Tumor HPV Research Articles

Determinants of the level of circulating-tumor HPV16 DNA in patients with HPV-associated oropharyngeal cancer at the time of diagnosis

Circulating tumor HPV DNA (ctHPV16) assessed in liquid biopsy may be used as a marker of cancer in patients with HPV-associated oropharyngeal cancer (HPV + OPC). Factors influencing the initial ctHPV16 quantity are not well recognized. In this study we aimed to establish what factors are related to the level of ctHPV16 at the time of diagnosis. 51 patients (37 men and 14 women, median age of 57 years old) with HPV + OPC prior to definitive treatment were included. ctHPV16 was measured by qPCR. Tumor and nodal staging were assessed according to AJCC8. Blood derived factors included squamous cell carcinoma antigen (SCC-Ag), serum soluble fragment of cytokeratin 19 (CYFRA 21-1), C-reactive protein (CRP), albumin level (Alb), neutrophils (Neut), thrombocytes (Plt) and lymphocyte (Lym) count, Neut/Lym ratio were assessed. The volumes of the primary tumor (TV) and involved lymph nodes (NV) were calculated using MRI, CT or PET-CT scans. Data were analysed using parametric and nonparametric methods. Variables for multivariable linear regression analysis were chosen based on the results from univariable analysis (correlation, univariable regression and difference). There were 9 (18%), 10 (19%) and 32 (63%) patients who had TV and NV assessed in MRI, CT or PET respectively. Primary tumor neither as T-stage nor TV was related to ctHPV16 level. Significant differences in the ctHPV16 between patients with high vs low pain (P = 0.038), NV (P = 0.023), TV + NV (P = 0.018), CYFRA 21-1 (P = 0.002), CRP (P = 0.019), and N1 vs N3 (P = 0.044) were observed. ctHPV16 was significantly associated with CYFRA 21-1 (P = 0.017), N stage (P = 0.005), NV (P = 0.009), TV + NV (P = 0.002), CRP (P = 0.019), and pain (P = 0.038). In univariable linear regression analysis the same variables predicted ctHPV16 level. In multivariable analyses, CYFRA 21-1 and CRP (both as categorical variables) were predictors of ctHPV16 level even above NV. ctHPV16 at presentation is driven by tumor volume measured mostly by N. CYFRA 21-1 and CRP are additional factors related to ctHPV16 prior to the treatment.

862P Clinical utility of circulating tumor HPV16 DNA detection in plasma from oropharyngeal squamous cell carcinoma patients

862P Clinical utility of circulating tumor HPV16 DNA detection in plasma from oropharyngeal squamous cell carcinoma patients

Real-World Experience of Intensity Modulated Radiation Therapy and Concurrent Chemotherapy for Anal Cancer with Long-Term Follow up and Clinical Outcomes

The standard treatment for epidermoid anal cancer (AC) is concurrent chemoradiation (CRT). Here we present real world evidence of the safety and outcomes of AC patients managed by IMRT and concurrent chemotherapy at a single academic cancer center. We retrospectively reviewed the outcomes of 180 AC patients treated with definitive CRT between 2011 and 2018. Patients were managed according to a prospectively designed protocol of IMRT with radiation dose escalated according to tumor stage: 50.4, 55.8 and 63 Gy for T1, T2 and T3/T4 disease respectively, and 36 Gy for elective nodal RT. Involved nodes were given the same dose based on T category. Concurrent chemotherapy consisted of two cycles of mitomycin C (MMC, 12 mg/m2) and 5-fluorouracil (5FU, 1000 mg/m2/day x 4 days) given on week 1 and 5. There was no planned treatment break. Univariate and multivariate analysis for outcomes were performed using Cox proportional hazard method and likelihood ratio statistics. Overall survival (OS) disease free (DFS), colostomy-free survival (CFS) and local failure rates (LFR) were described by Kaplan-Meier methods. There were 128 female and 52 male patients with a median age of 64 (IQR 55-74). The median size of the primary was 4.0 cm (0.6-11.0 cm). There were 18 T1, 91 T2, 38 T3 and 33 T4 lesions; 50.6% (91/180) of the patients had N0 disease. Thirteen (7.2%) did not receive concurrent chemotherapy, and 16 (8.9%) failed to complete treatment as planned. Forty-three (23.9%) patients had a treatment gap >5 (6-33) days. Eighteen of 147 (12%) with T1-3 disease failed locally, LF was observed in 13/33 (39%) T4 lesions (P = 0.0002). The 5-year OS, DFS, CFS and LFR were 85.1%, 75.6%, 87.6% and 15.5% respectively. On multivariate analysis, increasing age and N+ disease were significant for worse OS, and increasing size of the primary tumor was the only significant factor for worse DFS, CFS and LFR. Grade ≥3 acute toxicities were observed in 42.8% of patients, with grade ≥3 neutropenia and febrile neutropenia observed in 18.9% and 13.9% of patients respectively. Six patients (3.3%) died of acute toxicities. Thirteen (7%) patients experienced grade ≥3 late toxicities. Size of the primary appears to be the most important determinant of outcome following standard CRT using IMRT for AC. Despite IMRT, almost 1 in 4 patients required a treatment break, and over 40% experienced grade ≥3 acute toxicities including neutropenia and febrile neutropenia. Future studies with RT dose escalation or de-escalation, stratifying patients based on tumor size, HPV status and molecular markers are necessary to improve outcomes and decrease treatment related toxicity.

