Abstract Here we report a newly developed method utilizing long-range PCR and long-read Pacific Biosciences HiFi sequencing that successfully obtained two full-length and annotated mitochondrial genomes from Culex quinquefasciatus Say, 1823 and Culex tarsalis Coquillett, 1896, both from Maricopa County, Arizona, USA. Given the substantial burden of West Nile virus in Maricopa County over the past decade, and that these vectors are primarily responsible for spillover to human populations in the county, it is critical to better understand their distribution over time and space. This study begins to approach this need by contributing a novel approach that has resulted in the first West Nile virus vector mitochondrial genomes from Arizona. Our circular Cx. quinquefasciatus mitogenome is 15,587 bp in length, making it the first USA-based mitogenome sequenced through the AT-rich control region. The Cx. tarsalis mitochondrial genome is 16,416 bp long, longer than recently published California-based CTarK1 and Texas-based PQ585801 mitogenomes. The increased length of the Cx. tarsalis mitogenome is a result of a 905 bp insertion in the AT-rich control region, not present in the species’ publicly available mitogenomes. A maximum likelihood-based phylogenetic reconstruction supports the species designation of these newly-sequenced mitogenomes. The newly developed methodology offers a unique approach to study medically-important vector species around the globe, providing a solution to study populations through pooled vector pathogen surveillance programs.

Background: Adult T-cell leukemia/lymphoma (ATLL) is a distinct entity of peripheral T-cell lymphoma (PTCL) associated with chronic infection by human T-cell lymphotropic virus type 1 (HTLV-1). While ATLL and PTCL-not otherwise specified (PTCL-NOS) share overlapping clinical features, their underlying biology, response to therapy, and patient outcomes are markedly different. To date, there is a lack of comparative studies integrating clinical, sociodemographic, and molecular features are limited, and reliable markers for differentiation. Therefore, our aim was to compare the clinical, sociodemographic, and genetic features of ATLL and PTCL-NOS, and to identify transcriptomic signatures that may distinguish ATLL from other PTCLs, with the goal of improving diagnostic accuracy and therapeutic targeting. Materials and Methods: A retrospective cohort study was performed. Patients were classified according to the diagnosis of ATLL and PTCL-NOS. Descriptive statistics were used to describe the sociodemographics and clinical characteristics. Peripheral blood CD3⁺CD4⁺ T cells were isolated by negative selection and sorted by flow cytometry into CD7⁺CADM1⁻ (putative normal) and CD7⁻CADM1⁺ (leukemic ATLL) subsets. Bulk RNA sequencing was performed, and differential expression analysis was conducted using DESeq2. Key findings were validated at the protein level by Western blot or immunohistochemistry (IHC). Results: A total of 429 patients were included, comprising 220 with ATLL and 209 with PTCL-NOS. The median age at diagnosis was similar between groups (55 years vs. 58 years for ATLL and PTCL-NOS, respectively; p = 0.139). However, sex distribution differed significantly, with a higher proportion of females in the ATLL group (57% vs. 40%; p < 0.001). Most patients in both cohorts presented with advanced-stage disease (stage III–IV: 80% in ATLL vs. 75% in PTCL-NOS; p = 0.343). Functional status varied, a greater proportion of PTCL-NOS patients had ECOG 0–1 (90% vs. 76% in ATLL; p = 0.024). While CHOP-based regimens were common in both groups (ATLL vs. PTCL; 86% vs. 91%, p=0.497), PTCL patients were more likely to have pathologic complete response compared to ATLL (41% vs. 23%, p=0.028). Then RNA-seq analysis was performed on CD4⁺ T-cell subsets isolated from 8 PBMC samples (5 ATLL patients and 3 asymptomatic HTLV-1 carriers). Malignant CD7⁻CADM1⁺ T cells displayed a distinct transcriptional profile compared to CD7⁺CADM1⁻ (putative normal) counterparts. Notably, ZCCHC12 expression was significantly upregulated in leukemic cells (log₂ fold change ≈ 2; p = 0.0306), suggesting a potential role in ATLL pathogenesis. This differential expression was consistently observed across individual samples, highlighting ZCCHC12 as a candidate biomarker for malignant transformation in HTLV-1–associated T-cell populations. Conclusions 1 : There are crucial differences between ATLL and PCTL-NOS in terms of sociographic, clinical profiles, and treatment responses. However, Transcriptomic analysis further revealed distinct gene expression patterns in ATLL, with ZCCHC12 emerging as a potential biomarker of malignant transformation in HTLV-1–associated T-cell lymphomas. We propose this new biomarker as a promising target for tailored diagnosis and therapeutics strategies for patients with ATLL.

