CAR T-cell therapy has emerged as a transformative treatment for hematological malignancies, yet its potential to drive lymphomagenesis poses significant clinical concerns. In this study, we investigated the mechanisms underlying CAR T-cell-associated lymphomagenesis in the gastrointestinal (GI) tract on a single case, focusing specifically on the role of integrin a4b7 expression and a predisposing somatic SH2B3 mutation. We observed oligoclonal CAR T-cells homing to and clonally expanding in the GI tract, with the dominant expanded clone harboring both a pathogenic SH2B3 mutation and a CAR transgene integration within a TFCP2 locus. The clonal CAR T-cells subsequently transitioned beyond the GI tract into the peripheral blood, suggesting a potential pathway for systemic dissemination. We found clinical, histological, and molecular evidence demonstrating the efficacy of cyclosporine in reducing the expanded malignant clone and achieving durable clinical remission for more than a year. Our findings highlight the complex interplay between CAR T-cell therapy, pre-existing genetic vulnerabilities, and the GI microenvironment, emphasizing the need for vigilant monitoring and tailored therapeutic strategies to address the risks associated with CAR-T lymphomagenesis.

Background: Tumor-infiltrating lymphocyte (TIL) enrichment in primary breast cancers is predictive of treatment response, and specific CD4 and CD8 subsets are associated with neoadjuvant chemotherapy (NAC) and immunotherapy response. Spatially defined T-cell receptor (TCR) evolution patterns in resection tissues associated with NAC response and survival have not been described. Methods: To define such patterns, we applied high-throughput TCR profiling to pre- and post-NAC tissues and associated immune dynamics with tumor evolution and clinical outcomes. We generated TCR repertoires of 29 primary breast cancers and 768 spatially catalogued, post-NAC specimens from matched cases (median 24 post-treatment samples/case) using Sequence Affinity capture & analysis By Enumeration of cell-free Receptors (SABER) applied to tissue-based Cancer Personalized Profiling by Deep Sequencing (t-CAPP-Seq). To profile each primary tumor’s genomic and immunologic response to NAC, we designed personalized oligonucleotide panels using pretreatment whole exome sequencing and a fixed panel of recurrently mutated genes to capture emergent alterations, as well as a comprehensive panel of TCR-β regions. Leveraging information from fragmented TCRs in FFPE tissues, we spatially enumerated TCR clonotype count and composition in each post-NAC sample and compared the relative abundance of clones shared with each pretreatment tumor. For a subset of cases, we enumerated TILs and tumor cellularity in post-NAC samples (N = 119). To interrogate clonotype composition, we computed Jaccard similarity indices between each pre- and post-NAC sample pair. We associated post-NAC clonal abundance and clonotype composition with tMRD burden and genomic composition. Finally, we associated TCR repertoire dynamics with quantitative tMRD assessment and survival. Results: TIL density was strongly correlated with the presence of post-NAC residual disease (R=0.94, p<0.001 for histologic invasive carcinoma cellularity; R=0.6, p<0.001 for mean tMRD allele frequency (AF)). Because TILs are defined in the presence of histologic residual disease, we defined tMRD-TILs by TCRs sequenced in tMRD positive tissues, which demonstrated greater repertoire diversity than non-tMRD TCRs (greater ShannonE and inverse Simpson diversity indices, p=.007 and p=1.3e-5, respectively), suggesting a spatially defined tumor neoantigen response. Within cases with low tMRD burden (mean AF ≤3%), which experienced longer progression-free survival than those with high tMRD burden (p=0.034), we observed greater TCR clone count in the post-NAC tissue (p=.012), greater shared clone count with the pretreatment tumor (p=5.7e-8), and greater similarity of the post-NAC clonotypes to the pretreatment tumor (p=5.1e-6, Wilcoxon test of Jaccard indices). Presence of a pretreatment CSPP1 lesion was associated with decreased TCR repertoire diversity in chemotherapy-treated resection tissues (ShannonE index, p=0.00056), and CSPP1-mutated tMRD was associated with inferior PFS (HR 23.45, p=.0007) Conclusions: By spatially defining changes in the genomic and immunologic microenvironment following NAC, we found superior molecular response is correlated with preservation of the clonal relationship between tMRD-TILs and the primary tumor with simultaneous generation of a rich TCR repertoire. Ongoing investigations involve computationally predicting neoantigen targets of clonally dominant tMRD-TILs through NAC for in vivo validation, representing an avenue for refining selection of molecularly targeted adjuvant therapies. Citation Format: Julia Ransohoff, Mia A. Carleton, Sofia Miron Barroso, Gregory Bean, Alisha Maltos, James Ford, George Duran, Michael Khodadoust, Melinda L. Telli, Ash A. Alizadeh, David M. Kurtz. Spatially defining T-cell clonal evolution in post-neoadjuvant breast cancer and association with molecular residual disease [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-02-19.

