTLA-4), the chemotherapy gemcitabine (GEM), and their combinations on antitumor immune responses. Blockade of TGF-β and ICI in immune-competent mice bearing orthotopically injected murine PDA cells significantly inhibited tumor growth and was accompanied by antitumor M1 macrophage infiltration. In contrast, GEM treatment resulted in increased PDA tumor growth, decreased antitumor M1 macrophages, and decreased cytotoxic CD8+ T cell subpopulation compared to control mice. Together, these findings demonstrate the ability of TGF-β inhibition with GAL to prime antitumor immunity in the TME and the curative potential of combining GAL with dual ICI. These preclinical results indicate that targeted inhibition of TGF-β may enhance the efficacy of dual immunotherapy in PDA. Optimal manipulation of the immune TME with non-ICI therapy may enhance therapeutic efficacy.

Supplementary Figures and Legends

REporting recommendations for tumour MARKer prognostic studies (REMARK) checklist.

Cox regression for survival in miRNA-based subtypes and miRNAs individually associated with prognosis and HPV status in the TCGA cohort.

Baseline clinicopathological characteristics of patients with HNSCC in UNC and TCGA data sets.

PAGE enrichment z-scores and q-values.

Mutation frequency of top 50 cancer related genes in HNSCC miRNA subclasses.

Purpose:The objective of this study is to characterize the role of miRNAs in the classification of head and neck squamous cell carcinoma (HNSCC).Experimental Design:Here, we analyzed 562 HNSCC samples, 88 from a novel cohort and 474 from The Cancer Genome Atlas, using miRNA microarray and miRNA sequencing, respectively. Using an integrative correlations method followed by miRNA expression–based hierarchical clustering, we validated miRNA clusters across cohorts. Evaluation of clusters by logistic regression and gene ontology approaches revealed subtype-based clinical and biological characteristics.Results:We identified two independently validated and statistically significant (P < 0.01) tumor subtypes and named them “epithelial” and “stromal” based on associations with functional target gene ontology relating to differing stages of epithelial cell differentiation. miRNA-based subtypes were correlated with individual gene expression targets based on miRNA seed sequences, as well as with miRNA families and clusters including the miR-17 and miR-200 families. These correlated genes defined pathways relevant to normal squamous cell function and pathophysiology. miRNA clusters statistically associated with differential mutation patterns including higher proportions of TP53 mutations in the stromal class and higher NSD1 and HRAS mutation frequencies in the epithelial class. miRNA classes correlated with previously reported gene expression subtypes, clinical characteristics, and clinical outcomes in a multivariate Cox proportional hazards model with stromal patients demonstrating worse prognoses (HR, 1.5646; P = 0.006).Conclusions:We report a reproducible classification of HNSCC based on miRNA that associates with known pathologically altered pathways and mutations of squamous tumors and is clinically relevant.

Summary of functional characteristics of 50 miRNAs based on previously published reports and target gene ontology analysis.

Integrative analysis and unsupervised hierarchical clustering of microRNAs in two patient cohorts.

PURPOSE Hematologic toxic effects of peptide receptor radionuclide therapy (PRRT) can be permanent. Patients with underlying clonal hematopoiesis (CH) may be more inclined to develop hematologic toxicity after PRRT. However, this association remains understudied. MATERIALS AND METHODS We evaluated pre- and post-PRRT blood samples of patients with neuroendocrine tumors. After initial screening, 13 cases of interest were selected. Serial blood samples were obtained on 4 of 13 patients. Genomic DNA was analyzed using a 100-gene panel. A variant allele frequency cutoff of 1% was used to call CH. RESULT Sixty-two percent of patients had CH at baseline. Persistent cytopenias were noted in 64% (7 of 11) of the patients. Serial sample analysis demonstrated that PRRT exposure resulted in clonal expansion of mutant DNA damage response genes ( TP53, CHEK2, and PPM1D) and accompanying cytopenias in 75% (3 of 4) of the patients. One patient who had a normal baseline hemogram and developed persistent cytopenias after PRRT exposure showed expansion of mutant PPM1D (variant allele frequency increased to 20% after exposure from < 1% at baseline). In the other two patients, expansion of mutant TP53, CHEK2, and PPM1D clones was also noted along with cytopenia development. CONCLUSION The shifts in hematopoietic clonal dynamics in our study were accompanied by emergence and persistence of cytopenias. These cytopenias likely represent premalignant state, as PPM1D-, CHEK2-, and TP53-mutant clones by themselves carry a high risk for transformation to therapy-related myeloid neoplasms. Future studies should consider CH screening and longitudinal monitoring as a key risk mitigation strategy for patients with neuroendocrine tumors receiving PRRT.

