Shared and distinct biological circuits in effector, memory and exhausted CD8+ T cells revealed by temporal single-cell transcriptomics and epigenetics.

Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.

Human epigenetic and transcriptional T cell differentiation atlas for identifying functional T cell-specific enhancers.

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.

Developmental Relationships of Four Exhausted CD8+ T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms.

CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.

Paradoxical Role for Wild-Type p53 in Driving Therapy Resistance in Melanoma.

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.

TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion.

Exhausted CD8+ T (Tex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (Teff) or memory (Tmem) CD8+ T cells. Tex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, Tex cells are a distinct immune subset, with a unique chromatin landscape compared with Teff and Tmem cells. However, the mechanisms that govern the transcriptional and epigenetic development of Tex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of Tex cells in mice. TOX is largely dispensable for the formation of Teff and Tmem cells, but it is critical for exhaustion: in the absence of TOX, Tex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in Tex cells. Robust expression of TOX therefore results in commitment to Tex cells by translating persistent stimulation into a distinct Tex cell transcriptional and epigenetic developmental program.

Author Correction: PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.

Detection of Residual Peritoneal Metastases Following Cytoreductive Surgery Using Pegsitacianine, a pH-Sensitive Imaging Agent: Final Results from a Phase II Study.

BACKGROUND: For patients with peritoneal carcinomatosis, extent of disease and completeness of cytoreductive surgery (CRS) are major prognostic factors for long-term survival. Assessment of these factors could be improved using imaging agents. Pegsitacianine is a pH-sensitive polymeric micelle conjugated to the fluorophore indocyanine green. The micelle disassembles in acidic microenvironments, such as tumors, resulting in localized fluorescence unmasking. We assessed the utility of pegsitacianine in detecting residual disease following CRS. PATIENTS AND METHODS: NCT04950166 was a phase II, non-randomized, open-label, multicenter US study. Patients eligible for CRS were administered an intravenous dose of pegsitacianine at 1 mg/kg 24-72 h before surgery. Following CRS, the peritoneal cavity was reexamined under near-infrared (NIR) illumination to evaluate for fluorescent tissue. Fluorescent tissue identified was excised and evaluated by histopathology. The primary outcome was the rate of clinically significant events (CSE), defined as detection of histologically confirmed residual disease excised with pegsitacianine or a revision in the assessment of completeness of CRS. Secondary outcomes included acceptable safety and pegsitacianine performance. RESULTS: A total of 53 patients were screened, 50 enrolled, and 40 were evaluable for CSE across six primary tumor types. Residual disease was detected with pegsitacianine in 20 of 40 (50%) patients. Pegsitacianine showed high sensitivity and was well tolerated with no serious adverse events (SAEs). Transient treatment-related, non-anaphylactic infusion reactions occurred in 28% of patients. CONCLUSIONS: Pegsitacianine was well tolerated and facilitated the recognition of occult residual disease following CRS. The high rate of residual disease detected suggests that the use of pegsitacianine augmented surgeon assessment and performance during CRS.

Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response.

Tumour cells evade immune surveillance by upregulating the surface expression of programmed death-ligand 1 (PD-L1), which interacts with programmed death-1 (PD-1) receptor on T cells to elicit the immune checkpoint response1,2. Anti-PD-1 antibodies have shown remarkable promise in treating tumours, including metastatic melanoma2-4. However, the patient response rate is low4,5. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanomas release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. Stimulation with interferon-γ (IFN-γ) increases the amount of PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumour growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varies during the course of anti-PD-1 therapy. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of treatment, as an indicator of the adaptive response of the tumour cells to T cell reinvigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumour cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

Primary Hyperparathyroidism in Young Adult Patients.

