Representative flow cytometry histograms of STAT1 and P-STAT1 levels in patient PBMCs.

The ability of IL12 to stimulate production of IFNγ suggested it might improve the efficacy of low-dose IFNα. In this phase II trial, patients with metastatic malignant melanoma were administered recombinant human (rh) IL12 followed by IFNα2b. Primary endpoints were clinical response and progression-free survival. Secondary objectives were to evaluate the effect of endogenous IFNγ on JAK-STAT signaling and IFN-regulated genes in peripheral blood mononuclear cells (PBMC). Patients with advanced melanoma received rhIL12 on day 1 and IFNα2b on days 2 to 6 of a 14-day cycle. rhIL12 was given intravenously at 300 ng/kg. IFNα2b was dosed at 3 × 106 units subcutaneously. Plasma IFNγ was assayed by ELISA; JAK-STAT signaling was measured in PBMCs by flow cytometry. The proportion of responders was assessed via Simon two-stage design. Thirty-eight patients were enrolled. The regimen was well-tolerated. Two patients achieved a partial response lasting 6 months or longer (5.3%). IL12 administration led to an increase in mean plasma IFNγ from 33.57 pg/mL at baseline to a maximum of 564.86 pg/mL and increased expression of STAT1 and STAT2 in PBMCs. Generation of phosphorylated STAT1 and IFN-simulated gene product 15 in response to IFNα was enhanced following IL12. rhIL12 given prior to IFNα2b stimulated production of IFNγ, which led to increased levels of JAK-STAT signaling intermediates in patient PBMCs. Combination therapy was reasonably well-tolerated but conferred marginal benefit in patients with metastatic melanoma. These results can inform future studies that use recombinant IL12 or novel IL12 constructs.

INTRODUCTION:Next generation of Theranostics studies are looking at novel combinations with radiation sensitizers to enhance PRRT's efficacy. Here, we present safety and efficacy data from a multi-center phase 1 study of a [¹⁷⁷Lu]lutetium-dotatate -triapine combination in patients with GEP-NETs. METHODS:The study consisted of two phases (dose escalation and expansion) administering the recommended phase 2 doses (RP2D) for the two agents. All patients received 200 mCi of [¹⁷⁷Lu]lutetium-dotatate on day 1 of four 8-week cycles, in combination with oral triapine administered at assigned dose levels (100 mg, 150 mg or 200mg) from days 1 to 14 of each cycle. RESULTS:A total of 31 (A: 15 ; B: 16 ) patients received treatment. Nine patients experienced dose-limiting toxicities; Grade 3 anemia (n=1), Grade 5 cardiac arrest (n=1), and Grade 4 neutropenia (n=7). Based on the overall safety and pharmacokinetics data, a [¹⁷⁷Lu]lutetium-dotatate (200 mCi) plus triapine (150 mg) dose was selected as the RP2D for the expansion phase. Grade ≥3 treatment related adverse events were observed in 77% patients: anemia (23%), nausea and vomiting (3% ), lymphopenia ( 58%), neutropenia ( 35%) and leukopenia ( 39%). The majority of cytopenias were transient and resolved within two weeks. Among the 28 evaluable patients, the objective response rate (ORR) was 21.4% and the median progression-free survival (mPFS) has not been reached (median follow-up period: 23.7 months). CONCLUSIONS:The combination of [¹⁷⁷Lu]lutetium-dotatate (200 mCi) and oral triapine (150 mg D1-14) was well tolerated and demonstrated preliminary activity in patients with well-differentiated GEP-NETs. (NCT04234568).

Immune checkpoint inhibitors (ICIs) have improved outcomes for patients with melanoma and are now the standard of care for high-risk and advanced disease. However, long-term benefits are observed in only around 25% of patients, with significant risk for immune-related adverse events, highlighting the need for predictive biomarkers. To develop a minimally invasive, pre-treatment biomarker strategy, we profiled functional and subset-specific transcripts in peripheral blood T lymphocytes (PBTLs) and applied machine learning to identify predictive signatures. Patients were enrolled prior to receiving ICI monotherapy in the adjuvant (Exploratory n=61, Validation=78) or metastatic (Exploratory n=48, Validation=46) settings. Following feature selection, random forest models were trained and benchmarked against empirical null models. In the adjuvant setting, CD160 and GZMB predicted recurrence (95th percentile), while treatment-limiting toxicity was predicted by a signature comprising TNFRSF18, VTCN1, TIGIT, CCR4, and AHR (97th percentile). In the metastatic setting, baseline CD45RB, a marker of T-cell differentiation, most strongly predicted progression within one year (93.9th percentile). Distinct signatures in the adjuvant and metastatic settings suggest differences in T-cell programs associated with patient outcomes. These findings support further evaluation of pre-treatment circulating T-cell transcriptional profiles as predictors of ICI response and toxicity in melanoma.

