Patient samples used for single-cell RNA sequencing and whole exome sequencing.

Comparison to historical control comparing beta-binomial distributions.

Single-cell landscape of TME and epithelial cells from longitudinally collected tumor samples at baseline and during trial therapy.

List of Patient Mosaic Team and Rare Tumors Initiative collaborators

Operating characteristics under varying trial-limiting toxicities rates.

Treatment emergent adverse events at least possibly related to study therapy.

Overall survival from treatment initiation with ixazomib plus gemcitabine plus doxorubicin.

Number of tumor cells in scRNA-seq analysis from the 11 patients with RMC.

Tagraxofusp is a CD123-targeted therapy comprised of a recombinant human interleukin-3 (IL-3) fused to a truncated diphtheria toxin payload. It is the first approved treatment specifically for patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN). To identify biomarkers of response, bone marrow samples from 12 BPDCN patients who were treated with tagraxofusp in the pivotal phase II trial (NCT02113982) were profiled longitudinally using a gene panel and single-cell RNA sequencing. Residual tumor cells following tagraxofusp expressed lower levels of TXNRD1 that would reduce the efficacy of tagraxofusp. In support of this, enzymatic inhibition of TXNRD1 resulted in higher viability of CAL-1 BPDCN cells following tagraxofusp. Responders had either wild-type or missense TET2 mutations, while transient and non-responders had at least one truncating TET2 mutation. Examples of these mutations within the catalytic domain of TET2 were constructed and transduced into cells. Missense and truncating mutants displayed reduced sensitivities to hypomethylating agents and prolonged S-phase stasis. These results suggest that the levels of TXNRD1 interact with intrinsic TET2 truncating mutations within the bone marrow to modulate patient response to tagraxofusp.

Mucosal melanoma (MM) is a rare and aggressive melanoma subtype with poor outcomes and poorly understood risk factors, including the contribution of germline pathogenic variants (PVs). In this study, 346 MM patients treated at MD Anderson Cancer Center underwent germline sequencing of 322 known cancer susceptibility genes, with PV frequencies compared to population controls (gnomAD and TOPMed) using Fisher's exact test. The cohort had a median age at diagnosis of 64 years and was predominantly female (62%) and White (84.0%). Anatomical subtypes of MM included gastrointestinal (36%), head and neck (33%), and genitourinary (31%). Among patients with somatic testing results (n = 303), KIT mutations (26%) were most common, followed by NRAS (17%), BRAFNon-V600 (6.6%), and BRAFV600 (2.6%). Germline PVs were identified in 37 patients (10.7%), most frequently in CHEK2 (n = 9, 23.0%), ATM (n = 8, 20.5%), and MITF (n = 6, 15.4%). MITF p.E318K (OR 6.0; 95% CI 2.2-13.2) and CHEK2 c.1100delC (OR 6.7; 95% CI 1.8-17.5) were significantly enriched compared to population control rates. Patients with germline PVs were more likely to have two or more affected first-degree relatives (49.0% vs. 29.1%, p = 0.019). These findings highlight a meaningful germline contribution to MM risk and support the incorporation of genetic testing in this population.

Dose adjustments of trial therapy.

S1. Heatmap of unsupervised hierarchical clustering of the top 1000 most variable genes.S2. Violin plots showing the expression levels of genes previously identified as differentially expressed genes between primary and recurrent tumors in Haltia et al., 2020.S3. Gene set enrichment analysis performed with GO terms and KEGG pathways showing that primarily immune-related and hormone-regulated gene sets expression are altered between primary and recurrent aGCTs.S4. Gene set enrichment analysis results showing that top enriched gene sets are significantly enriched in recurrent tumors in comparison with primary tumors.S5. Boxplot showing the fraction of each noncancerous cell type identified by CIBERSORTx (A) and xCell (B) in primary and recurrent tumor samples.S6. Heatmap showing the correlation between immune cells fractions.