It is time to improve the diagnostic workup of oropharyngeal cancer with circulating tumor HPV DNA: Systematic review and meta-analysis.

The possibility of detecting circulating tumor HPV DNA (ctHPVDNA) in plasma in patients with oropharyngeal cancer has been demonstrated in several reports. However, these data are from small cohorts and available tests for detection of ctHPVDNA are not fully validated. The aim is to evaluate sensitivity, specificity, and accuracy of ctHPVDNA by ddPCR to define its efficacy in the clinical setting for the diagnosis of HPV + OPSCC. A comprehensive search of three different databases: MEDLINE, Embase, and Cochrane Library databases. A total of 998 patients were evaluated from the 13 studies. OPSSC p16+ were 729, while controls p16- were 269. The meta-analytic study estimated the diagnostic performance of ctHPVDNA as follows: pooled sensitivity and specificity of 0.90 (95% CI: 0.82-0.94) and 0.94 (95% CI: 0.85-0.98), respectively; positive and negative likelihood ratios of 12.6 (95% CI: 4.9-32.1) and 0.05 (95% CI: 0.02-0.13), respectively. ddPCR for ctHPVDNA has good accuracy, sensitivity, and specificity for diagnosis of HPV + OPSCC. ctHPVDNA kinetic represents a great reliable opportunity to improve diagnostic and therapeutic management of cancer patients and could open new perspectives for understanding tumor biology.

Abstract PO-021: TCGA differentially expressed genes between Caucasians and African Americans at variable analytic stringencies

Abstract Introduction: In interrogating large dimensional datasets, many methods have evolved to correctly identify differentially expressed genes at acceptable false discovery rates. We examined the NCI TCGA head and neck gene set with open source programs (at varying stringencie)s between Caucasian and African-Americans (AA) specimens and catalogued differentially expressed genes. Procedures: cBioPortal and UALCAN were utilized to investigate the Tumor Cancer Genome Atlas (TCGA). We compared AA and Caucasian samples for this analysis. Tumor samples for both groups were controlled for tumor stage (T3 and T4), HPV status (-), and age (40-75). Three different methods were utilized to identify potential genes at acceptable high stringency (defined as probability <2 false positives out of 20,000 genes being tested). First, those genes differentially expressed in AA vs Cau populations, as well as, those significantly differentially expressed in lymph node-positive (N+) vs negative (N0) patients were investigated (p<0.01 in both groups). Secondly, Benjamin-Hochberg (BH) analysis analyzed differentially expressed genes between just AA vs Cau groups (q<0.05). Finally, genes between these two groups possessing a p-value <0.0001, but not included in the BH analysis, were included in a third analysis. Results: Benjamin-Hochberg analysis identified four differentially expressed genes (q<0.05). There were 15 additional genes in this groups with p-values <0.0001. Analysis of genes differentially expressed in both AA N+ vs N0, as well as, those differentially expressed in AA vs Cau (p<0.01) identified one additional gene (PPIL2). Expression of this gene was found to be increased in a step-wise fashion with increased tumor grade (p<0.05). Three of the four genes identified by BH analysis were differentially expressed in HNSCC tumors vs control (LIN52, CTNNA2, PRMT6). Increased expression of LIN52 and PRMT6 were grade dependent (p<0.05). In the third analysis (AA vs Cau differentially expressed genes with p<0.0001), seven of 15 genes were overexpressed in tumor vs normal tissue; five of these were associated with increased tumor grade (p<0.05). All genes associated with increased tumor grade were demonstrated increased expression in AA groups vs Cau. Conclusions: We conclude open source TCGA programs could be readily employed to identify differentially expressed genes between both groups at varying stringencies. The high-stringency Benjamin-Hochberg analytical tool identified 4 genes differentially expressed between AA vs Cau T3/T4 HPV- tumors. Genes differentially expressed in both AA N+ vs N0, as well as, AA vs Cau revealed one potential gene of interest. When applying a p-value of < 0.0001, 15 additional genes were identified. The low overall number of AA specimens in this dataset was limiting and future biobanks should favor enriched collection of all stages of head and neck cancer in under represented groups. Citation Format: Seth Buryska, Jacob Tupa, Frank Ondrey. TCGA differentially expressed genes between Caucasians and African Americans at variable analytic stringencies [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-021.