Background: The different subtypes of ATLL, as per the Shimoyama classification, are consistently described as distinct diseases in terms of their clinical features, outcomes, and even distinctive molecular patterns. However, it is common to analyze the aggressive ATLL types (acute and lymphoma type) together, which can obscure the unique clinical and biological characteristics of each subtype thereby making it more challenging to understand this complex and difficult-to-treat disease. The objective of this study is to describe a cohort of patients with the lymphomatous type of ATLL in Latin America. Methods: We conducted a cohort study among patients ≥18 years with newly diagnosed ATLL and selected all lymphoma type from T-cell Brazil Project (TCBP, Ambispective, Clinicaltrials.gov ID= NCT03207789, 2015-2025, n= 51) and the Grupo de Estudio Latinoamericano de Linfoproliferativos (GELL, Retrospective, 2000-2023, n= 126). Clinical and biological data at diagnosis were collected, along with information on treatment, response, and follow-up. The Kaplan-Meier method was used to estimate survival, while the Log-Rank test was applied to compare the curves. Additionally, Cox regression models were used to identify prognostic factors for OS and EFS, with a significance level of 5%. Results: This analysis included a total of 177 cases, comprising 100 from Peru, 51 from Brazil, 13 from Chile, 7 from Argentina, and 6 from Colombia. The median age was 54 years (20-95), with 50.6% male, 83% having advanced disease (Ann Arbor stage III-IV), 44% with extra-nodal involvement, 61% having an IPI score of 3-5, and 59% having a PIT score of 2-4. The treatment was likely curative in almost 97% (72% chemotherapy (CT) alone, 18% CT + AZT and INF, 7% AZT + INF). CHOP-like was the most used (44% CHOP and 29% CHOEP). 28% had a complete response after 1st line therapy and 39% no response/progression. Only 5 (3%) received transplantation as consolidation, and 68% had progression/relapse. The median follow-up duration of the survivors was 14 months (1-182). 24-month OS was 28% and 18% for EFS. The final Cox model for OS was median age ≥ 54y (HR 2.35 95%CI 1.55-3.57, p< 0.0001), ECOG ≥2 (HR 1.90 95%CI 1.22-2.97, p=0.005), higher LDH (HR 2.77, 95%CI 1.50-5.10, p=0-.001) and B symptoms (HR 2.65, 95%CI 1.61-4.37, p<0.0001), and for EFS the final model was compound with the same prognosis factors.Conclusion: To our knowledge, this study represents the largest cohort of the ATLL lymphoma subtype in Latin America and suggests that OS and EFS for this malignancy remains poor, primarily due to advanced stage at diagnosis, high-risk clinical features, and limited access to adequate therapy. These findings underscore the need for a richer understanding of the condition to enable appropriate risk stratification and guide clinical trials exploring newer therapeutic approaches.