Background: Circulating tumor DNA (ctDNA) is undetectable after neoadjuvant chemotherapy (NAC) in most breast cancer patients with residual disease, limiting its utility to inform adjuvant treatment. The current standard-of-care for assessing NAC response is pathologic evaluation of resection tissues. Many patients without histologically detected post-NAC disease, however, go on to recur, and many with significant gross residual disease are cured, highlighting the need to more accurately quantify low-burden post-NAC MRD, when adjuvant treatment decisions are made. Methods: We describe spatial post-NAC MRD detection by tissue-based Cancer Personalized Profiling by Deep Sequencing (t-CAPP-Seq), profiling tissue-based MRD (tMRD) in breast and lymph node resection tissues. Using personalized oligonucleotide hybrid capture panels derived from whole exome sequencing (WES) along with a fixed panel of recurrently mutated and biologically relevant breast cancer genes, we established spatial tMRD detection across numerous spatially catalogued samples per case using barcode mediated error suppression and a Monte Carlo statistical framework. We compared tMRD to pathologic detection in matched samples and described patterns of quantitative tMRD measurement that predict recurrence risk. Results: We followed a median of 36 (range: 1-112) single nucleotide variants (SNVs)/case derived from WES and a fixed panel of 56 common breast cancer genes spanning 225kb across 29 tumors representing a range of breast cancer subtypes. Nine (33%) and 10 (37%) tumors harbored TP53 and PIK3CA mutations, respectively. We genotyped 797 individual spatially resolved post-surgical specimens spanning these cases (median 24 post-treatment blocks/case, range: 12-46) and established strong concordance between genomic and histologic MRD detection (considering histology as the gold standard, sensitivity=78.8%, specificity=83.3%), with excellent classification of histologic status by tMRD (AUC=0.92). tMRD positive samples undetected by pathology had lower mean allele frequencies (AFs) than those detected by both methods (p<0.001), suggesting a superior limit of detection by tMRD. With a cohort median progression-free survival (PFS) of 140 months, we identified 8 progression events, including two local recurrences adjacent to a tMRD-positive surgical margin that was histologically negative for invasive carcinoma. One of these recurrences was identified post-mastectomy in a pathologic complete response case. Both tMRD positive margins contained missense PIK3CA SNVs that were absent from the pretreatment tumor, representing identification of targetable (alpelisib, capivasertib) post-NAC driver lesions that were selected under the pressure of chemotherapy. In the overall cohort, higher mean tMRD AF across the resection tissues was associated with inferior PFS (HR 1.29, 95% CI 1.08-1.55, p = 0.004). We defined tMRD high status as a mean case AF >3%, which predicted inferior PFS (p=0.034) and identified four genes (CSPP1, POLE, DNAAF4, PCNT) containing SNVs in post-NAC tissues that were significantly correlated with inferior PFS across the cohort (Cox proportional hazards model, p<0.05). Conclusions: Here we introduce t-CAPP-Seq, a novel tMRD detection platform that outperforms surgical pathology for MRD assessment, quantitatively and spatially profiling patterns of genomic lesions under the selection pressure of treatment that are associated with clinical progression. We anticipate tMRD-based interventional adjuvant approaches will motivate movement of molecularly targeted therapies into the adjuvant setting, leading to superior survival outcomes and challenging current treatment paradigms. Citation Format: Julia Ransohoff , Mia A. Carleton, Sofia Miron Barroso, Gregory Bean, Alisha Maltos, James Ford, George Duran, Michael Khodadoust, Melinda L. Telli, Ash A. Alizadeh, David M. Kurtz. Enhanced spatial detection of post-neoadjuvant breast cancer minimal residual disease by tissue-based Cancer Personalized Profiling by Deep Sequencing [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-03-17.