Clinical Characteristics by Patient

The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal hematopoiesis is not well defined. We conducted whole-genome sequencing on 1,276 single-cell-derived hematopoietic stem and progenitor cell (HSPC) colonies from ten patients with multiple myeloma treated with chemotherapies and six normal donors. Melphalan treatment significantly increased the mutational burden, producing a distinctive mutation signature, whereas other chemotherapeutic agents had minimal effects. Consequently, the clonal diversity and architecture of post-treatment HSPCs resemble those observed in normal elderly individuals, particularly through the progression of oligoclonal hematopoiesis, thereby suggesting that chemotherapy accelerates clonal aging. Integrated phylogenetic analysis of matched therapy-related myeloid neoplasm samples traced their clonal origin to a single-HSPC clone among multiple competing clones, supporting a model of oligoclonal to monoclonal transformation. These findings underscore the need for further systematic research on the long-term hematological consequences of cancer chemotherapy.

Differentially Expressed Genes

Abstract Background: Mucosal melanoma (MM) is the rarest subtype of melanoma with markedly worse outcomes compared to cutaneous melanoma (CM). Unlike CM which is driven by somatic mutations due to UV exposure, risk factors for MM are less defined. While germline mutations in cancer susceptibility genes are well-studied in CM, they are less understood in MM. Herein, we aimed to identify the prevalence of germline mutations in cancer susceptibility genes in a large MM cohort. Methods: A cohort of 247 MM patients (pts) from MD Anderson Cancer Center with available blood for germline sequencing was identified. Pt characteristics were reflective of U.S. MM population with median age of 63, 60% females and 84.6% white. Clinicodemographic features of pts including tumor site, key somatic mutations (BRAF, NRAS, KIT) and personal history of other malignancies were abstracted, and their frequencies were compared in pts with and w/o germline mutation. Frequency of MITF and CHEK2 alleles were compared to expected population control rate based on TOPMED by Fisher’s exact test. Results: Among 247 pts, 10.9% had germline mutations, primarily in MITF (c.G952A:p.E318K) (2.4%) and CHEK2 (c.1100delC:p.T367fs) (1.6%) at OR of 13.9 (95%CI 5.0, 30.9) and 16.4 (4.4, 42.9) compared to population control rate (Table 1). MM sites of origin were gastrointestinal (36%), genitourinary (34%) and head & neck (30%). In terms of somatic mutations, KIT (26%) was most common, followed by NRAS (15%), non-V600E BRAF (6.5%) and BRAF V600E (2.4%). These variables did not differ between those with and w/o germline mutation. Pts with germline mutation had a higher incidence of non-melanoma malignancies (48% vs 29%; p=0.044). Table 1. Summary of identified germline mutations in the mucosal melanoma cohort. Germline mutation Protein MITF (N= 6) p.E318K CHEK2 (N=4) p.T367fs MUYTH p.G368D ATM splice ATM p.L263fs ATM p.R1875X ATM p.L1327X ATM p.A2067D ATM p.R2034X BRCA2 p.A1689fs CHEK2 p.S400T ERCC3 p.R109X MITF p.C29Y NF1 p.L1183R PALB2 p.V221X POLE p.D287E TP53 p.R306X TP53 p.V73Wfs*50 TSC1 p.Q844X Conclusions: This study reports a high prevalence of germline cancer susceptibility mutations in the largest cohort of MM to date, including mutations which are actionable. There is overlap with known MITF risk alleles for CM. We are currently performing a GWAS to comprehensively understand the genetic architecture of risk in MM. Citation Format: Afsaneh Amouzegar, Xiaogang Wu, James Long, Sabitha Prabhakaran, Latasha Little, Curtis Gumbs, Jared Malke, Julie Simon, Jianhua Zhang, Jennifer Wargo, Paul Scheet, Justin W. Wong, Priyadharsini Nagarajan, Jennifer L. McQuade, Andrew Futreal. Germline mutations in cancer susceptibility genes in patients with mucosal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7334.