Supplementary Data from Association between Ancestry-Specific 6q25 Variants and Breast Cancer Subtypes in Peruvian Women

The International Myeloma Society and International Myeloma Working Group recently updated the definition of high-risk multiple myeloma and for the first time this definition includes genomic features that can only be detected by DNA sequencing. As a consequence, the working group recommended clinical testing should transition from FISH to DNA sequencing-based technologies that can detect the translocations, copy number alterations, and coding mutations used to calculate risk along with beta-2-microglobulin levels. To support this transition, we characterized the translocation breakpoints in 68 commercially or publicly available myeloma cell lines to create a gold-standard reference set for the community. This was then used to evaluate a series of different structural callers or dedicated immunoglobulin translocation calling tools. Additionally, as part of the clinical validation of a rapid whole genome sequencing platform, we established the accuracy, limit of detection and precision of our platform and analytical workflow for the detection of immunoglobulin translocations. All cell lines and patient samples used for clinical validation were sequenced on a NovaSeq X Plus using PCR-free libraries sequenced to 30-40x and >118x, respectively. To confirm complex events in the cell lines, we performed long-read sequencing using a PacBio Revio with libraries selected to have inserts exceeding 15kb. We tested the ability of Manta v1.6, IgCaller v1.3, DRAGEN v4.4.6, and SCITAV v0.6.5 to call the individual derivative chromosomes or at least any one derivative from a given translocation. Within the panel of myeloma cell lines, we detected 160 junctions from 92 distinct balanced or unbalanced translocation events in 66 cell lines between a common target gene in myeloma (NSD2, CCND3, MYC, MAFA, CCND1, CCND2, MAF, MAFB) and one of the immunoglobulin loci (IgH, IgK, IgL). Of the individual junctions, we found 9 with breakpoints proximal but outside of the immunoglobulin loci, 15 with insertions of a tertiary part of the genome ranging from 42bp-338kb and one with an immunoglobulin breakpoint containing 565bp of novel sequence. Approximately, 5.6% of derivative chromosome junctions will be undetectable by standard short-read sequencing approaches given the 8/15 events with insertions exceeding a typical WGS library or the one with unmappable sequence. Although 7/15 events with insertions under 260bp could likely be detected with some whole genome sequencing tests, we limited our comparison of bioinformatics approaches to the 135 junctions from 82 translocations that are within the immunoglobulin loci and do not contain defined insertions at the junctions. Using each calling tool in a tumor-only mode, the individual derivative call rate ranged from 66.7% (Manta) to 97.8% (SCITAV), with IgCaller detecting 76.3% while DRAGEN detected 88.2%. Since the majority of translocations are balanced events with two detectable derivatives, we assessed which percentage of translocations are detectable when at least one derivative is detected. This raised the detection rate range from 81.7% (Manta) to 100% (SCITAV), with IgCaller detecting 87.8% and DRAGEN 91.5%. A common theme in undetected breakpoints was immunoglobulin windows with low mapping quality resulting in read pairs being distributed between homologous regions, which resulted in the same event being called multiple times or these reads being ignored, leading to a missed call. For clinical validation we compared the WGS results with gold standard FISH assays from each patient and SCITAV had a sensitivity and specificity of 100%, while DRAGEN had a sensitivity of 94.4% and specificity of 100%. The limit of detection for SCITAV was established to be 6% VAF by diluting tumor DNA into matched normal DNA and additional precision replicates at this VAF resulted in a 96% recovery of calls across all replicates. We have shown that the majority of immunoglobulin translocations are detectable with WGS, but comprehensive detection requires optimized approaches to overcome mapping quality issues existing within immunoglobulin loci. These gold standards and particularly the difficult to detect events can be used for the establishment of clinical sequencing assays for multiple myeloma, which will provide more uniform and complete risk assessments and hold the promise of impacting patient care by identifying novel therapeutic options or predicting if specific immunotherapies will be effective.

Introduction Peripheral T cell lymphoma (PTCL) accounts for 10-15% of all non-Hodgkin lymphomas. As previously described by our group (Fischer et al, 2023 ASH Meeting), Latin America has a distinct epidemiological PTCL distribution, with a high frequency of adult T cell leukemia/lymphoma and extranodal NKT cell lymphoma cases, likely influenced by distinct genetic profiles and viral epidemiology. Survival rates have not improved over the past 20 years, with an estimated OS of 40% at 3 years. Treatment advances have also been limited, except for BV-CHP which access is limited in low- and middle-income countries. Given the above, it is crucial to understand the risk factors related to poor outcomes in PTCL. The IPI, which includes extranodal (EN) site as a variable, has been validated for PTCL. However, the specific impact of EN involvement in nodal PTCL (such as PTCL-NOS, AITL, ALCL ALK+/ALK-) and its biological implications remain unclear. Simplification could improve the reproducibility and applicability of these models, especially in low- and middle-income countries. Objective We aim to evaluate the number of EN sites in nodal PTCL lymphomas