Abstract Abnormalities involving class I HLA are frequent in many lymphoma subtypes but have not yet been extensively studied in cutaneous T-cell lymphomas (CTCLs). We characterized the occurrence of class I HLA abnormalities in 65 patients with advanced mycosis fungoides or Sézary syndrome. Targeted DNA sequencing, including coverage of HLA loci, revealed at least 1 HLA abnormality in 26 of 65 patients (40%). Twelve unique somatic HLA mutations were identified across 9 patients, and loss of heterozygosity or biallelic loss of HLA was found to affect 24 patients. Although specific HLA alleles were commonly disrupted, these events did not associate with a decrease in the total class I HLA expression. Genetic events preferentially disrupted HLA alleles capable of presenting greater numbers of putative neoantigens. HLA abnormalities co-occurred with other genetic immune evasion events and were associated with worse progression-free survival. Single-cell analyses demonstrated that HLA abnormalities were frequently subclonal. Through analysis of serial samples, we observed that disrupting class I HLA events change dynamically over the disease course. The dynamics of HLA disruption are highlighted in a patient who received pembrolizumab and in whom resistance to pembrolizumab was associated with the elimination of an HLA mutation. Overall, our findings show that genomic class I HLA abnormalities are common in advanced CTCL and may be an important consideration in understanding the effects of immunotherapy in CTCL.

Despite advances in treatments, multiple myeloma (MM) remains an incurable cancer where relapse is common. We developed a circulating tumor DNA (ctDNA) approach in order to characterize tumor genomics, monitor treatment response, and detect early relapse in MM. By sequencing 412 specimens from 64 patients with newly diagnosed or relapsed/refractory disease, we demonstrate the correlation between ctDNA and key clinical biomarkers, as well as patient outcomes. We further extend our approach to simultaneously track CAR-specific cell-free DNA (CAR-cfDNA) in patients undergoing anti-BCMA CAR T-cell (BCMA-CAR) therapy. We demonstrate that ctDNA levels following BCMA-CAR inversely correlate with relative time to progression (TTP), and that measurable residual disease (MRD) quantified by peripheral blood ctDNA (ctDNA-MRD) was concordant with clinical bone marrow MRD. Finally, we show that ctDNA-MRD can anticipate clinical relapse and identify the emergence of genomically-defined therapy-resistant clones. These findings suggest multiple clinical uses of ctDNA for MM in molecular characterization and disease surveillance.

Localized cancers are increasingly treated with neoadjuvant therapies prior to resection. For operable breast cancers, this approach can aid in breast-conservation, therapeutic response assessment, and prognostication. For patients with residual disease, addition of adjuvant therapy can reduce recurrence risk and improve outcomes. Pathological assessments of neoadjuvant responses, however, imperfectly predict survival, resulting in under- and over-treatment of many patients. Current liquid biopsies are also falsely negative in most patients with residual disease, limiting their actionable utility. We developed a novel personalized strategy for detection of spatial molecular residual disease (sMRD) and applied it to monitoring neoadjuvant response in 767 spatially-cataloged resection tissues from 29 diverse breast cancers. We utilized Faxitron digital specimen mammography images to generate 3D spatial renderings of each tumor bed that we oriented within the breast and overlaid with sMRD and pathologic detection data from individual samples to evaluate disease distributions. We benchmarked sMRD against histopathological residual disease assessment of individual resection samples from each case, demonstrating strong concordance (sensitivity=88.9%, specificity=86.2%). Spatial case reconstructions captured local and distant recurrence patterns: we identified molecularly-involved, histologically negative margins at eventual local recurrence sites; metastatic progressors exhibited a broader maximal linear spread of sMRD relative to non-progressors (“Dmax”, 5.26cm vs 3.34cm, p=.041) and greater sMRD spatial dispersion interspersed with genomically undetected regions, while responders exhibited more compact disease foci (standard deviation of distances to highest detected sample per case, 1.43cm vs 0.96cm, p=.02). We additionally identified somatic alterations in multiple classes of genes selected through chemotherapy that were associated with clinical recurrence, including genes involved in DNA repair (BRCA1, FANCA), genomic stability (POLE), and ciliogenesis (CSPP1, DNAAF4, CFAP54), suggesting a novel role for loss of ciliogenesis as an emergent chemoresistance mechanism. Finally, we developed a multivariate predictive sMRD model of recurrence in which patients with high versus low sMRD scores experienced inferior recurrence-free survival (log-rank p=.001). We demonstrate that sMRD profiling permits ultrasensitive, quantitative assessment of genomic patterns of tumor evolution associated with clinical recurrence and outperforms routine histopathologic assessment. Our results suggest sMRD could guide risk-adapted personalized adjuvant therapies, heralding a shift of molecular therapeutics from the metastatic to the adjuvant setting. Julia D. Ransohoff, Mia A. Carleton, Sofia Miron Barroso, Nick Phillips, Alisha Maltos, Gregory Bean, Jordan Goldstein, Crystal Zhou, Ruwan Gunaratne, Hitomi Hosoya, George Duran, Michael Khodadoust, James Ford, Robert Tibshirani, Melinda L. Telli, David Kurtz, Ash A. Alizadeh. Defining spatial molecular residual disease by personalized tumor tissue profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6352.