Functional Genetic Variants in TGFβ1 and TGFβR1 in miRNA-Binding Sites Predict Outcomes in Patients with HPV-positive Oropharyngeal Squamous Cell Carcinoma.

TGFβ1 and TGFβ receptor 1 (TGFβR1) participate in regulation of the host's immune system and inflammatory responses and may serve as prognostic biomarkers for human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). This study included 1,013 patients with incident OPSCC, of whom 489 had tumor HPV16 status determined. All patients were genotyped for two functional polymorphisms: TGFβ1 rs1800470 and TGFβR1 rs334348. Univariate and multivariate Cox regression models were performed to evaluate associations between the polymorphisms and overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS). Patients with TGFβ1 rs1800470 CT or CC genotype had 70%-80% reduced risks of OS, DSS, and DFS compared with patients with TT genotype, and patients with TGFβR1 rs334348 GA or GG genotype had 30%-40% reduced risk of OS, DSS, and DFS compared with patients with AA genotype. Furthermore, among patients with HPV-positive (HPV+) OPSCC, the same patterns were observed but the risk reductions were greater: up to 80%-90% for TGFβ1 rs1800470 CT or CC genotype and 70%-85% for TGFβR1 rs334348 GA or GG genotype. The risk reductions were still greater (up to 17 to 25 times reduced) for patients with both TGFβ1 rs1800470 CT or CC genotype and TGFβR1 rs334348 GA or GG genotype compared with patients with both TGFβ1 rs1800470 TT genotype and TGFβR1 rs334348 AA genotype among patients with HPV+ OPSCC. Our findings indicate that TGFβ1 rs1800470 and TGFβR1 rs334348 may individually or jointly modify risks of death and recurrence in patients with OPSCC, particularly those with HPV+ OPSCC undergoing definitive radiotherapy, and may serve as prognostic biomarkers, which could lead to better personalized treatment and improved prognosis.

N-Acetyl-L-Cysteine Reduces Cervical Carcinogenesis by Promoting Apoptosis.

Cervical cancer is the fourth leading cause of cancer death in women, and is one of the most common malignant tumors of the reproductive system. However, more effective treatment for cervical cancer is needed. In this study, we aim to investigate whether N-acetyl-L-cysteine (NAC) could inhibit the proliferation of human papillomavirus (HPV)-positive cells, and reduce cervical carcinogenesis. The cervical cancer cell lines SiHa, HeLa, HPV-negative cell line C33A, and the immortalized human cervical keratinocyte cells S12 were used. The protein expression was determined using Western blot assay. mRNA expression was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was determined by Cell Counting Kit-8 assay. Cell apoptosis was evaluated using Annexin V-FITC apoptosis kits. The numbers of colonies were measured using colony-forming assay. Xenograft tumor necrosis and HPV16 E7 expression were determined using hematoxylin and eosin (H&E) staining and immunohistochemistry. Our results showed that NAC treatment at the concentration of 1.5mM significantly promoted cell apoptosis and reduced cell growth by inhibiting HPV16 E7 expression. NAC inhibited HPV16-oncoprotein-induced hypoxia-inducible factor (HIF)-1α protein expression and Akt activation invitro. Additionally, NAC suppressed tumor growth, as evidenced by the smaller tumor size in the xenograft mouse model and decreased HPV16 E7 expression in tumor tissues. Our findings demonstrate that NAC exhibits the potential to promote HPV-positive cell apoptosis, and suppress the proliferation of HPV-positive cells by inhibiting cell inhibitor of apoptosis protein 2 and HIF-1α.