Integration of viral DNA into the host genome has been implicated in human papillomavirus (HPV) associated tumorigenesis, and studies in HPV associated head and neck squamous cell carcinoma (HNSCC) attempting to classify viral integration status have reported associations among HPV status, viral load, and overall survival. However, few genomic studies have correlated HPV structure with commonly occurring somatic mutations in HNSCC. Here, we conducted hybrid-capture DNA sequencing using a panel of over 800 cancer related genomic targets, as well as probes specific for the full HPV16 and HPV18 genomes, in a novel cohort of 525 HNSCC cases. Paired reads were aligned to a human reference containing complete viral genomes, including all high-risk HPV types, which allowed for robust identification of HPV(+) tumors and specific HPV type. Variant calling and filtering, followed by pathway analysis of significantly mutated genes, defined the catalog of somatic driver alterations in HNSCC. Viral genome structure, integration status, and viral load were determined via initial copy number analysis of HPV-specific reads, followed by identification of viral/human breakpoints and indels with variant calling algorithms. Manual review was required in some cases due to variability in read coverage and alignment to HPV genome. We identified 252 HPV(+) tumors, the majority of which were HPV16 (n=228, 90%). Analysis of HPV16(+) tumors revealed five classes with variable viral loads: pure episome (EPI, n=54), episomal rearranged (EPI-R, n=51), episomal chimeric (EPI-C, n=22), integrated (INT, n=92), and integrated deleted (INT-A, n=12). Integrated tumors had at least one integration event (INT) and a few exhibited recurrent deletions in the viral genome (INT-A). Episomal samples had either complete HPV genomes (EPI), circular genomes with one or more indels (EPI-R), or circular genomes with HPV and human DNA (EPI-C). Interestingly, PIK3CA, the most frequently mutated gene in HPV(+) HNSCC, was mutated at a higher frequency in all integrated samples compared to all episomal types (44% vs 23%, p=0.005). We also noticed more deleterious mutations in innate immunity genes (HLA-A/B, B2M, TRAF3, BIRC3) in the episomal samples (16% vs 8%), which may allow cells with intact HPV genomes to evade anti-viral immune responses. Additionally, mutations in oxidative stress response genes that lead to the constitutive activation of NRF2 (NFE2L2, CUL3, KEAP1) were more frequent in integrated samples (11% vs 4%), which could confer radiotherapy resistance and a worse prognosis. Overall, using DNA sequencing and integrative genomic analysis in the largest cohort of HPV(+) HNSCC to date, we developed a novel classification method based on viral genome structure which associates with host somatic alterations that may contribute to variable outcomes in HNSCCs. Citation Format: Jeremiah Ray Holt, Xiaobei Zhao, Hyo Young Choi, Paul Little, Angela L. Mazul, Benjamin Wahle, Vonn Walter, Jose P. Zevallos, David Neil Hayes. Viral genome structure correlates with patterns of somatic alteration in human papillomavirus associated head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6062.