BACKGROUND: Primary hyperparathyroidism (PHPT) affects 2% of Americans over 55 years of age, and is less common in younger patients. Pediatric PHPT patients have higher rates of multigland disease (MGD). We studied young adult patients to determine whether they have similarly elevated rates of MGD and would benefit from routine bilateral neck exploration. METHODS: Retrospective chart review was performed on patients who underwent parathyroidectomy for PHPT (2000-2019). Cohorts were defined by age: Group A (18-40 years) and Group B (> 40 years). Univariate and multivariate logistic regression analyses were performed. RESULTS: Of 3889 patients with PHPT, 9.1% (n = 352) were included in Group A. On multivariate analysis, multiple endocrine neoplasia (odds ratio [OR] 6.3, 95% confidence interval [CI] 3.1-12.7), male sex (OR 1.3, 95% CI 1.0-1.5), family history of PHPT (OR 2.7, 95% CI 1.6-4.8), prior parathyroidectomy (OR 2.2, 95% CI 1.6-3.0), and non-localizing imaging (OR 1.8, 95% CI 1.5-2.1) were associated with MGD; younger age was not an independent risk factor. In patients with sporadic PHPT (n = 3833), family history was most strongly associated with MGD (OR 4.0, 95% CI 2.2-7.3). CONCLUSIONS: In our population of patients with sporadic PHPT, a positive family history of PHPT was strongly associated with MGD; additional associations were found with prior parathyroidectomy, non-localizing imaging, and male sex. Younger age was not an independent risk factor. Age alone in the absence of a family history should not raise suspicion for MGD nor determine the need for bilateral neck exploration.

Disparities in Presentation, Treatment, and Survival in Anaplastic Thyroid Cancer.

BACKGROUND: Disparities have been previously described in the presentation, management, and outcomes of other thyroid cancer subtypes; however, it is unclear whether such disparities exist in anaplastic thyroid cancer (ATC). METHODS: We identified patients with ATC from the National Cancer Database (2004-2020). The primary outcomes were receipt of surgery, chemotherapy, and radiation. The secondary outcome was 1-year survival. Multivariable logistic and Cox proportional hazards regressions were used to assess the associations between sex, race/ethnicity, and the outcomes. RESULTS: Among 5359 patients included, 58% were female, and 80% were non-Hispanic white. Median tumor size was larger in males than females (6.5 vs. 6.0 cm; p < 0.001) and in patients with minority race/ethnicity than in white patients (6.5 vs. 6.0 cm; p < 0.001). After controlling for tumor size and metastatic disease, female patients were more likely to undergo surgical resection (odds ratio [OR]: 1.20; p = 0.016) but less likely to undergo chemotherapy (OR: 0.72; p < 0.001) and radiation (OR: 0.76; p < 0.001) compared with males. Additionally, patients from minority racial/ethnic backgrounds were less likely to undergo chemotherapy (OR: 0.69; p < 0.001) and radiation (OR: 0.71; p < 0.001) than white patients. Overall, unadjusted, 1-year survival was 23%, with differences in treatment receipt accounting for small but significant differences in survival between groups. CONCLUSIONS: There are disparities in the presentation and treatment of ATC by sex and race/ethnicity that likely reflect differences in access to care as well as patient and provider preferences. While survival is similarly poor across groups, the changing landscape of treatments for ATC warrants efforts to address the potential for exacerbation of disparities.

Radiation, Lymph Node Dissection, or Both: Management of Lymph Node Micrometastases from Merkel Cell Carcinoma.

BACKGROUND: Regional lymph node micrometastases from Merkel cell carcinoma (MCC) can be treated with completion lymph node dissection (CLND) and/or radiation therapy (RT). It is unclear how these options compare in terms of survival benefits for patients. PATIENTS AND METHODS: This retrospective cohort study used data from years 2012-2019 of the National Cancer Database. Patients with MCC and clinically negative, but pathologically positive, lymph node metastases who received RT to and/or CLND of the regional lymph node basin were included. Inverse probability weight balancing was performed using covariates followed by Cox proportional hazards modeling for survival analysis. RESULTS: A total of 962 patients were included [median (interquartile range) age, 74 (67-80) years, 662 (68.8%) male patients, 926 (96.3%) white patients]. The majority (63%, n = 606) had a CLND only, while 18% (n = 173) had RT only, and 19% (n = 183) had both CLND and RT. From 2016 to 2019, usage of RT only increased from 10% to 31.8%. Multivariate analysis demonstrated that treatment modality was not associated with survival [RT versus CLND, hazard ratio (HR) 0.842, 95% confidence interval (CI) 0.621-1.142, p = 0.269, RT+CLND versus CLND, HR 1.029, 95% CI 0.775-1.367, p = 0.844]. This persisted after balancing weights (RT versus CLND, HR 0.837, 95% CI 0.614-1.142, p = 0.262, RT+CLND versus CLND, HR 1.085, 95% CI 0.801-1.470, p = 0.599). CONCLUSIONS: The usage of RT for nodal micrometastasis in MCC is increasing as compared with CLND. This strategy appears to be safe, with no significant difference in survival outcomes.

Neoadjuvant Chemotherapy in Retroperitoneal Sarcoma: A National Cohort Study.