Rapid establishment of patient-derived organoids from treatment-naive head and neck cancer patients for drug and radiosensitivity screening.

e18043 Background: Patient-derived organoids (PDOs) have been shown to be powerful tools for modeling disease and patient-individualized drug sensitivity screening, opening a novel avenue to precision medicine. A major challenge for guidance of clinical decisions is the extended time frame for establishing these models. Here, we report the feasibility of a refined protocol for PDO generation from head neck squamous cell carcinoma (HNSCC) tumors, suitable to guide personalized treatment decisions in the curative setting. Methods: After informed consent, tumor tissue specimens from 145 newly diagnosed HNSCC patients were collected during diagnostic biopsy or curative surgery. Following mechanic / enzymatic dissociation and initial expansion in 2D cultures, tumor cells were seeded in extracellular matrix (Matrigel) to form PDOs. Primary endpoint of the study was the PDO engraftment rate. In additio, the time needed for ex vivo expansion of PDO cultures for personalized drug/radiosensitivity testing was determined. Interference of patient and tumor characteristics with PDO engraftment was analyzed using SPSS version 27. Results: The PDO engraftment rate in the entire cohort was 51% (n = 74 of 145 patients). Neither tumor stage, grading, anatomical sublocalisation, HPV status nor sample type (biopsy vs. surgical specimen) were significantly associated with PDO establishment. Of note, Kaplan-Meier analysis revealed a trend between successful ex vivo engraftment and reduced overall survival (log-rank: p = 0.09), suggesting preferential engraftment of aggressive poor prognosis tumors. Engraftment rate in the subgroup of patients with recommendation for adjuvant chemoradiation was 58%. Time for expansion of PDO cultures for subsequent drug/radiation screening was 18.5 ± 9.6 days (median ± SD). Expansion was completed before start of adjuvant chemoradiation in all but one cases. Conclusions: Our study suggests feasibility of PDO generation for locally advanced aggressive HNSCC within a timeframe suitable for guiding treatment decisions for adjuvant therapy. Planned future directions include clinical validation of drug testing results for individual patients and clinical study cohorts.

Platinum/taxane/pembrolizumab vs platinum/5FU/pembrolizumab in patients with recurrent/metastatic (r/m) head and neck squamous cell carcinoma (HNSCC).

6039 Background: Pembrolizumab +/- chemotherapy is standard of care for patients (pts) with r/m HNSCC. Despite approval of platinum/5FU/pembrolizumab based upon the KN-048 study, a taxane is often substituted for 5FU due to ease of administration and tolerability. No studies have directly compared these approaches. We describe nationwide patterns of taxane vs 5FU chemoimmunotherapy in r/m HNSCC and compare survival and time on treatment between the two. Methods: This study used the nationwide Flatiron Health electronic health record (EHR)-derived de-identified database, and included pts treated with frontline pembrolizumab plus platinum-based chemotherapy (either taxane or 5FU) for r/m HNSCC. Demographic, cancer, and clinical outcomes were summarized. Categorical variables were compared using Pearson’s chi-square test; continuous variables were compared using T-test. Overall survival (OS) was estimated using Kaplan Meier methodology, and compared using log-rank test. Multivariable Cox regression for overall survival adjusting for age, sex, race, year of diagnosis, ECOG performance status (PS), smoking history, primary tumor site, PD-L1, HPV status, cisplatin use, socioeconomic status, insurance, and practice type was performed, with multiple imputation by chained equations for missing variables. Results: Of 444 pts, 321 (72%) received 5FU and 123 (28%) received taxane (108 paclitaxel, 15 docetaxel). Use of cisplatin rather than carboplatin was higher in the 5FU group than the taxane group (16.8% vs 4.9%, p=0.001). Taxane use was higher in academic vs community practices (50.7% vs 23.9%, p<0.001); within community sites, Northeast states had highest taxane use (27.5%) and Western states had lowest taxane use (16.7%). Pts with Medicare or Medicaid were more likely to receive taxane (35.5%) vs commercial (23.6%) or other (26.7%) insurance. OS did not differ between taxane and 5FU groups (mOS 12.7 vs 13.5 months, p=0.743). On multivariable Cox regression, HR for death associated with taxane vs 5FU was 1.01 (95%CI 0.73-1.41). HPV positive status was associated with improved survival (HR 0.58, 95%CI 0.40-0.83). PS>1 (HR 1.38, 95%CI 0.96-1.98) and cisplatin use (HR 1.37, 95%CI 0.95-1.98) showed non-significant trends towards worse survival. Taxane-treated pts received more treatment cycles (mean 11.3 vs 8.8 cycles, p=0.015) and were on treatment slightly longer (mean 6.9 vs 6.0 months, p=0.289) than 5FU-treated pts. Conclusions: In US pts with r/m HNSCC undergoing chemoimmunotherapy, rates of taxane vs 5FU use vary by academic setting, geographic region, and insurance status. There was no difference in survival between 5FU and taxane, and taxane-treated pts received more cycles of therapy. Platinum-taxane appears to be a non-inferior alternative to platinum-5FU with pembrolizumab for r/m HNSCC, and should be allowed in clinical trials.

The Role of the p16 and p53 Tumor Suppressor Proteins and Viral HPV16 E6 and E7 Oncoproteins in the Assessment of Survival in Patients with Head and Neck Cancers Associated with Human Papillomavirus Infections.