as a risk factor or surrogate for outcomes such as OS and PFS in a Latin America cohort. Methodology We compiled data from patients (pts) aged ≥18 years with newly diagnosed nodal PTCL-NOS, AITL and ALCL ALK+/ALK-, from the retrospective registry of the Grupo de Estudio Latinoamericano de Linfoproliferativos (n=339, 2000-2023), the T-cell Brazil Project (n=427, 2015-2022, ambispective). Clinical information included coded patient and site identifiers, gender, age, date and site of diagnostic biopsy, sites of disease, EN (number) and Ann Arbor stage. Initial therapy and response; details on response and relapse rates; subsequent lines of therapy and vital status were also recorded. Our primary endpoints were OS and PFS. REDcap Platform (by Vanderbilt) has been used to collect and store data and for descriptive analysis the IBM-SPSS version 24 was applied. Kaplan-Meier method estimated the OS, whereas Log-Rank tests to compare its curves. This trial is registered at Clinical trials (NCT03207789). Results We enrolled 766 pts (427 TCBP; 339 GELL). We grouped pts according to the number of EN involvement: No EN (n=383); one EN (EN1 n=168) and ≥2 EN involvement (EN2 n=215). In the entire cohort, there was a slight male predominance (61%); median age 56 years (range 15-92 y/o); 74% stage III/IV; 69% IPI ≥2. Most frequently histology was PTCL-NOS (60%), followed by ALCL ALK- (19%) and AITL (12%). The majority had B symptoms (61%) and elevated LDH (55%). CHOEP was the most frequent chemotherapy used (47%), followed by CHOP (34%). Transplant was performed as consolidation in only 16% of the cases. Almost half of patients achieved complete response (CR) after first line (47%), although 33% relapsed. Clinical characteristics were similar between No EN, EN1 and EN2 groups except for stage III/IV (58% vs 79% vs 96%; p<.0001); IPI ≥2 (58% vs 59% vs 99%; p<.0001); ECOG>1 (58% vs 92% vs 99%; p<.0001); bone marrow involvement (16% vs 24% vs 63%%; p <.0001), elevated LDH (54% vs 47% vs 61%; p=.03) and albumin <3.5 g/dL (33% vs 38% vs 58%; p <.0001), respectively. As previously reported, better OS and PFS were seen in ALCL ALK+ compared to ALK- and PTCL-NOS. Among No EN, EN1 and EN2 groups, the CR rates after 1st line (54% vs 46% vs 37%, p=0.002), 60-month OS (45% vs. 44% vs. 27%, p <.0001) and PFS (31% vs. 34% vs. 18%; p <.0001) were inferior in the EN2 group, suggesting the number of EN sites involved assessed by CT and PET-CT matters and may be a feasible clinical surrogate for outcomes in this patient population. Conclusion PTCL remains an unmet medical need due to suboptimal treatment responses and outcomes. Thus, new biomarkers are essential to better stratify patients' risk for PTCL mortality. In this large cohort of PTCL patients, we found the number of EN involvement as a useful and practical prognostic tool to predict response to therapy and outcomes. EN2 was associated to inferior response rates and shorter OS and PFS. Our findings indicate distinct biological characteristics in EN PTCL presentation that merit further molecular investigation, potentially enhancing tailored therapies. Registries are essential given the rarity and poor prognosis associated with these diseases as well as for hypotheses generation.

Background Children with relapsed central nervous system (CNS tumors), neuroblastoma, sarcomas, and other rare solid tumors face poor outcomes. This prospective clinical trial examined the feasibility of combining genomic and transcriptomic profiling of tumor samples with a molecular tumor board (MTB) approach to make real‑time treatment decisions for children with relapsed/refractory solid tumors. Methods Subjects were divided into three strata: stratum 1—relapsed/refractory neuroblastoma; stratum 2—relapsed/refractory CNS tumors; and stratum 3—relapsed/refractory rare solid tumors. Tumor samples were sent for tumor/normal whole-exome (WES) and tumor whole-transcriptome (WTS) sequencing, and the genomic data were used in a multi-institutional MTB to make real‑time treatment decisions. The MTB recommended plan allowed for a combination of up to 4 agents. Feasibility was measured by time to completion of genomic sequencing, MTB review and initiation of treatment. Response was assessed after every two cycles using Response Evaluation Criteria in Solid Tumors (RECIST). Patient clinical benefit was calculated by the sum of the CR, PR, SD, and NED subjects divided by the sum of complete response (CR), partial response (PR), stable disease (SD), no evidence of disease (NED), and progressive disease (PD) subjects. Grade 3 and higher related and unexpected adverse events (AEs) were tabulated for safety evaluation. Results A total of 186 eligible patients were enrolled with 144 evaluable for safety and 124 evaluable for response. The average number of days from biopsy to initiation of the MTB-recommended combination therapy was 38 days. Patient benefit was exhibited in 65% of all subjects, 67% of neuroblastoma subjects, 73% of CNS tumor subjects, and 60% of rare tumor subjects. There was little associated toxicity above that expected for the MGT drugs used during this trial, suggestive of the safety of utilizing this method of selecting combination targeted therapy. Conclusions This trial demonstrated the feasibility, safety, and efficacy of a comprehensive sequencing model to guide personalized therapy for patients with any relapsed/refractory solid malignancy. Personalized therapy was well tolerated, and the clinical benefit rate of 65% in these heavily pretreated populations suggests that this treatment strategy could be an effective option for relapsed and refractory pediatric cancers. Trial registration ClinicalTrials.gov, NCT02162732. Prospectively registered on June 11, 2014.