INTRODUCTION: Management of retroperitoneal sarcoma (RPS) remains controversial, with the mainstay of treatment being surgery. While neoadjuvant radiation demonstrated no improvement in recurrence-free survival in a prospective randomized trial (STRASS), the role of neoadjuvant chemotherapy (NCT) remains unknown and is the subject of ongoing study (STRASS2). METHODS: Patients who underwent surgical resection of high-grade RP leiomyosarcoma (LMS) or dedifferentiated liposarcoma (DDLS) were identified from the National Cancer Database (2006-2019). Predictors of NCT were analyzed using univariate and multivariate logistic regression analyses. Differences in 5-year survival were examined using the Kaplan-Meier (KM) method and by Cox proportional hazard modeling. RESULTS: A total of 2656 patients met inclusion criteria. Fifty-seven percent of patients had DDLS and 43.5% had LMS. Six percent of patients underwent NCT. Patients who received NCT were younger (median age 60 vs 64 years, p < 0.001) and more likely to have LMS (OR 1.4, p = 0.04). In comparing NCT with no-NCT patients, there was no difference in 5-year overall survival (OS) on KM analysis (57.3% vs 52.8%, p = 0.38), nor was any difference seen after propensity matching (54.9% vs 49.1%, p = 0.48, N = 144 per group). When stratified by histology, there was no difference in OS based on receipt of NCT (LMS: 59.8% for NCT group, 56.6% for no-NCT, p = 0.34; DDLS: 54.2% for NCT group, 50.1% for no-NCT, p = 0.99). CONCLUSION: In patients undergoing surgical resection of RP LMS or DDLS, NCT does not appear to confer an OS advantage. Prospective randomized data from STRASS2 will confirm or refute these retrospective data.

Impact of Patient Comorbidities on Presentation Stage of Breast and Colon Cancers.

BACKGROUND: While patients with multiple comorbidities may have frequent contact with medical providers, it is unclear whether their healthcare visits translate into earlier detection of cancers, specifically breast and colon cancers. METHODS: Patients diagnosed with stage I-IV breast ductal carcinoma and colon adenocarcinoma were identified from the National Cancer Database and stratified by comorbidity burden, dichotomized as a Charlson Comorbidity Index (CCI) Score of <2 or ≥2. Characteristics associated with comorbidities were analyzed by univariate and multivariate logistic regression. Propensity-score matching was performed to determine the impact of CCI on stage at cancer diagnosis, dichotomized as early (I-II) or late (III-IV). RESULTS: A total of 672,032 patients with colon adenocarcinoma and 2,132,889 with breast ductal carcinoma were included. Patients with colon adenocarcinoma who had a CCI ≥ 2 (11%, n = 72,620) were more likely to be diagnosed with early-stage disease (53% vs. 47%; odds ratio [OR] 1.02, p = 0.017), and this finding persisted after propensity matching (CCI ≥ 2 55% vs. CCI < 2 53%, p < 0.001). Patients with breast ductal carcinoma who had a CCI ≥ 2 (4%, n = 85,069) were more likely to be diagnosed with late-stage disease (15% vs. 12%; OR 1.35, p < 0.001). This finding also persisted after propensity matching (CCI ≥ 2 14% vs. CCI < 2 10%, p < 0.001). CONCLUSIONS: Patients with more comorbidities are more likely to present with early-stage colon cancers but late-stage breast cancers. This finding may reflect differences in practice patterns for routine screening in these patients. Providers should continue guideline directed screenings to detect cancers at an earlier stage and optimize outcomes.

Delayed time to radiation and overall survival in Merkel cell carcinoma.

