James Jabalee

Response to anti-PD-1 immunotherapy in advanced HPV-associated anal squamous cell carcinoma (ASCC) varies and is assessed by infrequent imaging that lags behind tumor biology. Circulating tumor DNA (ctDNA) can be sampled frequently and reflects near-real-time tumor activity yet is rarely built into mechanistic models of treatment response. Using longitudinal tumor volume and HPV ctDNA from 26 patients treated with pembrolizumab, we found that baseline ctDNA scores tracked tumor burden but did not distinguish responders from non-responders, whereas ctDNA after one treatment cycle had a higher relative decrease in responders, six weeks before the next scan. We developed an ordinary differential equation model linking tumor volume to the ctDNA released by tumor cells. Sensitivity, identifiability, and model-selection analyses reduced it to two patient-specific parameters, proliferation and shedding rates, which reproduced individual volume and ctDNA trajectories, including a transient ctDNA spike at treatment onset. Proliferation was faster in non-responders. This framework supports model-based, ctDNA-guided monitoring of immunotherapy response.

Background: TTMV‐HPV DNA testing is prognostic during post‐treatment surveillance of HPV‐associated oropharyngeal squamous cell carcinoma (OPSCC), but the significance of testing before conventional 3‐month response assessment remains unclear. Methods: We retrospectively analyzed 332 patients across 8 US sites with biopsy‐proven HPV‐associated OPSCC who underwent initial definitive treatment and had ≥ 1 TTMV‐HPV DNA test performed 1 to 89 days after treatment completion. Event‐free survival (EFS) was evaluated using a 90‐day landmark approach. Results: The first TTMV‐HPV DNA test obtained 1–89 days after treatment was negative in 310 patients (93%), indeterminate in 3 (1%), and positive in 19 (6%). Among 59 patients with repeat testing after an initially negative result, 57 (97%) remained negative and 2 (3%) converted to positive; both patients who converted subsequently developed recurrence. Of 13 patients with an initially positive result and sufficient follow‐up for trajectory analysis, 6 (46%) subsequently resolved to negative and none developed recurrence, whereas 7 (54%) remained persistently positive and 6 (86%) developed recurrence. In a 90‐day landmark analysis, the last early post‐treatment TTMV‐HV DNA result was associated with subsequent event‐free survival. Conclusions: TTMV‐HPV DNA testing during the 1–89‐day post‐treatment interval was associated with subsequent clinical outcomes in HPV‐associated OPSCC. Persistent positivity identified patients at increased risk, whereas positive results that resolved were not followed by recurrence in this cohort.

Background/Objectives: Surveillance for anal squamous cell carcinoma (ASCC) recurrence relies on clinical examination and imaging. Post-treatment edema, fibrosis, and inflammation can result in clinically indeterminate findings (CIFs) that delay diagnosis and increase patient and system burden. Circulating tumor tissue-modified viral (TTMV)-HPV DNA offers a biologically specific, noninvasive biomarker that may clarify equivocal assessments. Methods: In this multi-center retrospective study, 233 patients with HPV-associated ASCC were evaluated, including 185 with ≥1 post-treatment TTMV-HPV DNA test. CIFs were defined as exams or imaging results not definitively positive or negative for disease, and were paired with subsequent TTMV-HPV DNA tests. Concordance was defined by prespecified follow-up windows comparing TTMV-HPV DNA results with subsequent clinical outcomes. Results: Ninety patients (39%) experienced 214 CIFs, arising from exams (46%, 98) or imaging (54%, 116). Indeterminate rates by assessment were 7% for exams, 17% for imaging, and 1.3% for TTMV-HPV DNA testing. Overall, 52 CIF/TTMV-HPV DNA pairs were eligible for analysis, and TTMV-HPV DNA resolved disease status accurately for 48/52 (92%, 95% CI: 81.5–97.9). Negative tests predicted cancer-free status for 37/41 CIFs (90%), while 100% of positive tests (11/11) were concordant with clinically confirmed recurrence. In 73% of positive cases (8/11), TTMV-HPV DNA was the first indication of recurrence (median lead-time 29 days; IQR 25–147). Conclusions: TTMV-HPV DNA testing reliably clarifies clinically indeterminate findings during ASCC surveillance, demonstrating high accuracy (92%) and earlier detection of recurrence. These data support integration into post-treatment management to reduce diagnostic uncertainty and guide timely care.