Purpose: Previous studies implicated viral oncoproteins E6 and E7, of which targets include but are not limited to tumor suppressors p53 and pRb, in human papillomavirus (HPV) associated tumorigenesis. However, HPV-associated tumors must accumulate additional genomic alterations as HPV oncoproteins alone are insufficient for tumor formation in vivo. Experimental Design: Targeted sequencing was conducted with a panel of over 800 cancer related genomic regions and genes in a novel cohort of 525 head and neck squamous cell carcinoma (HNSCC) patients, including 252 HPV(+) and 273 HPV(-) tumors. Variant calling and filtering, followed by analysis with computational tools such as MutSig for somatic mutations and DiNAMIC.Duo for recurrent copy number alterations (CNAs), defined the catalog of driver alterations that are specific to HPV(+) and HPV(-) tumors. Pathway analysis was then used to elucidate the functional role of somatic alterations in the context of previously described HNSCC perturbations and HPV pathophysiology. Results: Integrative genomic analysis in one of the largest HPV(+) HNSCC cohorts to date revealed differential patterns of somatic mutations and CNAs in E6/E7 targets between HPV(+) and HPV(-) tumors. Specifically, EP300, an activator of p53 and target of E6, and retinoblastoma (RB) family members RB1 (pRb) and RBL2 (p130), which are known targets of E7 mediated degradation, were selected as significantly mutated by MutSig in HPV(+) samples only (EP300 & RB1 P Conclusions: Previous studies suggest HPV-associated tumors lack significant levels of somatic alterations in canonical tumor suppressor genes that are known targets of viral oncoproteins. Here, we show additional selective pressure exists for somatic alteration of E6/E7 target genes in at least a subset of HPV(+) HNSCCs, which further deregulate cell cycle and apoptosis pathways in these tumors. Citation Format: Jeremiah Holt, Heejoon Jo, Xiaobei Zhao, Hyo Young Choi, Vonn Walter, Paul Little, Benjamin Wahle, Angela Mazul, Jose P. Zevallos, David Neil Hayes. Integrative genomic analysis of human papillomavirus associated head and neck squamous cell carcinoma reveals selective pressure for somatic alteration of E6 and E7 targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 49.

Gene expression analyses of head and neck cancer have revealed four molecular subtypes: basal (BA), mesenchymal (MS), atypical (AT), and classical (CL). We evaluate whether gene expression subtypes in oral cavity squamous cell carcinoma (OCSCC) and laryngeal squamous cell carcinoma (LSCC) can be used to predict nodal metastasis and prognosticate survival.Retrospective cohort study and genomic analysis.OCSCC and LSCC cases were identified from the The Cancer Genome Atlas (TCGA) head and neck cancer cohort. RNA-seq by expected maximization (RSEM) was used to quantify gene expression levels from TCGA RNA-seq data and to assign each case to one of four subtypes. Descriptive statistics were used to describe patient, disease, and treatment characteristics in each subtype. Cox regression and Kaplan-Meier analyses were used to determine associations with survival.OCSCC cases were comprised primarily of the MS and BA subtypes, whereas LSCC was comprised primarily of CL and AT subtypes. In OCSCC, the MS subtype was significantly associated with higher risk of nodal metastasis. In a subset analysis of clinically T1-2N0M0 OCSCC, we demonstrate that the MS subtype was predictive of occult nodal metastasis (relative risk = 3.38, 95% confidence interval [CI]: 1.08-10.69). In LSCC, the CL subtype was associated with significantly worse overall survival (hazard ratio = 4.32, 95% CI: 1.77-10.54, P = .001).Gene expression analysis reveals potential novel markers of nodal metastasis and survival in human papillomavirus-negative head and neck cancer. Future studies will continue to refine and validate these markers, with the goal of providing molecular risk assessments that guide therapy and improve patient outcomes.2b Laryngoscope, 129:154-161, 2019.

