Dynamic Risk Profiling Using Serial Tumor Biomarkers for Personalized Outcome Prediction.

Accurate prediction of long-term outcomes remains a challenge in the care of cancer patients. Due to the difficulty of serial tumor sampling, previous prediction tools have focused on pretreatment factors. However, emerging non-invasive diagnostics have increased opportunities for serial tumor assessments. We describe the Continuous Individualized Risk Index (CIRI), a method to dynamically determine outcome probabilities for individual patients utilizing risk predictors acquired over time. Similar to "win probability" models in other fields, CIRI provides a real-time probability by integrating risk assessments throughout a patient's course. Applying CIRI to patients with diffuse large B cell lymphoma, we demonstrate improved outcome prediction compared to conventional risk models. We demonstrate CIRI's broader utility in analogous models of chronic lymphocytic leukemia and breast adenocarcinoma and perform a proof-of-concept analysis demonstrating how CIRI could be used to develop predictive biomarkers for therapy selection. We envision that dynamic risk assessment will facilitate personalized medicine and enable innovative therapeutic paradigms.

Circulating tumor DNA in head and neck cancer: Early successes and future promise.

LAY SUMMARY: The genetic components (DNA) of human papillomavirus-related throat cancer (in the oropharynx) might be measured after surgery to help to predict whether treatment has been successful.

A Toolbox to Characterize Human Induced Pluripotent Stem Cell-Derived Kidney Cell Types and Organoids.

BACKGROUND: The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible. METHODS: We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics. RESULTS: Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments. CONCLUSIONS: We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.

Modified V-Y Fasciocutaneous Flap Reconstruction After Abdominoperineal Resection in Irradiated Patients Prevents Wound Dehiscence and Associated Complications: A Retrospective Analysis and Benchtop Confirmation.

BACKGROUND: Primary perineal closure following abdominal perineal resection (APR) is reported to have a wound complication rate as high as 66%, whereas flap reconstruction reduces wound complications to 15% to 35%. A modified de-epithelialized V-Y fasciocutaneous flap aims to further improve results in this patient population. METHODS: To study the breaking force of a simple interrupted suture in either skin or subcutaneous fat, various quantitative assessments were performed in a porcine flap model using uniaxial static tensile testing with an Instron tensiometer, with a single or triple row of 3 Vicryl sutures in both skin and fat.An outcomes analysis was performed in 24 patients who underwent modified V-Y flap reconstruction after APR. Primary outcome was wound complications including infection, dehiscence, seroma, hematoma, and pelvic fluid collections. RESULTS: Tensile strength of sutures anchored in skin was found to be up to 8 times stronger than sutures anchored in subcutaneous fat in a single row and 3 times as strong in 3 rows (breaking force, 500.2 N vs 263.7 N). In our patient cohort of 24 irradiated cancer patients, 10 (42%) had wound healing complications. Wound dehiscence of various degrees accounted for 80% of these complications. Five patients with wound complications (50%) had associated pelvic fluid collections (infection, 1; wound dehiscence, 4). Minor dehiscence was more likely to occur after suture removal and less likely to be associated with pelvic collections compared to patients with major dehiscence. Our study yields total complication rates lower than what is reported in the literature for anterolateral thigh or gracilis flap including much lower infection rates, and almost similar results to the commonly used vertical rectus myocutaneous muscle. CONCLUSION: Tension-free de-epithelialized V-Y flap use after APR effectively reconstructs the defect while eliminating an additional donor site. Benchtop studies suggest enhanced flap integrity yielded by layered closure. Wound complications can be managed with local care in their majority (90%). Staggering or delaying suture removal can decrease minor dehiscence. Based on analysis of our results, review of the literature and consideration of donor site morbidity, we believe that modified V-Y flap is the best approach for APR reconstruction in irradiated patients.

Effects of Short-term Electronic(e)-Cigarette Aerosol Exposure in the Mouse Larynx.

OBJECTIVES: The effects of electronic cigarettes (e-cigarettes) on the larynx are relatively unknown. This study examined the short-term effects of e-cigarette inhalation on cellular and inflammatory responses within the mouse laryngeal glottic and subglottic regions after exposure to pod-based devices (JUUL). METHODS: Male C57BL6/J mice (8-9 weeks) were assigned to control (n = 9), JUUL flavors Mint (JMi; n = 10) or Mango (JMa; n = 10). JUUL mice were exposed to 2 h/day for 1, 5, and 10 days using the inExpose inhalation system. Control mice were in room air. Vocal fold (VF) epithelial thickness, cell proliferation, subglandular area and composition, inflammatory cell infiltration, and surface topography were evaluated in the harvested larynges. Mouse body weight and urinary nicotine biomarkers were also measured. Chemical analysis of JUUL aerosols was conducted using selective ion flow tube mass spectrometry. RESULTS: JUUL-exposed mice had reduced body weight after day 5. Urinary nicotine biomarker levels indicated successful JUUL exposure and metabolism. Quantitative analysis of JUUL aerosol indicated that chemical constituents differ between JMi and JMa flavors. VF epithelial thickness, cellular proliferation, glandular area, and surface topography remained unchanged after JUUL exposures. Acidic mucus content increased after 1 day of JMi exposure. VF macrophage and T-cell levels slightly increased after 10 days of JMi exposures. CONCLUSIONS: Short-term e-cigarette exposures cause minimal flavor- and region-specific cellular and inflammatory changes in the mouse larynx. This work provides a foundation for long-term studies to determine if these responses are altered with multiple e-cigarette components and concentrations. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1316-1326, 2024.