Supplementary Table S1. Representativeness of Study Participants.

The phase 2 SWOG S1512 trial ( NCT02775851 ) was designed to evaluate the response to pembrolizumab (anti-PD-1) in individuals with desmoplastic melanoma. Here we report the results of cohort A of the trial, evaluating the pathological complete response (pCR) rate of neoadjuvant PD-1 blockade in surgically resectable desmoplastic melanoma. Secondary endpoints included clinical response rate, overall survival and toxicities. Twenty-eight eligible individuals with resectable desmoplastic melanoma received intravenous pembrolizumab (200 mg) every 3 weeks three times, followed by excision. Tissue samples before treatment, at 3-5 weeks after treatment initiation and at the time of surgery were reviewed. The primary endpoint of pCR rate by local pathological review was 71% (95% confidence interval, 51-87%; P < 0.001), which met the prespecified endpoint. There were two (7%) grade 3 treatment-related adverse events. At three years of follow-up, four participants have died, none known to be from melanoma or adverse events. In conclusion, neoadjuvant pembrolizumab in individuals with resectable desmoplastic melanoma results in a high pCR rate with acceptable safety profile. Clinicaltrials.gov: NCT02775851 .

Supplementary Table S2. Flow cytometry gating strategy relevant to Figure 3.

Circulating tumor DNA (ctDNA) 'tumor fraction' (TF) may correlate with therapy response, but little is known regarding TF dynamics immediately after therapy initiation. Plasma samples from a single-arm clinical trial enrolling patients with triple-negative breast cancer were collected at a variety of time points following infusion of onalespib (day -7), paclitaxel (day 1), and onalespib + paclitaxel (day 8). 313 samples from 14 patients underwent shallow whole genome sequencing and TF determination. The primary objective was to evaluate TF change from pre-infusion to 6 h and 24 h post-infusion. There was significant TF decline from pre-infusion to 6 h for paclitaxel (p = 0.03) but no change for onalespib or onalespib+paclitaxel at 6hl. There was no ctDNA TF surge within minutes to 24 h of onalespib, paclitaxel, or combination, despite an overall decline in TF during the first therapy cycle. However, there was significant decline in TF from pre-infusion to D9 (16% to 6.5%, p = 0.004). These findings support further research on ctDNA dynamics immediately after therapy initiation.

Abstract Diamond magnetometry (DM), using fluorescent nitrogen-vacancy center nanodiamonds (FND), is an enzyme-free, quantum-based platform that can detect and monitor cancer biomarkers, such as microRNAs. Fluorescence is due to the nitrogen vacancy center (NV-center), a crystallographic defect where a nitrogen atom is adjacent to a vacant (empty) site within the diamond lattice. Importantly, NV-center fluorescence can be optically manipulated and is sensitive to nanoscale magnetic fields. In the present study, we used diamond magnetometry to rapidly detect picomolar amounts of microRNAs in human melanoma cell lines without requiring the Polymerase Chain Reaction (PCR) or amplification or preamplification steps. Method. FNDs and magnetic nanoparticles were functionalized with biomolecules to create NV-Biosensors for miR-486-5p, miR-363-3p, miR-196-5p, miR-135b-5p, and miR-21-5p. Synthetic RNA and DNA oligonucleotides were synthesized by IDT and quantified on a Nanodrop spectrophotometer. Total RNA was prepared from the melanoma cell lines A375, MEL-39, CHL-1 HT-144, 18105 Mel, and MEL1174. Following isolation, nucleic acid sensors were mixed with 2 μL of total RNA in TBS reaction buffer containing 0.05% SDS, 5 mM MgCl2, and 1 nM of a random 50-mer DNA sequence. Reactions were run for 15 minutes, then spotted onto a glass coverslip, gently rinsed, then mounted on a widefield fluorescence microscope set-up, and analyzed by a technique called optically detected magnetic resonance (ODMR). In the absence of a microRNA target, the FND and magnetic particle are associated with DNA hybridization and show low ODMR contrast. If a microRNA target is present, it displaces the FND-MNP hybrid, and the FND shows increased ODMR contrast. Results were collected in a few minutes at room temperature, with between 200 and 600 independent ODMR data points recorded for each sample. The large number of data points allowed robust data analysis. Results. We demonstrated the specific detection of synthetic RNA and DNA oligonucleotides for miR-21-5p and miR-486-5p down to a concentration of 1 pmole. Our NV-Biosensors also detected miR target molecules in total RNA preparations from cell lines. We could not detect miR-486-5p in total RNA from HT-144 and this was consistent with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) results. We used total RNA from HT-144 in spiking studies to demonstrate specific detection of miR-486-5p. Finally, we benchmarked our technology against the gold standard, RT-PCR, and showed that DM was as sensitive as RT-PCR for microRNA detection. DM has the advantage of using single microliter sample volumes and is a simple, accurate assay that can be performed in a fraction of the time needed for RT-PCR Citation Format: Isaac Rampersaud, Charles Fletcher, Colin Angell, William E. Carson, Arfaan Rampersaud. Sensitive detection of microRNAs from human melanoma cell lines and FFPE tissue using fluorescent nitrogen-vacancy center nanodiamonds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7628.