10595 Background: Mucosal melanoma (MM) is a rare and aggressive subtype of melanoma. Outcomes for MM are significantly worse than for cutaneous melanoma (CM). While somatic mutations induced by UV radiation are known to cause CM, less is known about environmental and genetic alterations associated with MM. High penetrance alleles have been identified in familial cases of CM but the contribution of germline mutations to development of MM is not understood. Further, whilst population-based risk has been elaborated in CM, little is known for MM. The aim of this study is to characterize the germline genetic architecture of MM from high penetrance alleles to patient population genetics. Methods: A large retrospective cohort of MM patients (n=247) seen at MD Anderson Cancer Center were identified. Demographics, primary tumor sites, and presence of somatic mutations in BRAF, NRAS and KIT were extracted. Germline sequencing was performed on PBMC derived genomic DNA from the cohort using a gene panel of 322 major cancer genes. Frequency of identified germline alleles were compared to expected population control rate based on TOPMED using Fisher’s exact test. For genome wide association analysis (GWAS), genotyping will be performed on the MM cohort and the control group using the Infinium Global Screening Array. Cancer-free controls matched 3:1 based on MM cohort demographics have been assembled from existing case-control studies at MD Anderson. Following standard quality control procedures, we will impute variants from TOPMed using the Michigan Imputation Server. SNPtest v2.5.268 software will be utilized to perform association tests, using a multivariate logistic regression model incorporating gender and principal components. In addition to variants that may surpass a genome-wide significance score, we will inspect results for an enrichment of cancer genes. Results: Median age of MM cohort is 63, female predominant (60%), primarily White (84.6%), with gastrointestinal (36%), genitourinary (34%) and head & neck (30%) being the most common MM subtypes. Characteristics of the cohort were representative of the US MM population. Somatic mutations in KIT (26%) were most common. Germline mutations were found in 10.9% of the cohort, with most common detected variations in MITF (c.G952A:p.E318K) (2.4%) at OR of 13.9 (95%CI 5.0, 30.9), and CHEK2 (c.1100delC:p.T367fs) (1.6%) at OR of 16.4 (4.4, 42.9) compared to population control rate. Conclusions: Our study is the first to demonstrate the distribution of germline mutations in the largest cohort of MM patients. The MITF E318K allele is a known risk factor for CM, while CHEK2 1100delC allele has mixed evidence for conferring CM risk. GWAS data are currently being analyzed and these updated results will be presented in the context of the comprehensive analyses of germline risk alleles in MM patients.

Therapy-related myeloid neoplasms (t-MNs) represent one of the most devastating complications associated with cancer chemotherapy. The incidence of t-MNs is notably high following autologous stem cell transplantation (ASCT) for multiple myeloma (MM) or lymphoma. Previous studies have investigated peripheral blood stem cells (PBSCs) in patients undergoing ASCT, revealing an association between the presence of clonal hematopoiesis (CH) and an increased risk of t-MNs post-ASCT. However, it remains unclear whether the CH mutations identified in PBSCs ultimately evolve into the t-MN clone, due to the absence of analyses that examine matched PBSC and t-MN samples. Furthermore, patients without CH in their PBSCs can still develop t-MNs, and the origin of t-MNs in these individuals remains unidentified. To elucidate the clonal origin and evolutionary trajectory of t-MNs post-ASCT, we studied 9 patients with MM who developed t-MNs following ASCT, with a median latency of 3 years (range: 1-8). We generated single-cell derived colonies from mobilized PBSCs of these patients and performed whole-genome sequencing (WGS) on these. A median of 86 colonies per patient was sequenced, totaling 1,032 colonies. For the matched t-MN samples, we conducted bulk WGS on bone marrow samples with a median coverage of 50x. Utilizing genome-wide somatic mutations identified in each colony, we constructed phylogenetic trees of PBSCs. Subsequently, we integrated the genome of t-MN samples within the PBSC phylogeny to identify the clonal origin (i.e., most recent common ancestor [MRCA]) of t-MNs at the single stem cell resolution. In the phylogenetic trees derived from PBSC samples, parallel evolution of distinct TP53 and PPM1D mutations was pervasive. Single-cell WGS data facilitated clone-specific analyses of mutation burden and mutation signatures. The number of somatic mutations was comparable among TP53, PPM1D, and wild-type colonies. Cells harboring TP53 or PPM1D mutations did not exhibit specific mutation signatures. Among 84 TP53-mutated cells, 82 (97.6%) exhibited normal copy number profiles, with only 2 cells displaying copy number alterations in 17p. These findings suggest that TP53-mutated cells do not yet manifest genomic instability at the clonal hematopoiesis stage. Integrated phylogenetic analysis of PBSC colonies and t-MN genomes identified the MRCA of t-MNs in 5 of 9 (56%) patients' PBSC samples. In these patients, the origin of t-MNs could be traced to a single stem cell carrying TP53 mutations, which later acquired chromosomal abnormalities or biallelic alteration of TP53 at the time of transformation. The MRCA was not identifiable in the PBSCs of 4 patients. In two of these patients, melphalan-related mutation signatures were detected in their t-MN samples. Since these patients did not receive melphalan therapy other than during ASCT conditioning, the presence of melphalan-related signatures in t-MN samples suggests that their clonal origin lies in bone marrow HSCs that were not mobilized. Our findings indicate the existence of two distinct pathways for t-MN development post-ASCT: one originating from mutant stem cells in mobilized PBSCs, and the other from bone marrow stem cells that were not mobilized. Further studies are warranted to elucidate the clinical implications of these distinct evolutionary pathways in t-MNs.