The role of HPV in the survival prognosis of patients with head and neck squamous cell carcinoma, especially patients with laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC), is still somewhat ambiguous. The present study aimed to explore the significance of tumor suppressor proteins and HPV16 E6 and E7 oncoproteins in the assessment of survival in patients with oropharyngeal squamous cell carcinoma (OPSCC), LSCC, and HPSCC associated with high-risk (HR-) and low-risk (LR-) HPV infections. By utilizing molecular and immunohistochemical investigations of HNSCC samples and patient data, univariate and multivariate survival analyses were conducted. The presence of HPV DNA (LR- and HR-HPV) was associated with a better 5-year OS and DSS for OPSCC and LSCC. The IHC overexpression of HPV16 E6 protein and p16 protein was associated with better survival in the univariate (for OPSCC) and multivariate (OPSCC and HPSCC) survival analyses. The overexpression of p53 was associated with better survival in OPSCC. HPV infection plays a significant role in the tumorigenesis of HNSCC, and the immunohistochemical assessment of HPV16 E6 protein expression should be interpreted as a useful prognostic marker for OPSCC and HPSCC.

PES1 is a biomarker of head and neck squamous cell carcinoma and is associated with the tumor microenvironment.

As a nucleolar protein associated with ribosome biogenesis in multiple cancer types, PES1 has been reported to be overexpressed, promoting cancer cell proliferation and invasion. However, in head and neck squamous cell carcinoma (HNSCC), the role of PES1 on the prognosis and immune infiltration remains unknown. Multiple databases and qRT-PCR evaluated the expression of PES1 in HNSCC. The prognostic potential of PES1 in HNSCC patients was analyzed by Cox regression and Kaplan-Meier curves. Then, we used LASSO regression and stepwise multivariate Cox regression to construct the PES1-related risk assessment model. In addition, the association between PES1 and tumor immune microenvironment and drug sensitivity was explored by R packages. Finally, we used cell function assays to explore in HNSCC if PES1 influences tumor growth and metastasis. PES1 was significantly up-regulated in HNSCC and closely correlated with HPV status, tumor stage, clinical grade, and TP53 mutation status. Survival analysis suggested that PES1 is associated with worse survival outcomes, acting as an independent prognostic indicator for HNSCC. Our model also performed well in terms of prognosis prediction. Furthermore, tumor-infiltrating immune cells and antitumor drug susceptibility were negatively related to PES1 expression. Functionally, as for HNSCC cell lines in vitro, the knockdown of PES1 could inhibit proliferation, migration, and invasion. We have demonstrated that PES1 may be a promoter of tumor growth. PES1 holds excellent promise as a novel biomarker to assess the prognosis of patients with HNSCC and may guide immunotherapy.

Abstract 6062: Viral genome structure correlates with patterns of somatic alteration in human papillomavirus associated head and neck squamous cell carcinoma

Abstract 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.

Abstract 4572: 3D-EXpress ex vivo platform using a biorepository of characterized fresh patient tumoroids allows development of rational combinations with drugs targeting DNA damage response and immune checkpoint blockade

Abstract Introduction: Nilogen Oncosystems’ 3D-EXpress platform allows for the detailed characterization offreshly resected patient tumor tissue for inclusion in studies investigating rational combinations of IOtherapeutics. Incorporating data on immune cell composition, tumor cell target expression, mutationalstatus, and HPV infection within tumoroids generated from HNSCC tissues provides tremendous input toidentify candidate tissues for therapeutic testing. The 3D-EXpress platform employed here providesdetailed observations investigating tumor cell viability using confocal microscopy and immune cellactivation indicated by increases in cytokine release. Materials and Methods: 3D tumoroids measuring 150 microns in size were generated from fresh patientHNSCC resection samples collected with proper patient consent and relevant IRB approval. Tumoroidswere never enzymatically dissociated, propagated, or reassembled to maintain the intact tumormicroenvironment (TME). The resulting tumoroids were cryopreserved in the Biorepository, where eachsample had associated patient demographic data, tumor grade, stage, mutational and HPV status inaddition to a detailed tumor immune profile and a FFPE representative sample in TMA format for targetselection. Pooled tumoroids for each patient tumor were treated ex vivo both singly with a selectiveWEE1 inhibitor, AZD1775 and an ATR blocker AZD6738 alone and in combination with a PD-1 immunecheckpoint inhibitor antibody nivolumab for 72h. Results and Summary: To quantify treatment-mediated tumor cell killing we used 3D high-contentconfocal imaging and a proprietary algorithm for data analysis. Additionally, culture supernatantsisolated from treated tumoroids were used for multiplex cytokine detection to monitor changes in theTME upon ex vivo treatment. Responses to treatments were further correlated with the composition ofinnate and adaptive TIL populations within each tumor as assessed by 21-color flow cytometry inaddition to tumor mutational status, HPV infection, smoking status, as well as EGFR and PD-L1expression levels analyzed by multiplex IF on associated TMA slides. Conclusion: These results demonstrate that targeting DNA damage response and immune checkpointblockade may provide a potential combination strategy for the treatment of HNSCC. Furthermore, weshowed that our 3D-EXpress ex vivo tumoroid model provides a unique platform to rapidly assess theefficacy of drugs and drug combinations in fresh patient tumor samples with intact TME. Correlation ofex vivo drug responses with characteristics of each tumor’s immune microenvironment and detailedclinicopathological data may allow to identify clinically relevant biomarkers to enable the most effectivetreatment strategies for individual patients in the clinic. Citation Format: Jared Ehrhart, Seth Currlin, Sharon Camacho, Samantha Hoffman, Angie Rivera, Soner Altiok. 3D-EXpress ex vivo platform using a biorepository of characterized fresh patient tumoroids allows development of rational combinations with drugs targeting DNA damage response and immune checkpoint blockade. [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 4572.