Cytokine expression in partial responders.

The adenosinergic pathway represents a critical immunometabolic checkpoint within the tumor microenvironment of non-small cell lung cancer (NSCLC), contributing to immune suppression and therapeutic resistance. PBF-1129, an oral, selective A2B adenosine receptor (A2BAR) antagonist, was evaluated in a phase 1, open-label, dose-escalation trial (NCT03274479) in patients with advanced/metastatic NSCLC who had progressed on standard therapies. All patients had previously received chemotherapy and immune checkpoint blockade. Twenty-one patients received escalating doses (40–320 mg once daily), with no dose-limiting toxicities observed. The most frequently reported treatment related adverse events of any grade were lymphocytopenia (n = 8, 38.1

Water plot of best response in lung NEN.

Rates of breast cancer are increasing among young adult (YA) women aged ≤ 40 years. YAs face unique challenges, including being at high risk for financial hardship. Treatment-related adverse events may represent a modifiable and often overlooked source of financial hardship. In this interview-based study, the narratives of YAs with breast cancer were analyzed to understand how treatment-related adverse events contributed to medical and non-medical costs and long-term economic burden. In this secondary analysis of semi-structured interviews characterizing financial toxicity among adult women with stage I–IV breast cancer treated at The Ohio State University Comprehensive Cancer Center (OSUCCC) between 1/1/2015 and 12/31/2019, previously transcribed and coded data from women ≤ 40 years old was analyzed using inductive and deductive approaches. Twenty breast cancer survivors aged ≤ 40 years participated. Treatment-related adverse events emerged as an important factor contributing to financial toxicity. Participants described complications in nearly every organ system, many of which were disabling and required intervention. While indirect (e.g., job loss, reduced work hours) and direct sources (e.g., compression garments for lymphedema) of costs were noted to cause psychological distress and impact treatment adherence, participants did articulate possible solutions for reducing financial hardship (e.g., direct cash transfer, financial navigation). Treatment-related adverse events can contribute to financial toxicity after breast cancer through direct and indirect costs. Among young adults, indirect costs can include those that result from vocational disruption. Strategies to reduce the risk of financial toxicity should be included in care pathways to address complications of treatment itself.

Myeloid-derived suppressor cells (MDSCs) are expanded in cancer patients, have an intrinsic immunosuppressive function, and thus may play a role in resistance to immunotherapy. Ulceration of the melanoma primary is associated with more aggressive disease and is an independent prognostic factor for melanoma-specific survival. However, the underlying factors contributing to this more aggressive phenotype are not completely understood. The current study aims to correlate changes in circulating MDSC during immunotherapy in patients with ulcerated vs non-ulcerated melanoma primary tumors. Longitudinal changes in levels of circulating MDSCs were analyzed via flow cytometry in melanoma patients receiving immune checkpoint inhibitors (ICIs) and stratified by ulceration status. Following the initiation of therapy, the percentage of total MDSCs increased significantly in patients with both ulcerated ( P = 0.003) and non-ulcerated ( P < 0.001) tumors. When MDSCs were stratified by subset, the proportion of granulocytic MDSC (PMN-MDSC) decreased in patients with non-ulcerated tumors ( P = 0.023), while the proportion remained stable in patients with ulcerated tumors ( P = 0.121). The reduction in the proportion PMN-MDSC in non-ulcerated patients coincided with a statistically significant increase in the proportion of CD14 + /CD15 + MDSC ( P = 0.008), resulting in a greater proportion of CD14 + /CD15 + MDSC in non-ulcerated patients as compared to ulcerated melanoma patients following two infusions of ICIs (27.3 ± 19.2% vs 16.1 ± 19.2%; P = 0.008). The trajectories of the MDSC populations described here provide insight into the altered tumor microenvironment in ulcerated melanoma and highlight key changes in a cell population that could contribute to immunotherapy resistance.