Introduction: Peripheral T-cell lymphomas (PTCL) encompass a diverse group of mature T-cell neoplasms characterized by unfavorable prognosis, with limited-stage (LS) disease presentation being particularly rare. Current clinical practice in LS PTCL varies, with some clinicians employing abbreviated courses of chemotherapy in conjunction with radiotherapy (RT) (combined modality therapy, CMT), while others adopt more aggressive approaches. This variability is due to the high relapse rates observed in PTCL and the lack of supporting evidence from clinical trials. Most evidence for management arises from real-world studies encompassing all stages of PTCL, with the limited available studies involving small cohorts, leaving the optimal management of LS PTCL undefined. To reduce this knowledge gap, we describe the presentation and management of LS PTCL, and compared outcomes across therapy approaches. Methods: We conducted a retrospective international pooled cohort study of patients (pts) aged ≥18 with newly diagnosed LS PTCL during 2000-2023. Data were obtained from the Grupo de Estudio Latinoamericano de Linfoproliferativos (GELL) registry, the Brazilian T Cell Project (BTCP), and two academic US cancer centers. Primary and secondary endpoints were overall survival (OS) and progression-free survival (PFS). We stratified PTCL subtypes as anaplastic large cell lymphoma (ALCL, regardless of ALK status) and non-ALCL groups (i.e., PTCL not otherwise specified [NOS]; angioimmunoblastic T cell lymphoma [AITL]; and lymphomatous type adult T cell leukemia/lymphoma [L-ATL]). Chemotherapy regimens were classified as brentuximab vedotin (BV)-based (i.e., BV-CH[E]P); CHOP and intensive chemotherapy [iCT] (e.g., CHOEP, EPOCH, HyperCVAD) with or without RT. A pre-pooled analysis confirmed no heterogeneity in endpoints based on regions (i.e., US vs Latin America), suggesting the feasibility of combining the data. Outcomes of first-line therapy are presented by PTCL stratification. Time-to-event endpoints were analyzed using Kaplan-Meier. Results: We enrolled 333 pts (GELL 44%, BTCP 30%, US 26%). The overall median age at diagnosis was 56 years (18-95); most were male (60%), had stage II disease (62%), nodal presentation (65%), ECOG 0-1 (86%), lacked B symptoms (65%), normal serum LDH (54%) and serum albumin >3.5 g/dL (81%). PTCL NOS was the most common non-ALCL subtype (53% vs 8% AITL and 8% L-ATL); 31% were ALCL (ALK+ 15%, ALK- 14%, unknown 2%). Most pts (91%) received first-line therapy with curative intent. CHOP (40%) and iCT (40%) were the most common approaches; only 12% received BV-based therapy and were generally treated at US centers. CMT was given to 27% of LS PTCL. With a median follow-up of 36.2 months in all pts, the 3-year OS and PFS rates were 67% and 52%, respectively. Better survival was seen in ALCL than non-ALCL pts (OS 87% [95% CI 79-94] vs 56% [48-65]; PFS 70% [61-81] vs 42% [35-51], both p<0.001, respectively). The overall response and complete response (CR) rates of BV-, CHOP- and iCT-based approaches were 91% (CR 77%), 81% (CR 62%) and 80% (CR 57%), respectively. None of the chemotherapy regimens offered OS or PFS advantage for ALCL and non-ALCL subtypes. However, in ALCL pts, CMT conferred superior OS (100% [100-100] vs 80% [71-92], p=0.025) and PFS (100% [100-100] vs 57% [45-73], p<0.001) than chemotherapy alone. This effect remains positive even in those managed with abbreviated courses (e.g. 3-4 chemo cycles followed by RT). A non-statistically significant trend towards improved OS (p=0.072) and PFS (p=0.10) was seen in non-ALCL pts treated with CMT; yet, pts aged >70 had better OS (83% [58-100] vs 31% [14-67] p=0.027) and PFS (42% [15-100] vs 21% [8-55] p=0.025) with CMT. Conclusion: In this large study evaluating LS PTCL, most pts were middle-aged, fit, and presenting with nodal disease. Our findings suggest no survival benefit of BV-based over other chemotherapy approaches in LS PTCL, contrasting previous studies that included all PTCL stages. However, RT consolidation provided a survival advantage in ALCL and elderly non-ALCL pts. This research provides critical insights into the management and outcomes of this rare subset of aggressive lymphomas, contributing to optimizing therapeutic strategies in LS PTCL. Future prospective studies should validate our findings. Additional data, including response rates in specific PTCL subtypes, will be presented at the meeting.