Low-Dose Aspirin Has Antiproliferative and Apoptosis Effects in HPV16 Tumor Cells and Delays Tumor Development and Growth in an Experimental Model

Objectives: The purpose of this study was to investigate the effect of aspirin on epithelial HPV16-transformed cells and its antitumor effects, in an experimental HPV16-positive tumor model. Design: The design of the study is experimental (in vitro and in vivo). Participants/Materials, Setting, and Methods: SiHa and BMK-16/myc cells were treated with aspirin and cell proliferation was determined by MTT; Caspase-Glo 3/7 Assay was used to determine apoptosis. The tumor-bearing mice group was treated with 50 mg/gr/day of aspirin (orally) during 30 days and the antitumor effect was determined. Results: Here, we provide evidence that aspirin has a negative effect on proliferation and induces apoptosis in the human (SiHa) and murine (BMK-16/myc) HPV16 cells. Furthermore, aspirin showed inhibition of tumor growth, and in mice treated with aspirin prior to implantation of tumor cells, the tumor growth was delayed. Also, the effect of aspirin increased survival in tumor-bearing mice and in mice pre-treated with aspirin. Limitations: It is necessary to carry out in vitro and in vivo studies of the molecular mechanisms involved in the effects of aspirin on tumor cells. Conclusion: Aspirin showed antiproliferative effects in tumor cells and inhibited tumor progression and could be effective as a chemopreventive agent. Thus, aspirin deserves further exploration for the treatment of cervical cancer and other neoplasms.

Association of Human Papillomavirus Status With Suicide Risk Among Patients With Head and Neck Cancer

Human papillomavirus (HPV) is strongly associated with head and neck cancer, and HPV status is considered a prognostic factor. Being a sexually transmitted infection, HPV-related cancers may have greater risk of stigma and psychological distress; however, the potential association of HPV-positive status with psychosocial outcomes, such as suicide, is understudied in head and neck cancer. To investigate the association between HPV tumor status and suicide risk among patients with head and neck cancer. This population-based retrospective cohort study included adult patients with clinically confirmed cases of head and neck cancer based on HPV tumor status from the Surveillance, Epidemiology, and End Results database from January 1, 2000, to December 31, 2018. Data analysis was conducted from February 1 to July 22, 2022. The outcome of interest was death by suicide. Primary measure was HPV status of tumor site, dichotomized as positive or negative. Covariates included age, race, ethnicity, marital status, cancer stage at presentation, treatment modality, and type of residence. Cumulative risk of suicide among patients with HPV-positive and HPV-negative head and neck cancer was assessed using Fine and Gray competing risk models. Of 60 361 participants, the mean (SD) age was 61.2 (13.65) years, and 17 036 (28.2%) were women; there were 347 (0.6%) American Indian, 4369 (7.2%) Asian, 5226 (8.7%) Black, 414 (0.7%) Native Hawaiian or Other Pacific Islander, and 49 187 (81.5%) White individuals. A competing risk analysis showed a significant difference in the cumulative incidence of suicide between HPV-positive cancers (5-year suicide-specific mortality, 0.43%; 95% CI, 0.33%-0.55%) and HPV-negative cancers (5-year suicide-specific mortality, 0.24%; 95% CI, 0.19%-0.29%). Tumor status that was HPV positive was associated with increased suicide risk in the unadjusted model (hazard ratio [HR], 1.76; 95% CI, 1.28-2.40), but not the fully adjusted model (adjusted HR, 1.18; 95% CI, 0.79-1.79). Among people with oropharyngeal cancer only, HPV status was associated with increased suicide risk, but the width of the confidence interval prevented definitive conclusion (adjusted HR, 1.61; 95% CI 0.88-2.94). The results of this cohort study suggest that patients with HPV-positive head and neck cancer have similar risk of suicide as patients with HPV-negative cancer, despite differences in overall prognosis. Early mental health interventions may be associated with reduced suicide risk in all patients with head and neck cancer and should be assessed in future work.