The combination of cisplatin, 5-fluorouracil, and cetuximab is a standard treatment for patients with recurrent/metastatic head and neck cancer, with a high rate of toxicity. Identifying less toxic, equally effective regimens is imperative. Therefore, in the current study, the authors investigated first-line treatment with an all-oral regimen of capecitabine and lapatinib.Patients were required to have incurable head and neck cancer of any primary site other than the nasopharynx, an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 to 2, and no prior exposure to capecitabine or lapatinib. Subjects were treated with capecitabine at a dose of 1000 mg/m(2) twice daily and lapatinib at a dose of 1250 mg daily. Capecitabine was administered for 14 days of each 21-day cycle for 4 cycles. Lapatinib was administered daily until disease progression. The primary outcome was overall survival.A total of 44 subjects were accrued between November 13, 2009 and April 29, 2014. Approximately 38.6% of the sample had an ECOG PS of 0, 52.3% had an ECOG PS of 1, and 9.1% had an ECOG PS of 2. Approximately 81.8% were male and the median age of the patients was 62 years. Prior attempts at curative treatment with chemotherapy had been used in 68.2% of patients (platinum was used in 55.8%). There was no grade 5 toxicity noted (toxicity was graded according to National Cancer Institute Common Terminology Criteria for Adverse Events [version 3.0]). The most common adverse events were diarrhea (18.2% of patients with grade 3) and rash (13.6% of patients with grade 3). The primary objective was met; the median overall survival was 10.7 months (90% confidence interval [90% CI], 8.7-12.9 months). The overall response rate was 25% (90% CI, 15%-38%). The median progression-free survival was 4.2 months (90% CI, 3.6-5.1 months). The results were not substantially different when subdivided by p16 status. Only 2 patients were positive for human epidermal growth factor receptor 2 by immunohistochemistry.The current study met its primary objective of survival comparable to the combination of cisplatin, 5-FU and cetuximab regimen, and the toxicity of this all-oral regimen was tolerable. Cancer 2016;122:2350-2355. © 2016 American Cancer Society.

Cetuximab is a monoclonal antibody against epidermal growth factor receptor with activity against head and neck cancer and colorectal cancer. Anaphylaxis in response to cetuximab is a significant clinical problem in the Southeastern United States with a grade 3/4 infusion reaction rate of 14%. Previous retrospective data have suggested that the presence of preformed immunoglobulin E antibodies against galactose-α-1,3-galactose in serum can predict anaphylaxis in response to cetuximab.Sixty patients were prospectively screened as part of the entry criteria for a phase 2 study of neoadjuvant carboplatin, nab-paclitaxel, and cetuximab. Patients were recruited at 2 academic medical centers known to have high anaphylaxis rates: the University of North Carolina and Vanderbilt. Only patients with a negative laboratory result were treated on the clinical protocol.No patient experienced anaphylaxis; the negative predictive value was thus 100%. Other than smoking history, the demographics were similar for assay-positive subjects and assay-negative subjects.Subjects with a negative test result can be safely treated with cetuximab. Further research is required regarding the optimal cutoff for positivity and the positive predictive value. Cancer 2016;122:1697-701. © 2016 American Cancer Society.

Cluster analysis has proved to be an invaluable tool for the exploratory and unsupervised analysis of high-dimensional datasets. Among methods for clustering, hierarchical approaches have enjoyed substantial popularity in genomics and other fields for their ability to simultaneously uncover multiple layers of clustering structure. A critical and challenging question in cluster analysis is whether the identified clusters represent important underlying structure or are artifacts of natural sampling variation. Few approaches have been proposed for addressing this problem in the context of hierarchical clustering, for which the problem is further complicated by the natural tree structure of the partition, and the multiplicity of tests required to parse the layers of nested clusters. In this article, we propose a Monte Carlo based approach for testing statistical significance in hierarchical clustering which addresses these issues. The approach is implemented as a sequential testing procedure guaranteeing control of the family-wise error rate. Theoretical justification is provided for our approach, and its power to detect true clustering structure is illustrated through several simulation studies and applications to two cancer gene expression datasets.

EGFR inhibitors provide benefit in patients with advanced and metastatic head and neck squamous cell carcinoma (HNSCC). Mass spectrometry profiling has been used to predict outcome in patients with HNSCC after EGFR inhibitor treatment, and may enable prior identification of patients most likely to benefit from these therapies. However, further validation in prospective studies is needed.