Background: T-cell lymphoma (TCL) derived from chimeric antigen receptor (CAR)-T cells have been reported as a rare event (2 cases) in multiple myeloma (MM) patients receiving ciltacabtagene autoleucel (cilta-cel; Harrison et al., ASH 2023; Ozdemirli et al., NEJM 2024), one of which was limited to the gastrointestinal tract. Here, we present the second reported case of enterotropic CAR+ TCL post cilta-cel, presenting as persistent diarrhea. Methods: Diagnostic workup for TCL included routine labs, serial EGD/colonoscopies (EGD/C) with biopsies with immunohistochemistry and FDG-PET scan. The presence of BCMA-CAR+ cells in tissue and blood was assessed by flow cytometry using fluorochrome labeled human BCMA/TNFRSF17 protein (CAR FACS). T-cell receptor (TCR) clonality was assessed by high-throughput sequencing (TCR-HTS). Mutational profiling of tissue and blood was performed by a clinically validated targeted next-generation sequencing, Stanford Tumor Actionable Mutation Panel for Hematopoietic and Lymphoid Neoplasms (Heme-STAMP). Results: A 50 y/o male with a history of kappa light chain MM received cilta-cel as 5th line of therapy. Upon evaluation for CAR-T, his free kappa light chain (FKLC) was 33.8 mg/dL, bone marrow plasma cells (BMPCs) >90%. After a bridging therapy with high-dose cyclophosphamide, the BMPCs decreased to 1-5% and FKLC to 11.5 mg/dL. After CAR-T infusion, he had grade 1 CRS on day (D)8 lasting 24 hours with no ICANS. On D59, he developed profuse watery diarrhea with negative work-up for infectious etiology and was treated with antibiotics with moderate improvement. On D86, he presented with 4-6 liters (L) stool/day. Biopsies of the stomach and duodenum showed crypt apoptotic activity. Of note, he did not have prior history of allogeneic transplant or inflammatory bowel disease (IBS). He received budesonide, corticosteroids, and infliximab (10 mg/kg) on D102/D111 with improvement to 4 formed stools/day. However, by D133, he had recurrent 4-6L diarrhea/day with 17 kg weight loss. Repeat EGD/C D137 revealed duodenal erosions and mild atrophy and edema in the proximal small intestine with dense T-cell infiltration in the lamina propria (predominately CD8+/TCRb+/TRBC1- cells positive for TIA1, granzyme, and perforin). H. pylori and celiac tests were negative. He received 2 more doses of infliximab. Repeat EGD/C on D151 showed villous blunting and foveolar metaplasia in duodenum and ileum with decreased density of lymphocytes. There was no evidence of chronic intestinal inflammation to suggest IBS. Due to persistent diarrhea (2L/day) he was started on IL-12/23 antibody ustekinumab on D178. However, due to minimal improvement, cyclosporine 5 mg/kg/day was started on D196. After attainment of target level (300-400 ng/mL), he began to have 4 formed stools/day D226, which is stable to date. A EGD/C D246 showed significant improvement of the duodenal architecture with reduced lymphocytes infiltration in the lamina propria. FDG-PET on D210 showed no FDG-avid lesions. CAR FACS of D199 duodenum biopsy showed 35% CD8+CAR+ cells, 16% CD4+CAR+ cells of the total CD45+cells, respectively. Similar findings were observed from ileum (CD8+CAR+ 26%; CD4+CAR+ 13%). TCR-HTS showed 2 dominant nonproductive TCRg sequences, Vg2-Jg1/2 (40%) and Vg5-Jg1/2 (27%). In TCRb, 2 dominant sequences were identified, Db1-Jb2-1 (46%, nonproductive), Vb19-Jb2-3 (13%, productive). Retrospectively, these sequences were also dominant in D97 ileum and D144/D151 duodenum biopsy. Interestingly, these TCRb and TCRg sequences were also detected as dominant from D207 peripheral blood. Heme-STAMP on D144 duodenal biopsy revealed SH2B3 L390P at allele frequency of 26%, which was mapped to the signaling-inhibitory SH2 domain and the variant was predicted to be pathologic by 7 out of 7 variant effect prediction algorithms (Li et al., Sci Adv 2022). Genomic changes of SH2B3 are reported to contribute to oncogenesis in TCL (Bastidas Torres et al., Haematologica 2022). The clonal CAR+ T-cell proliferation within the GI tract was consistent with indolent CAR+ TCL . Conclusions: CAR+ TCL after CAR-T therapy is a rare entity. This is the second reported case of an enterotropic CAR+ TCL. A missense mutation in SH2B3 potentially contributed to the oncogenesis of TCL, but further investigation is needed to elucidate key mechanisms underlying the pathogenesis of this TCL.

Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA+CD39+ T cells. We develop an unbiased multi-omic profiling approach enabling discovery of immune modules stratifying patients. We identify an enrichment of activated regulatory CLA+CD39+ T cells in non-responders and activated cytotoxic CLA+CD39+ T cells in leukemic patients. Our results provide insights into the effects of immunotherapy in CTCL patients and a generalizable framework for multi-omic analysis of clinical trials.

Abstract Background: Neoadjuvant chemotherapy (NAC) for locally advanced breast cancer can down-stage disease prior to surgery, monitor chemo-sensitivity, and provide key prognostic information via pathologic response that guides adjuvant treatment. Greater residual disease as assessed by the residual cancer burden (RCB) index is associated with inferior outcomes but imperfectly predicts recurrence, particularly in triple negative breast cancer (TNBC) where there is heterogeneity of outcomes across RCB scores. As an alternative to RCB and PCR, blood-based minimal residual disease (MRD) from circulating tumor DNA (ctDNA) has been explored. However, poor sensitivity limits ctDNA, which is typically negative post-NAC when treatment decisions are made in patients who later recur. Methods: To address this lack of sensitivity, we developed tissue-based MRD (t-MRD) to assess molecular disease burden in post-NAC breast and lymph node resection tissue. We performed whole exome sequencing on diagnostic tumor and matched germline tissues from five pilot patients with early-stage TNBC with a range of responses to NAC. For each patient, we designed a personalized assay to track tumor-specific SNVs, combined with limited panel of genes recurrently mutated in breast cancer, to enable MRD detection. We then applied this assay to DNA from individual pathology blocks from the tumor resection via ultra-deep targeted hybrid capture. We assessed each sample for the presence of MRD using a Monte Carlo statistical framework to integrate all tumor mutations as previously described (Newman et al, Nature Biotechnology 2016). We aggregated results across all pathology blocks from each case to define each patient’s t-MRD profile and assessed the performance of t-MRD versus gold standard pathologic assessment. Results: We tracked a mean of 136 MRD reporters per case across 119 post-NAC blocks derived from five patients. Twenty-five samples had pathologic residual disease and 28 had detectable t-MRD. There was strong concordance between t-MRD mean allele frequency (AF) and tumor cellularity as assessed by pathology, defined as percent cellularity within the region containing viable tumor (Spearman r = 0.9052, p< 0.0001), and excellent classification of true positive pathology samples by genomic sequencing (AUC = 0.9675, p < 0.0001). Samples (n = 6) where t-MRD but not pathologic residual disease was detected had a lower mean AF than those where there was also histologic residual disease (p=0.0001). Among the five studied cases, we confirmed a molecular complete response with no t-MRD detected in the RCB-0 case (0/44 t-MRD positive blocks). In the two RCB-III cases, we observed more diffuse t-MRD than that assessed by pathology (n = 4 additional blocks with t-MRD but not pathologic disease detected), and in the RCB-II case, we detected minimal t-MRD in only a single block where pathology did not detect residual disease. Significance: By applying blood-based MRD assessment tools to tumor tissue, we developed t-MRD to profile molecular residual disease at the landmark post-NAC timepoint when disease burden is low and adjuvant treatment decisions are made. We demonstrated high concordance with gold standard pathologic assessment, with increased sensitivity and minimal false positive signal. Further studies assessing the additional prognostic value of t-MRD beyond stage-based prognostication and post-NAC RCB scores are ongoing. Citation Format: Julia Ransohoff, Mia Carleton, Alisha Birk, Gregory Bean, Melinda Telli, Ash Alizadeh, James Ford, George Duran, Michael Khodadoust, David Kurtz. Predicting outcomes in locally advanced breast cancer through profiling post-neoadjuvant tissue-based minimal residual disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-10.