HPV Molecular Genotyping as a Differential Diagnosis Tool in Cervical Cancer Metastasis

Differentiating metastatic cervical cancer from another primary tumor can be difficult in patients with a history of cervical cancer and a distant lesion. The use of routine HPV molecular detection and genotyping tests could help in these cases. The objective of this study was to identify if an easy-to-use HPV molecular genotyping assay would allow differentiating between HPV tumor metastasis and a new independent primary non-HPV-induced tumor. Between 2010 and 2020, we identified patients with a primary cervical carcinoma who also had another secondary lesion. This identification included a clinical and histologic differential diagnosis of metastatic cervical cancer versus a new primary cancer or metastatic cancer from another site. We used a routine multiplex real-time PCR (rt-PCR) AnyplexTM II HPV28 (Seegene, Seoul, Republic of Korea) to detect the high-risk (HR)-HPV genome in the distant lesions in these patients. Eight cases of cervical cancer with a new secondary lesion were identified. In seven, HR-HPV DNA was detected in the biopsy of the distant lesion, which confirmed the diagnosis of cervical cancer metastasis. In the remaining case, no HPV was detected in the secondary lung biopsy, confirming the diagnosis of new primary lung cancer. Our results pave the way for HPV molecular genotyping use in cases of newly diagnosed distant lesions in patients with a history of HPV cervical neoplasia by using a routine diagnosis process to complete the clinical and histologic differential diagnosis when confronted with ambiguous situations.

Endogenous Retrovirus RNA Expression Differences between Race, Stage and HPV Status Offer Improved Prognostication among Women with Cervical Cancer.

Endogenous human retroviruses (ERVs) are remnants of exogenous retroviruses that have integrated into the human genome. Using publicly available RNA-seq data from 63 cervical cancer patients, we investigated the expression of ERVs in cervical cancers. Four aspects of cervical cancer were investigated: patient ancestral background, tumor HPV type, tumor stage and patient survival. Between the racial subgroups, 74 ERVs were significantly differentially expressed, with Black Americans having 30 upregulated and 44 downregulated (including MER21C, HERV9-int, and HERVH-int) ERVs when compared to White Americans. We found that 3313 ERVs were differentially expressed between HPV subgroups, including MER41A, HERVH-int and HERVK9. There were 28 downregulated (including MLT1D and HERVH-int) and 61 upregulated (including MER41A) ERVs in locally advanced-stage compared to early-stage samples. Tissue microarrays of cervical cancer patients were used to investigate the protein expression of ERVs with protein coding potential (i.e., HERVK and ERV3). Significant differences in protein expression of ERV3 (p = 0.000905) were observed between early-stage and locally advanced-stage tumors. No significant differential expression at the protein level was found for HERVK7 (p = 0.243). We also investigated a prognostic model, supplementing a baseline prediction model using FIGO stage, age and HPV positivity with ERVs data. The expression levels of all ERVs in the HERVd were input into a Lasso-Cox proportional hazards model, developing a predictive 67-ERV panel. When ERVs expression levels were supplemented with the clinical data, a significant increase in prognostic power (p = 9.433 × 10-15) relative to that obtained with the clinical parameters alone (p = 0.06027) was observed. In summary, ERV RNA expression in cervical cancer tumors is significantly different among racial cohorts, HPV subgroups and disease stages. The combination of the expression of certain ERVs in cervical cancers with clinical factors significantly improved prognostication compared to clinical factors alone; therefore, ERVs may serve as future prognostic biomarkers and therapeutic targets. Novelty and Impact: When endogenous retroviral (ERV) expression signatures were combined with currently employed clinical prognosticators of relapse of cervical cancer, the combination outperformed prediction models based on clinical prognosticators alone. ERV expression signatures in tumor biopsies may therefore be useful to help identify patients at greater risk of recurrence. The novel ERV expression signatures or adjacent genes possibly impacted by ERV expression described here may also be targets for the development of future therapeutic interventions.

Pipeline to characterize antigen-specific TCR repertoires in tumors: Examples from an HPV16 tumor model.