Evaluation of circulating CD8+ cell subsets. (A) Changes in levels of circulating CD8+ naïve (CD45RA+ CCR7+), central memory (CM; CD45RA- CCR7+), effector memory (EM; CD45RA- CCR7-), and terminal effector memory (TE; CD45RA+ CCR7-) subsets at screening and cycle 1, day 15 (C1D15) of nivolumab and temozolomide treatment in the entire study cohort. (B) Levels of co-inhibitory molecules PD-1, LAG3, TIM3, and KLRG1 on circulating CD8+ T cells on study treatment compared to screening. Each symbol represents one patient (n = 9). Line indicates mean, *p<0.05

Desmoplastic melanoma is a distinct subtype of melanoma known to have preexisting immune infiltrates and high ultraviolet light damage, resulting in a high tumor mutational burden. We hypothesized that this may result in high response rates with single-agent anti-programmed death protein 1 (PD-1) therapy. SWOG S1512 was a two-cohort clinical trial testing the activity of pembrolizumab in patients with surgically resectable (cohort A) and unresectable (cohort B) desmoplastic melanoma. Here we report on the cohort B single-arm clinical trial, which enrolled 27 patients with unresectable desmoplastic melanoma receiving pembrolizumab 200 mg intravenously every 3 weeks for up to 2 years, with the primary endpoint of complete response rate. The complete response rate was 37% (95% confidence interval: 19-58%), and the post hoc endpoint of objective response rate was 89% (95% confidence interval: 71-98%). The estimated secondary endpoints of 3-year melanoma-specific progression-free survival and overall survival were 84% and 96%, respectively, with only one patient having died from melanoma progression. Ten patients (37%) experienced grade 3 or 4 adverse events, and nine patients (33%) discontinued treatment because of adverse events. Patients with advanced desmoplastic melanoma have a high response rate to single-agent PD-1 blockade therapy, supporting single-agent anti-PD-1 as the treatment of choice, but are limited by a frequency of toxicities that is numerically higher than in other patient populations. ClinicalTrials.gov identifier: NCT02775851.

Immune checkpoint blockers (ICB) targeting the PD-1/PD-L1 axis represent established therapies for many cancers. However, resistance occurs in most patients due to complex immune-suppressive mechanisms in the tumor microenvironment. NK cells can play effector roles in tumor control, but their impact on T-cell dysfunction and ICB efficacy remains controversial. Through genetic and antibody-mediated NK cell depletion, we found that a subset of tumor-associated NK cells plays a negative role in ICB sensitivity; they further impede CD8+ T-cell differentiation toward a CD69+ BCL2+ EOMES+ GZMB+ TIM3- GITR- phenotype. Mechanistically, the retinoic acid receptor α-dependent differentiation program in CD8+ T cells is hindered by tumor-infiltrating NK cells via competition for IFNα and IL-2. Finally, we observed that lower frequencies of NK cells correlate with better clinical responses to ICBs in patients with cancer. These findings suggest potential avenues for enhancing CD8+ T cell-centered immunotherapy by targeting regulatory NK cells. SIGNIFICANCE:Although NK cells are traditionally viewed as antitumor effectors, our study uncovers their unexpected suppressive role in CD8+ T cell-based immunotherapy. By competing for cytokines, they disrupt retinoic acid receptor α-driven CD8+ T-cell differentiation and limit ICB efficacy. Clinically, reduced NK cell presence is associated with an enhanced immunotherapy response. See related commentary by Galvez-Cancino et al., p. 1777 See related article by Pozniak et al., p. 1819.