Introduction: Immunotherapy is an effective treatment for cutaneous T-cell lymphoma (CTCL) and an active area of investigation. Genomic biomarkers may aid in identifying CTCL patients likely to benefit from immunotherapies. Somatic mutations involving HLA have been previously reported in CTCL. However, there has not yet been a dedicated study of HLA in CTCL, so the frequency of genomic aberrations of HLA remains unknown. We thus aimed to characterize the occurrence of class I HLA abnormalities in patients with advanced CTCL. Methods: We sequenced skin and/or blood specimens with matched germline from a cohort of 49 patients with advanced mycosis fungoides (MF) or Sézary syndrome (SS). Targeted DNA sequencing was performed with a 225-gene panel including coverage of HLA loci. Somatic mutations of class I HLA were called using POLYSOLVER and validated by Sanger sequencing. Loss Of Heterozygosity in Human Leukocyte Antigen (LOHHLA) was used to identify loss of heterozygosity (LOH). Single-cell RNA-sequencing (scRNA-seq) was performed on 8 samples from 6 patients. Allele-specific expression of class I HLA at a single-cell level was used to validate LOH. Progression-free survival (PFS) was determined from the time of initial diagnosis until the first progression event. Results: Nine unique somatic HLA mutations were identified among 6 patients. In contrast, only one patient had a coding mutation involving B2M. LOH of class I HLA was common, affecting 20 patients (41%), and was due to both focal and non-focal deletions (Figure 1A). In two patients with SS, we validated HLA LOH through HLA allele-specific scRNA-seq analysis. Imbalanced expression of HLA alleles occurred exclusively in the malignant T-cell population. In one patient, LOH involving HLA appeared to be clonal (Figure 1B-C), but in the other, LOH was identified in a phenotypically distinct subclonal population. scRNA-seq data for a third patient confirmed expression of an HLA-B splice site mutation restricted to malignant T-cells. In total, at least one HLA abnormality was detected in 24/49 patients (45%). Patients were categorized by HLA status for correlation with clinical characteristics and outcomes. There were no differences in CTCL subtype (MF vs. SS), stage, or age. There were also no differences in estimated tumor mutational burden. The solitary patient with an abnormality ofB2M had a hypermutated phenotype with 131 mutations per megabase. Patients with genomic abnormalities of HLA had significantly worse PFS than those without (median PFS 31.7 months vs. 98.9 months, respectively; P = 0.016). Conclusions: Our findings show that genomic class I HLA abnormalities are common in advanced CTCL. Further investigation is necessary to explore potential links between HLA abnormalities, disease prognosis, and responses and/or resistance to immunotherapy. The research was funded by: Grant K08 CA207882 and Haas Family Foundation. Keywords: Cutaneous non-Hodgkin lymphoma, Genomics, Epigenomics, and Other -Omics, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

The supplementary data include one table and 5 figures. Table S1 presents cabazitaxel cytotoxic activity in the human breast cancer cell line MCF-7, uterine sarcoma cell line MES-SA, and ovarian cancer cell lines ES-2, MES-OV, and OVCAR-3. Figure S1 presents the molecular structure of cabazitaxel and cytotoxicity curves for three taxanes in MCF-7 cells. Figure S2 shows an immunoblot of drug transporter proteins in parental and drug resistant variants of MCF-7. Figure S3 shown rhodamine123 and BODIPY-paclitaxel accumulation by flow cytometry in parental and resistant variants. Figure S4 shows polymerized versus soluble tubulin in parental vs resistant variants. Figure S5 shows expression of apoptotic regulators.

Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III β-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other β-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking β-tubulin isotype compensation visible at the level of total β-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.

Background Measurable residual disease (MRD) is an important predictor of survival outcomes in both newly diagnosed and relapsed/refractory multiple myeloma (MM). Currently, it is assessed by bone marrow aspirate, either using next-generation sequencing or multi-color flow cytometry. However, not only does it require an invasive procedure, it may not capture the spatial heterogeneity of the tumor. Here, we apply cell-free DNA liquid biopsies via Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) to detect circulating tumor DNA (ctDNA) and quantitatively track in MM patients undergoing anti-BCMA CAR T-cell therapy. Methods We performed CAPP-Seq for the detection of ctDNA using a MM-specific gene panel, which we previously developed.1 A total of 252 plasma, germline and tumor samples from 28 patients receiving BCMA directed standard of care CAR-T therapy (idecabtagene vicleucel, n=23 or ciltacabtagene autoleucel, n=5) were sequenced to assess the dynamics of ctDNA. Clinical MRD was performed on day 90 (+/- 15 days) after CAR infusion by ClonoSeq (Adaptive Biotechnologies), or by multiparametric flow cytometry (Mayo Clinic) in patients without a trackable sequence for ClonoSeq. The clinical MRD and ctDNA levels at matched timepoints were assessed for correlation. These metrics were also correlated with patient outcomes. Results Using CAPP-Seq, a median of 90 SNVs (range 2–264) were detected per case; 24/28 patients (86%) had a sufficient number of variants for tumor monitoring. On day 90, 14 patients had evaluable plasma. For clinical MRD, 24/28 patients were submitted for identification of trackable sequences by ClonoSeq including 6 patients who already had identified clones during prior therapy; 8/24 patients (33%) failed the identification of dominant sequences. For these patients, MRD flow cytometry was sent on day 90. Overall, 16 patients had clinical MRD data (ClonoSeq, n=13; flow, n=3). For patients who had both day 90 ctDNA and clinical MRD (n=14), there was a significant correlation in quantification by respective measures (rho=0.71, p=0.007; figure 1A). Kaplan-Meier analysis showed progression-free survival (PFS) significantly correlated with day 90 ctDNA level (p=0.03; figure 1B) as well as clinical MRD (p=0.008). Conclusions MRD has emerged as an important prognostic biomarker in MM. However, current methods depending on bone marrow aspirates limit the frequency of assessment. ctDNA assessment by CAPP-Seq can quantitatively follow the disease burden and assess MRD. ctDNA-MRD levels correlate with clinical MRD, and ctDNA negativity on day 90 is associated with improved PFS. ctDNA-MRD can potentially provide comparable information to clinical bone marrow MRD. Reference Hosoya H, Carleton M, Tanaka K, Sworder B, Hovanky V, Duran G, Zhang T, Khodadoust M, Miklos D, Arai S, Iberri D, Liedtke M, Sidana S, Kurtz D. Disease Characterization and Response Prediction in Myeloma Patients Undergoing Conventional and Cellular Therapies from Circulating Tumor DNA. Blood 2022;140(Supplement 1):1546–1548. Ethics Approval This study has obtained ethics approval by Stanford IRB under protocol number of #18329 and #5019. All the participants gave informed consent before taking part.