Normal hematopoietic stem and progenitor cells (HSPCs) inherently accumulate somatic mutations and lose clonal diversity with age, processes implicated in the development of myeloid malignancies 1 . The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal HSPCs is not well defined. We conducted whole-genome sequencing on 1,032 single-cell-derived HSPC colonies from 10 patients with multiple myeloma (MM), who had undergone various chemotherapy regimens. Our findings reveal that melphalan treatment distinctly increases mutational burden with a unique mutation signature, whereas other MM chemotherapies do not significantly affect the normal mutation rate of HSPCs. Among these therapy-induced mutations were several oncogenic drivers such as TET2 and PPM1D . Phylogenetic analysis showed a clonal architecture in post-treatment HSPCs characterized by extensive convergent evolution of mutations in genes such as TP53 and PPM1D . Consequently, the clonal diversity and structure of post-treatment HSPCs mirror those observed in normal elderly individuals, suggesting an accelerated clonal aging due to chemotherapy. Furthermore, analysis of matched therapy-related myeloid neoplasm (t-MN) samples, which occurred 1-8 years later, enabled us to trace the clonal origin of t-MNs to a single HSPC clone among a group of clones with competing malignant potential, indicating the critical role of secondary mutations in dictating clonal dominance and malignant transformation. Our findings suggest that cancer chemotherapy promotes an oligoclonal architecture with multiple HSPC clones possessing competing leukemic potentials, setting the stage for the selective emergence of a singular clone that evolves into t-MNs after acquiring secondary mutations. These results underscore the importance of further systematic research to elucidate the long-term hematological consequences of cancer chemotherapy.

Abstract T-prolymphocytic leukemia (T-PLL) is a rare mature T-cell neoplasm defined by rearrangements involving TCL1 or MTCP1. Cases showing some overlapping features with T-PLL but lacking TCL1 and MTCP1 rearrangements have been rarely reported but are not well characterized. Whether these neoplasms belong within the category of T-PLL or represent a distinct entity is unknown. Here, we fully characterize 20 such cases. The median survival for this cohort was 34.7 months. Clinically, 40% of patients were diagnosed incidentally and 65% of patients presents with an indolent phase that was associated with a better survival. Leukemic cells were small to medium sized with a mature morphology. They were CD4-positive with TCRαβ subtype and maintained the expression of other pan-T antigens. A complex karyotype, 11q22.3/ATM deletion and chromosome 8q abnormalities were common, present in 70%, 45% and 35% of patients, respectively. The most common mutations involved ATM and JAK/STAT pathway genes, identified in 40% and 38% of patients, respectively. When this cohort was compared to 42 cases of prototypical T-PLL, they shared many overlapping clinicopathological features and had a similarly poor prognosis. We therefore propose that the neoplasms in this cohort are best classified as TCL1-family negative T-PLL.