Introduction: Our PETAL consortium group recently characterized the largest global cohort of relapsed/refractory TNKL patients demonstrating statistically significant survival differences between patients in high-income and developing countries like LATAM (Koh MJ, et al. 2023). Estimating outcomes for newly diagnosed (ND) TNKL patients in developing countries can be challenging due to the lack of comprehensive lymphoma registries, limited clinical and molecular data, and substantial missing information. This missing data often does not occur randomly and may contain crucial insights regarding patient characteristics and prognoses. However, traditional analyses often exclude these patients, which ultimately affects the validity of the conclusions. Therefore, developing cutting edge machine learning (ML) tools to estimate survival with high precision is warranted. Methods: We conducted a retrospective cohort study of 487 patients with ND TNKL from 9 LATAM countries (Argentina, Chile, Colombia, Guatemala, Mexico, Paraguay, Uruguay, and Venezuela). Eligible patients were aged ≥18 years and diagnosed between 2000-2022. Overall survival (OS) was calculated from diagnosis to death or loss to follow-up using the Cox proportional-hazards method, based on two covariates: histologic subtype and IPI or PIT score at diagnosis. The analysis was performed using both complete and imputed data. To adjust for unobserved confounders arising from missing data in retrospective datasets, OS was estimated using these covariates with a novel causal inference method called survival synthetic intervention (SSI). SSI utilizes available data to estimate survival for a specific group of patients by first converting the data to panel data with the Kaplan-Meier estimator. In the panel data setting, SSI leverages principal component regressions to estimate outcomes using observations from (a) all patient groups for a reference intervention and (b) observations under an intervention of interest for a “representative” subset of patient groups. Specifically, SSI learns from the high-dimensional survival information of the group of patients with missing data to better characterize the OS of the entire dataset. We evaluated the empirical performance of SSI and compared it to the Cox method. The data were randomly split into a training set (80%) to build the model and a testing set (20%) to test performance. Results: We included 475 patients with a median age of 52.8 years (range, 15-95), of whom 283 (60%) were male. ATLL was the predominant histology, observed in 183 (39%) patients, followed by PTCL-NOS in 157 (33%) and ENKTCL in 75 (16%). Using histologic TNKL subtype and IPI or PIT scores at diagnosis as covariates, we found that SSI outperformed multivariate Cox models in both the training and testing sets. For Model I (incorporating histologic subtype and IPI score), the testing concordance index (CI) for predicting OS was 0.595 for the Cox model trained with patients who had complete data only, 0.612 for the Cox model trained with imputed data, and 0.651 for SSI. For Model II (incorporating histologic subtype and PIT score), the testing CI for predicting OS was 0.556 for the Cox trained with patients who had complete data only, 0.612 for the Cox model trained with imputed data, and 0.635 for SSI. Conclusions: Low survival rates for TNKL in LATAM highlight the urgent need for improved prognostication tools and access to novel therapies. We applied SSI, a novel machine learning method, to correct biases in a retrospective real-world dataset from LATAM, achieving accurate outcome estimates. Excluding patients from analyses due to missing data leads to biased outcomes. SSI learns from patients with missing information, enhancing overall outcome predictive accuracy.