Impact of Electronic Cigarettes on the Upper Aerodigestive Tract: A Comprehensive Review for Otolaryngology Providers.

Objective: The use and effects of electronic (e)-cigarettes (e-cigs) are particularly relevant for otolaryngology providers as tobacco plays a major role in benign and malignant diseases of the upper aerodigestive tract. This review aims to (1) summarize the recent policies regarding e-cigs and important patterns of use and (2) serve as a comprehensive resource for clinical providers on the known biologic and clinical effects of e-cigs on the upper aerodigestive tract. Data Sources: PubMed/MEDLINE. Review Methods: We conducted a narrative review on (1) general information on e-cig use and informative findings in the lower respiratory system and a comprehensive review on (2) the effects of e-cigs on cell and animal models and the clinical implications of these products on human health as is relevant to otolaryngology. Conclusions: Although e-cigs are likely less harmful than conventional cigarettes, preliminary research on e-cigs suggest several deleterious effects including in the upper aerodigestive tract. Due to this, there has been increased interest in restricting e-cig usage, particularly among the adolescent population, and caution in recommending e-cigs to current smokers. Implications for Practice: Chronic e-cig use is likely to have clinical implications. It is critical for otolaryngology providers to be aware of the rapidly changing regulations and use patterns regarding e-cigs and how e-cigs influence human health, particularly with regards to the upper aerodigestive tract, to accurately council patients regarding potential risks and benefits of use.

Tracing Founder Mutations in Circulating and Tissue-Resident Follicular Lymphoma Precursors.

Follicular lymphomas (FL) are characterized by BCL2 translocations, often detectable in blood years before FL diagnosis, but also observed in aging healthy individuals, suggesting additional lesions are required for lymphomagenesis. We directly characterized early cooperating mutations by ultradeep sequencing of prediagnostic blood and tissue specimens from 48 subjects who ultimately developed FL. Strikingly, CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, and largely distinguished patients developing FL (14/48, 29%) from healthy adults with or without detected BCL2 rearrangements (0/13, P = 0.03 and 0/20, P = 0.007, respectively). CREBBP variants were detectable a median of 5.8 years before FL diagnosis, were clonally selected in FL tumors, and appeared restricted to the committed B-cell lineage. These results suggest that mutations affecting the CREBBP KAT domain are common lesions in FL cancer precursor cells (CPC), with the potential for discriminating subjects at risk of developing FL or monitoring residual disease. SIGNIFICANCE: Our study provides direct evidence for recurrent genetic aberrations preceding FL diagnosis, revealing the combination of BCL2 translocation with CREBBP KAT domain mutations as characteristic committed lesions of FL CPCs. Such prediagnostic mutations are detectable years before clinical diagnosis and may help discriminate individuals at risk for lymphoma development. This article is highlighted in the In This Issue feature, p. 1275.

Inferring gene expression from cell-free DNA fragmentation profiles.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.

Short Diagnosis-to-Treatment Interval Is Associated With Higher Circulating Tumor DNA Levels in Diffuse Large B-Cell Lymphoma.

PURPOSE: Patients with Diffuse Large B-cell Lymphoma (DLBCL) in need of immediate therapy are largely under-represented in clinical trials. The diagnosis-to-treatment interval (DTI) has recently been described as a metric to quantify such patient selection bias, with short DTI being associated with adverse risk factors and inferior outcomes. Here, we characterized the relationships between DTI, circulating tumor DNA (ctDNA), conventional risk factors, and clinical outcomes, with the goal of defining objective disease metrics contributing to selection bias. PATIENTS AND METHODS: We evaluated pretreatment ctDNA levels in 267 patients with DLBCL treated across multiple centers in Europe and the United States using Cancer Personalized Profiling by Deep Sequencing. Pretreatment ctDNA levels were correlated with DTI, total metabolic tumor volumes (TMTVs), the International Prognostic Index (IPI), and outcome. RESULTS: Short DTI was associated with advanced-stage disease (P < .001) and higher IPI (P < .001). We also found an inverse correlation between DTI and TMTV (RS = -0.37; P < .001). Similarly, pretreatment ctDNA levels were significantly associated with stage, IPI, and TMTV (all P < .001), demonstrating that both DTI and ctDNA reflect disease burden. Notably, patients with shorter DTI had higher pretreatment ctDNA levels (P < .001). Pretreatment ctDNA levels predicted short DTI independent of the IPI (P < .001). Although each risk factor was significantly associated with event-free survival in univariable analysis, ctDNA level was prognostic of event-free survival independent of DTI and IPI in multivariable Cox regression (ctDNA: hazard ratio, 1.5; 95% CI [1.2 to 2.0]; IPI: 1.1 [0.9 to 1.3]; -DTI: 1.1 [1.0 to 1.2]). CONCLUSION: Short DTI largely reflects baseline tumor burden, which can be objectively measured using pretreatment ctDNA levels. Pretreatment ctDNA levels therefore have utility for quantifying and guarding against selection biases in prospective DLBCL clinical trials.

Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA.

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors.

Advances in predictive in vitro models of drug-induced nephrotoxicity.

In vitro screens for nephrotoxicity are currently poorly predictive of toxicity in humans. Although the functional proteins that are expressed by nephron tubules and mediate drug susceptibility are well known, current in vitro cellular models poorly replicate both the morphology and the function of kidney tubules and therefore fail to demonstrate injury responses to drugs that would be nephrotoxic in vivo. Advances in protocols to enable the directed differentiation of pluripotent stem cells into multiple renal cell types and the development of microfluidic and 3D culture systems have opened a range of potential new platforms for evaluating drug nephrotoxicity. Many of the new in vitro culture systems have been characterized by the expression and function of transporters, enzymes, and other functional proteins that are expressed by the kidney and have been implicated in drug-induced renal injury. In vitro platforms that express these proteins and exhibit molecular biomarkers that have been used as readouts of injury demonstrate improved functional maturity compared with static 2D cultures and represent an opportunity to model injury to renal cell types that have hitherto received little attention. As nephrotoxicity screening platforms become more physiologically relevant, they will facilitate the development of safer drugs and improved clinical management of nephrotoxicants.

Circulating Tumor DNA Measurements As Early Outcome Predictors in Diffuse Large B-Cell Lymphoma.

PURPOSE: Outcomes for patients with diffuse large B-cell lymphoma remain heterogeneous, with existing methods failing to consistently predict treatment failure. We examined the additional prognostic value of circulating tumor DNA (ctDNA) before and during therapy for predicting patient outcomes. PATIENTS AND METHODS: We studied the dynamics of ctDNA from 217 patients treated at six centers, using a training and validation framework. We densely characterized early ctDNA dynamics during therapy using cancer personalized profiling by deep sequencing to define response-associated thresholds within a discovery set. These thresholds were assessed in two independent validation sets. Finally, we assessed the prognostic value of ctDNA in the context of established risk factors, including the International Prognostic Index and interim positron emission tomography/computed tomography scans. RESULTS: Before therapy, ctDNA was detectable in 98% of patients; pretreatment levels were prognostic in both front-line and salvage settings. In the discovery set, ctDNA levels changed rapidly, with a 2-log decrease after one cycle (early molecular response [EMR]) and a 2.5-log decrease after two cycles (major molecular response [MMR]) stratifying outcomes. In the first validation set, patients receiving front-line therapy achieving EMR or MMR had superior outcomes at 24 months (EMR: EFS, 83% v 50%; P = .0015; MMR: EFS, 82% v 46%; P < .001). EMR also predicted superior 24-month outcomes in patients receiving salvage therapy in the first validation set (EFS, 100% v 13%; P = .011). The prognostic value of EMR and MMR was further confirmed in the second validation set. In multivariable analyses including International Prognostic Index and interim positron emission tomography/computed tomography scans across both cohorts, molecular response was independently prognostic of outcomes, including event-free and overall survival. CONCLUSION: Pretreatment ctDNA levels and molecular responses are independently prognostic of outcomes in aggressive lymphomas. These risk factors could potentially guide future personalized risk-directed approaches.

A Rare Case of Malignant Transformation of Oral Lichen Planus of the Mandible.

Oral lichen planus (OLP) is an immune-mediated mucocutaneous disease associated with an increased risk in oral squamous cell carcinoma (OSCC). Nearly all cases of malignant transformation have been reported in patients >40 years old. We report the case of a 37-year-old woman with a 5-year history of erosive OLP who presented with malignant transformation to OSCC. Delineating the margins of the disease was impossible at presentation given her OLP, and she was initially treated with concurrent chemoradiation therapy. She then developed a recurrence of the mandibular alveolar ridge. The patient was successfully treated with a composite resection including a segmental mandibulectomy, buccal mucosa resection, partial glossectomy, and ipsilateral neck dissection. This was reconstructed with a free fibula osteo-septo-cutaneous flap. Mandibular OSCC is a rare complication of OLP with few reports on effective reconstructive interventions. The case represents the youngest reported patient with mandibular OSCC arising in the context of OLP and highlights the utility of the free vascularized fibula graft in the treatment of these patients.