Supplementary Figure S1: Case examples of false negative TTMV-HPV DNA results identified during surveillance

Background: The incidence and mortality of anal squamous cell carcinoma (ASCC) are rising, with greater than 80% of cases linked to human papillomavirus (HPV), primarily HPV16. Post-treatment surveillance can be challenging due to the limitations of anoscopy, digital anal rectal exam (DARE), and imaging. Plasma tumor tissue modified viral (TTMV)-HPV DNA has shown strong sensitivity, specificity, and predictive value in detecting the recurrence of HPV-driven oropharyngeal cancer. Here, we investigate the ability of TTMV-HPV DNA for the early recurrence detection of ASCC. Methods: This retrospective clinical case series included 117 patients with HPV-driven ASCC across 7 U.S. centers, monitored with TTMV-HPV DNA during routine clinical care between March 2020 and June 2024. Physician-reported clinical data and biomarker testing data were combined to create a comprehensive, longitudinal dataset for evaluating test performance metrics. Results: Patients had a median age of 63 years and median post-diagnosis follow-up of 19 months. HPV status was primarily confirmed by TTMV-HPV DNA (52%) or p16 immunohistochemistry (39%). Of those tested for TTMV-HPV DNA pretreatment, 85% had a positive result. TTMV-HPV DNA clearance during or within three months post-treatment was associated with significantly better recurrence-free survival. The per-patient surveillance sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 82.8%, 98.4%, 96.0%, and 92.5%. Of 24 patients with a documented recurrence and a positive TTMV-HPV DNA test, the test was the first evidence of recurrence in 14 patients (58.3%), with a median lead time of 59 days (range: 10–536). TTMV-HPV DNA accurately resolved 94.3% of cases with indeterminate clinical findings. Conclusions: TTMV-HPV DNA testing provides a sensitive and specific approach for detecting patients with recurrent ASCC and resolving the status of patients with indeterminate clinical findings.

Supplementary Figure S1: Case examples of false negative TTMV-HPV DNA results identified during surveillance

Supplementary Figure S1: Case examples of false negative TTMV-HPV DNA results identified during surveillance

Purpose: Human papillomavirus (HPV) is causally linked to oropharyngeal squamous cell carcinoma (OPSCC). Consensus guidelines recommend clinical exams and imaging in decreasing frequency as part of posttreatment surveillance for recurrence. Plasma tumor tissue modified viral (TTMV)-HPV DNA testing has emerged as a biomarker which can inform disease status during surveillance. Experimental Design: This retrospective observational cohort study involved 543 patients who completed curative-intent therapy for HPV-associated OPSCC between February 2020 and January 2022 at eight U.S. cancer care institutions. We determined the negative predictive value (NPV) of TTMV-HPV DNA for recurrence when matched to physician-reported clinical outcome data (median follow-up time: 27.9 months; range: 4.5–154). Results: The cohort included mostly men with a median age of 61 who had locoregionally advanced disease. HPV status was determined by p16 positivity in 87% of patients, with a positive HPV PCR/ISH among 55%; while pretreatment TTMV-HPV DNA status was unknown for most (79%) patients. Patients had a mean of 2.6 tests and almost half had three or more TTMV-HPV DNA results during surveillance. The per-test and per-patient sensitivity of the assay was 92.5% [95% confidence interval (CI): 87.5–97.5] and 87.3% (95% CI: 79.1–95.5), respectively. The NPV for the assay was 99.4% (95% CI: 98.9–99.8) and 98.4% (95% CI: 97.3–99.5), respectively. Conclusions: TTMV-HPV DNA surveillance testing yields few false negative results and few missed recurrences. These data could inform decisions on when to pursue reimaging following first disease restaging and could inform future surveillance practice. Additional study of how pretreatment TTMV-HPV DNA status impacts sensitivity for recurrence is needed.

Although advances in targeted therapies have driven great progress in cancer treatment and outcomes, drug resistance remains a major obstacle to improving patient survival. Several mechanisms are involved in developing resistance to both conventional chemotherapy and molecularly targeted therapies, including drug efflux, secondary mutations, compensatory genetic alterations occurring upstream or downstream of a drug target, oncogenic bypass, drug activation and inactivation, and DNA damage repair. Extracellular vesicles (EVs) are membrane-bound lipid bilayer vesicles that are involved in cell–cell communication and regulating biological processes. EVs derived from cancer cells play critical roles in tumor progression, metastasis, and drug resistance by delivering protein and genetic material to cells of the tumor microenvironment. Understanding the biochemical and genetic mechanisms underlying drug resistance will aid in the development of new therapeutic strategies. Herein, we review the role of EVs as mediators of drug resistance in the context of cancer.

Extracellular vesicles (EVs) play a key role in the communication between cancer cells and stromal components of the tumor microenvironment (TME). In this context, cancer cell-derived EVs can regulate the activation of a CAF phenotype in TME cells, which can be mediated by several EV cargos (e.g., miRNA, proteins, mRNA and lncRNAs). On the other hand, CAF-derived EVs can mediate several processes during tumorigenesis, including tumor growth, invasion, metastasis, and therapy resistance. This review aimed to discuss the molecular aspects of EV-based cross-talk between CAFs and cancer cells during tumorigenesis, in addition to assessing the roles of EV cargo in therapy resistance and pre-metastatic niche formation.