BACKGROUND: Clinically localized Merkel cell carcinoma (MCC) is commonly treated with surgical excision and radiotherapy. The relationship between time to adjuvant radiotherapy and overall survival (OS) remains understudied. METHODS: This retrospective study used data from the National Cancer Database (2006-2019). Patients with clinically localized MCC who received surgical excision and adjuvant radiotherapy were included. Multivariate regressions were used to account for various patient and tumor factors. The primary outcome was 5-year OS, and the secondary outcome was time from diagnosis to adjuvant radiation (TTR). RESULTS: Of the 1965 patients included, most were male (n = 1242, 63.2%) and white (n = 1915, 97.5%), and the median age was 74 years (interquartile range [IQR]: 66-81). The median TTR was 83 days (IQR: 65-106). A total of 83.6% of patients received radiotherapy to the primary site, 21.3% to the draining nodal basin, 17.1% to both, and 12.2% whose target location of radiotherapy was not recorded in the data. TTR of ≥79 days (the 45th percentile) was associated with worse OS on both univariate and multivariate analyses (log-rank p = 0.0014; hazard ratio [HR]: 1.258, 95% confidence interval [CI]: 1.055-1.500, p = 0.010). This persisted on sub-analyses of patients <80 years old (n = 1407; HR: 1.380, 95% CI: 1.080-1.764, p = 0.010) and of patients with Charlson comorbidity index (CCI) of 0 (n = 1411; HR: 1.284, 95% CI: 1.034-1.595, p = 0.024). Factors associated with delayed TTR included greater age (p = 0.039), male sex (p = 0.04), CCI > 1 (p = 0.036), academic facility (p < 0.001), rural county (p = 0.034), AJCC T2 stage (p = 0.010), negative margins (p = 0.017), 2+ pathologically positive regional nodes (p = 0.011), and margin size >2 cm (p = 0.015). CONCLUSIONS: Delayed radiotherapy (≥79 days) was associated with worse OS of MCC patients. Further study in controlled cohorts is needed to ascertain this relationship.

Quality and readability assessment of online patient information on cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND AND OBJECTIVES: We aim to assess the quality and readability of online information available to patients considering cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). METHODS: The top three search engines (Google, Bing, and Yahoo) were searched in March 2022. Websites were classified as academic, hospital-affiliated, foundation/advocacy, commercial, or unspecified. Quality of information was assessed using the JAMA benchmark criteria (0-4) and DISCERN tool (16-80), and the presence of a Health On the Net code (HONcode) seal. Readability was evaluated using the Flesch Reading Ease score. RESULTS: Fifty unique websites were included. The average JAMA and DISCERN scores of all websites were 0.72 ± 1.14 and 39.58 ± 13.71, respectively. Foundation/advocacy websites had significantly higher JAMA mean score than commercial (p = 0.044), academic (p < 0.001), and hospital-affiliated websites (p = 0.001). Foundation/advocacy sites had a significantly higher DISCERN mean score than hospital-affiliated (p = 0.035) and academic websites (p = 0.030). The HONcode seal was present in 4 (8%) websites analyzed. Readability was difficult and at the level of college students. CONCLUSIONS: The overall quality of patient-oriented online information on CRS-HIPEC is poor and available resources may not be comprehensible to the general public. Patients seeking information on CRS-HIPEC should be directed to sites affiliated with foundation/advocacy organizations.

Neoadjuvant chemotherapy in patients undergoing neoadjuvant radiation for trunk and extremity soft tissue sarcoma.

INTRODUCTION: Many patients with high-risk soft tissue sarcoma (STS) develop distant metastases. Meta-analyses suggest that chemotherapy confers a small survival benefit, though few studies focus on neoadjuvant chemotherapy (NCT). There has been more frequent use of neoadjuvant radiation therapy (NRT) in STS, but the utility of NCT for these patients remains unclear. METHODS: Patients with stage II-III trunk/extremity STS who underwent NRT and resection were identified using the National Cancer Database (2006-2019). Predictors of NCT were analyzed using logistic regression. Change in rate of NCT use over time was assessed using log-linear regression modeling. Survival was examined using Kaplan-Meier (KM) and Cox proportional hazard modeling. RESULTS: Of 5740 patients, 25% underwent NCT. The overall median age was 62, 55% of patients were male, and 67% had stage III disease. The most common histological subtypes were fibrosarcoma/myxofibrosarcoma (39%) and liposarcoma (16%). Use of NCT decreased by 4.0% per year throughout the study period (p < 0.01). Predictors of NCT included younger age (median 54, IQR 42-64 vs. median 65, IQR 53-75, p < 0.01), treatment at an academic center (odds ratio [OR] 1.5, p < 0.01), and stage III disease (OR 2.2, p < 0.01). Histologic predictors of NCT included synovial sarcoma (52%) and angiosarcoma (45%). With a median follow-up time of 77 months, NCT was associated with improved 5-year survival compared to NRT alone on KM analysis (70% vs. 63%, p < 0.01). This difference persisted on multivariate analysis (hazard ratio 0.86, p = 0.027) and after propensity matching (70% vs. 65%, p = 0.0064). CONCLUSION: Despite risk of distant failure in high-risk STS, use of NCT has decreased over time in patients receiving NRT. In this retrospective analysis, NCT was associated with a modestly improved overall survival.

Regional control after precision lymph node dissection for clinically evident melanoma metastasis.