Supplementary Information from Genetic and Epigenetic Modeling of the Origins of Multidrug-Resistant Cells in a Human Sarcoma Cell Line

Background: Multiple myeloma (MM) is an incurable disease with a heterogenous clinical course and genomic landscape. Autologous anti-BCMA chimeric antigen receptor (CAR) T-cells are a promising new therapy, but determinants of response and resistance are not well known. Cell-free DNA (cfDNA) is a useful tool to study MM as it allows for repeated, non-invasive tumor assessment. We apply a novel method for simultaneously tracking tumor mutations and CAR T-cells from cfDNA using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq). Methods: We designed a 480kb CAPP-Seq hybrid capture panel to identify mutations, track tumor burden and minimal residual disease, and detect cfDNA derived from the CAR transgene (CAR-cfDNA) in patients receiving idecabtagene vicleucel (ide-cel). Flow cytometry (FC) for enumeration of CAR T-cells was performed from peripheral blood mononuclear cells (PBMCs) when available. Results: We profiled 153 biologic samples, including plasma, PBMCs, and bone marrow mononuclear cells, from 15 patients receiving ide-cel and 18 healthy controls. We observed a median of 84 SNVs (range 30-277) prior to therapy. Patients with prolonged responses (>90 days) had significantly lower circulating tumor DNA (ctDNA) at day 28 post-infusion than patients with early progression (<90 days) (0.6 vs. 4.6 log haploid genome equivalents (hGE)/mL; p=0.002). Additionally, higher ctDNA at D28 was prognostic for time to progression (TTP) (HR=1.67, p=0.019). We validated CAR-cfDNA detection by comparison with FC from PBMCs at matched timepoints (n=38), finding a significant correlation (rho=0.79, p=3E-09). CAR-cfDNA typically reached its peak level around D14 (median 332 hGE/mL), with ctDNA declining at the same time-point. Thus, CAR-cfDNA levels and ctDNA burden were inversely correlated (rho= -0.3, p=0.019). Surprisingly, peak CAR expansion was not associated with TTP (HR=1, p=0.463). However, lower CAR-cfDNA at D28 was prognostic for inferior TTP (HR=2.68, p=0.011). This suggests CAR persistence may play a more important role in clinical outcomes. Furthermore, among progressors, time until loss of detectable CAR-cfDNA correlated with TTP (rho=0.81, p=0.02). Patients who progressed before day 90 had a median CAR persistence of 28 days. In contrast, patients who progressed after day 90 had a median of 137 days and often had emergent copy number alterations in ctDNA at relapse. This included one case with emergent loss of chr16, where TNFRSF17 (BCMA), resides. This event was detected 36 days prior to clinical relapse; BCMA loss was validated via whole-genome sequencing and immunohistochemistry staining of the tumor. Conclusions: Cell-free DNA is a promising biomarker for mutational genotyping, disease monitoring, and tracking CAR T-cells in MM. The persistence of CAR-cfDNA has particular prognostic importance and novel strategies to increase CAR persistence should be explored. Citation Format: Mia Carleton, Hitomi Hosoya, Kailee L. Tanaka, Brian Sworder, Vanna Hovanky, Bita Sahaf, Matthew J. Frank, George E. Duran, Tian Y. Zhang, Sally Arai, David Iberri, Michaela Liedtke, David B. Miklos, Michael S. Khodadoust, Surbhi Sidana, David M. Kurtz. Tumor and immune determinants of response to anti-BCMA CAR T-cell therapy in multiple myeloma using cell-free DNA. [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 5707.

Mogamulizumab is a humanized anti-CCR4 antibody approved for the treatment of mycosis fungoides and Sézary Syndrome. Despite almost universal expression of CCR4 in these diseases, most patients eventually develop resistance to mogamulizumab. We tested whether resistance to mogamulizumab is associated with loss of CCR4 expression. We identified 17 patients with mycosis fungoides or Sézary syndrome who either were intrinsically resistant or acquired resistance to mogamulizumab. Low expression of CCR4 by immunohistochemistry or flow cytometry was found in 65% of patients. Novel emergent CCR4 mutations targeting the N-terminal and transmembrane domains were found in 3 patients after disease progression. Emerging CCR4 copy number loss was detected in 2 patients with CCR4 mutations. Acquisition of CCR4 genomic alterations corresponded with loss of CCR4 antigen expression. We also report on outcomes of three cutaneous T-cell lymphoma patients with gain-of-function CCR4 mutations treated with mogamulizumab. Our study indicates that resistance to mogamulizumab in CTCL frequently involves loss of CCR4 expression and emergence of CCR4 genomic alterations. This finding has implications for management and monitoring of CTCL patients on mogamulizumab and development of future CCR4-directed therapies.