Immunotherapies that improve T cell-based anti-tumor immunity have revolutionized cancer. However, the underlying mechanisms of cancer immune responsiveness are still not fully understood. Using immune competent mice for preclinical development of novel mono and combination therapies is a common strategy, and to monitor the T cell response inside tumors and in the periphery offers valuable insight. T cells recognize target cells by based on the binding between the T cell receptor (on T cells) and peptides presented on MHC-I (on tumor cells). As such, the T cell receptor can be used as a "barcode" for a specific T cell clone. Via TCR sequencing, the sequence of this "barcode" can be identified, and eventually, the TCR repertoire in a sample can be assessed as a whole. This information can be useful in multiple ways, including but not excluded to: (i) tracing specific clones in tissues and in blood, and (ii) determine clonal expansion of a specific clone in the tumor microenvironment which suggest anti-tumor activity of the clone in question. This protocol can be used as a guide from experimental design through TCR-sequencing to analysis of the repertoire. Instead of being specifically focused on one type of TCR-sequencing, this protocol can be used as a resource and contains links and references to useful information that has to be considered. Lastly, certain common metrics when analyzing the TCR repertoire are given and discussed.

Predictive markers of immunotherapy in cervical cancer

Study of PD-L1 expression in squamous and adenosquamous cell cervical cancer (CC) by immunohistochemical (IHC) method, assessment of the relationship between PD-L1 tumor status and its clinical and morphological characteristics, TILs, MSI/dMMR, and HPV tumor status. Surgical material was obtained from 41 patients with CC, on which the expression of PD-L1, proteins of the MMR system and p16 was studied by the IHC method, the TILs index was determined. Positive PD-L1 status was found in 51.2% of the studied CC samples. In the study sample, the level of PD-L1 expression depended on the severity of lymphoid infiltration of the tumor (p=0.038), it was shown that a positive PD-L1 status of CC can be expected with a TILs value greater than or equal to 50%. The age of the patients, the histological variant of the tumor, the pT and pN stage, the presence of lymphovascular invasion, and the HPV status did not statistically significantly affect the level of PD-L1 expression, however, there was an association between the PD-L1 status and the grade of CC malignancy (p=0.027). The presence of the MSI/dMMR phenomenon was detected in a small percentage of carcinomas (4.9%), the PD-L1 status of these tumors was determined as positive. A positive PD-L1 status is determined in a significant number of cases of CC, regardless of most of the studied clinical and morphological characteristics; there is a statistically significant relationship between PD-L1 expression and the degree of tumor differentiation and TILs. It has been shown that CC with the MSI/dMMR phenomenon is characterized by a positive PD-L1 status. The authors consider it necessary to study the expression of PD-L1 in patients with cervical carcinomas in order to determine the possibility of prescribing personalized therapy with immune checkpoint inhibitors.

HPV status represents dominant trait driving delineation of survival-associated gene co-expression networks in head and neck cancer

Integration of high-dimensional tumor gene expression data with clinicopathological data can increase our understanding of disease diversity, enable retrospective patient stratification, and identify new potential biomarkers and therapeutic targets. Using a systems biology approach, we provide a holistic overview of gene co-expression networks in head and neck squamous cell carcinomas (HNSCC). Weighted gene co-expression network analysis of HNSCC RNA sequencing data from 519 patients from The Cancer Genome Atlas (TCGA) was used to determine correlates of 5-year survival, using regression tree-based optimal threshold calculations. Survival-associated gene sets were transformed to gene set scores that were assessed for correlation with clinicopathological data. We identified 8 gene co-expression modules for HNSCC tumors, each of which contained co-expressed genes associated significantly with 5-year survival. Survival-associated co-expression gene signatures correlated dominantly with tumor HPV and p16 status. Network analysis identified that survival was associated with signaling networks of infection, immunity, epithelial-mesenchymal transition (EMT), hypoxia, glycolysis, focal adhesion, extracellular matrix, MYC signaling, autophagy and transcriptional regulation. EMT-associated gene signatures were expressed dominantly in fibroblasts, and cancer-associated fibroblasts were inversely correlated with immune activity. Interestingly, a high Immune Suppression Score based on expression of 21 genes associated with immune inhibition and including immune checkpoints, cytokines and regulatory T cell factors, was also associated with increased survival probability, and was significantly higher in HPV+ HNSCC. Networks associated with HNSCC survival were further associated with survival in cervical cancer, melanoma and lung cancer. This study defines 5129 genes associated with HNSCC survival, organized into co-expressed networks, their correlation with clinicopathological data, and with gene expression data from other malignant diseases, and provides a source for the discovery of biomarkers and novel therapies for HNSCC.