INTRODUCTION: Completion lymph node dissection (CLND) for microscopic lymph node metastases has been replaced by observation; however, CLND is standard for clinically detectable nodal metastases (cLN). CLND has high morbidity, which may be reduced by excision of only the cLN (precision lymph node dissection [PLND]). We hypothesized that same-basin recurrence risk would be low after PLND. METHODS: Retrospective review at four tertiary care hospitals identified patients who underwent PLND. The primary outcome was 3-year cumulative incidence of isolated same-basin recurrence. RESULTS: Twenty-one patients underwent PLND for cLN without synchronous distant metastases. Reasons for forgoing CLND included patient preference (n = 11), comorbidities (n = 5), imaging indeterminate for distant metastases (n = 2), partial response to checkpoint blockade (n = 1), or not reported (n = 2). A median of 2 nodes (range: 1-6) were resected at PLND, and 68% contained melanoma. Recurrence was observed in 33% overall. Only 1 patient (5%) developed an isolated same-basin recurrence. Cumulative incidences at 3 years were 5.0%, 17.3%, and 49.7% for isolated same-basin recurrence, any same-basin recurrence, and any recurrence, respectively. Complications from PLND were reported in 1 patient (5%). CONCLUSIONS: These pilot data suggest that PLND may provide adequate regional disease control with less morbidity than CLND. These data justify prospective evaluation of PLND in select patients.

Sentinel lymph node biopsy status improves adjuvant therapy decision-making in patients with clinical stage IIB/C melanoma: A population-based analysis.

BACKGROUND: Given the results of the recent KEYNOTE-716 trial, the performance of sentinel lymph node (SLN) biopsy for patients with clinical stage IIB/C melanoma has been questioned. OBJECTIVE: Determine the utility of SLN status in guiding the recommendations for adjuvant therapy. METHODS: Patients with clinical stage IIB/C cutaneous melanoma who underwent wide local excision and SLN biopsy between 2004 and 2011 were identified from the Surveillance, Epidemiology, and End Results database. Two prognostic models, with and without SLN status, were developed predicting risk of melanoma-specific death (MSD). The primary outcome was net benefit at treatment thresholds of 20% to 40% risk of 5-year MSD. RESULTS: For the 4391 patients included, the 5-year MSD rate was 46%. The model estimating 5-year MSD risk that included SLN status provided greater net benefit at treatment thresholds from 30% to 78% compared to the model without SLN status. The added net benefit for the SLN biopsy-containing model persisted in subgroup analysis of patients in different age groups and with various T stages. LIMITATIONS: Retrospective study. CONCLUSIONS: A prognostic model with SLN status estimating patient risk for 5-year MSD provides superior net benefit compared to a model with primary tumor staging factors alone for threshold mortality rates ≥30%.

Sentinel lymph node biopsy in patients with T1a cutaneous malignant melanoma: A multicenter cohort study.

BACKGROUND: Sentinel lymph node biopsy is not routinely recommended for T1a cutaneous melanoma due to the overall low risk of positivity. Prognostic factors for positive sentinel lymph node (SLN+) in this population are poorly characterized. OBJECTIVE: To determine factors associated with SLN+ in patients with T1a melanoma. METHODS: Patients with pathologic T1a (<0.80 mm, nonulcerated) cutaneous melanoma from 5 high-volume melanoma centers from 2001 to 2020 who underwent wide local excision with sentinel lymph node biopsy were included in the study. Patient and tumor characteristics associated with SLN+ were analyzed by univariate and multivariable logistic regression analyses. Age was dichotomized into ≤42 (25% quartile cutoff) and >42 years. RESULTS: Of the 965 patients identified, the overall SLN+ was 4.4% (N = 43). Factors associated with SLN+ were age ≤42 years (7.5% vs 3.7%; odds ratio [OR], 2.14; P = .03), head/neck primary tumor location (9.2% vs 4%; OR, 2.75; P = .04), lymphovascular invasion (21.4% vs 4.2%; OR, 5.64; P = .01), and ≥2 mitoses/mm2 (8.2% vs 3.4%; OR, 2.31; P = .03). Patients <42 years with ≥2 mitoses/mm2 (N = 38) had a SLN+ rate of 18.4%. LIMITATIONS: Retrospective study. CONCLUSION: SLN+ is low in patients with T1a melanomas, but younger age, lymphovascular invasion, mitogenicity, and head/neck primary site appear to confer a higher risk of SLN+.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.