Background. As we move towards the adoption of sequencing-based assays for classification, prognosis, and treatment prediction in multiple myeloma it is essential to establish high quality reference sets for usage by the community. The sensitivity and specificity of detecting copy number changes and translocations monitored today by FISH can vary based on the technology and analysis platforms used. To create a reproducible set of defined breakpoint junctions for assay validation and technology assessment requirements we have defined to the best of our ability the breakpoint sequences of derivative chromosomes involved in classic and non-classic translocations in the MMRF CoMMpass study and 68 cell lines. Classic translocations are defined as immunoglobulin translocations commonly tested for in clinical FISH panels that bring an immunoglobulin (IgH, IgK, or IgL) enhancer in proximity with a known target genes leading to the overexpression of the gene. The non-classic translocations were separated into two groups: non-classic enhancer, bringing a target genes into proximity with a non-immunoglobulin enhancer; or non-classic target, events where an immunoglobulin enhancer is dysregulating a non-classic myeloma oncogene. Methods. The MMRF CoMMpass whole genomes and RNAseq data was generated and analyzed at TGen. PCR-free whole genome sequencing libraries were created for the entire cell line panel and sequenced to >40x on a NovaSeq X Plus instrument. A subset of 8 cell lines were independently sequenced to >200x for sensitivity testing of two-fold dilutions of tumor content from 100% down to 0.78%. Translocations were called using four independent methods; Manta (default), TGen-Manta (IgH mapping optimized), Pairoscope, and GaMMiT. Additionally, the 8 cell lines used in the sensitivity efforts and those with difficult to define breakpoint locations where sequenced to >20x with long-read sequencing on a PacBio Revio or Oxford Nanopore PromethION. Results. To define a final comprehensive translocation call set in the CoMMpass cohort we integrated the results between all callers, manually reviewed any single tool calls and compared calls with matched RNA expression data to confirm overexpression. This process identified immunoglobulin (IgH, IgK, and IgL) translocations dysregulating NSD2 (13.5%), CCND3 (2.1%), MYC (14.5%), MAFA (1.0%), CCND1 (21.1%), CCND2 (1.4%), MAF (4.9%), and MAFB (1.3%) in this large patient cohort. Unexpectedly, the classic high risk translocations t(4;14) and t(14;16) did not significantly decrease time to second line therapy or overall survival in this cohort. To determine how frequently an expected FISH positive translocation call would be missed by sequencing we compared and reviewed our calls on the cell lines with published results. This identified 9 uncalled derivative chromosomes out of 91 (9.9%) expected events. In all cases there was an unexpected piece of DNA ranging from 0.2-338kb inserted between the enhancer and target gene containing chromosomes. These complex events were limited to events targeting MYC. In CoMMpass similar events were observed during manual curation but in all cases a second derivative chromosome existed and was detected. When comparing translocation detection sensitivity, we found event detection varied depending on the copy number state, number of derivatives, and breakpoint sequence context. At 100% tumor content all events were called at 50x coverage, however, at 50% tumor content two events required >100x coverage to be detectable and at 25% tumor one event required over 150x to be detected. Finally, by integrating the available breakpoints and target gene overexpression we have defined high confidence regions from which an event detected in a DNA sample is expected to cause target gene dysregulation. Conclusion. The adoption of sequencing-based detection of translocations in the clinical work-up of multiple myeloma patients will expand the number of detected events over current FISH assays. However, in some rare cases with a single derivative the junction structure can prevent direct detection of a reportable event due to the insertion of unexpected DNA sequences. Most other detection limitations can be overcome with optimized analysis tools, increased insert or sequencing read lengths, and sequencing coverage.

1037 Background: AURKA is a key regulator of the mitotic spindle, G2/M transition and epithelial-mesenchymal transition. AURKA is amplified and/or overexpressed in breast cancer and is associated with therapy resistance and worse survival. A randomized phase II trial in hormone receptor (HR)-positive, HER2-negative and triple negative (TN) MBC pts showed that addition of A to weekly P significantly improved progression-free survival (PFS) compared with P alone (O’Shaughnessy J et al. JAMA N etwork Open, 2021). Pts’ primary or metastatic disease tissues were analyzed for biomarkers associated with clinical benefit from A. Methods: Retrospective analysis of tumor whole exome and whole transcriptome sequencing of formalin-fixed paraffin embedded pre-treatment tissues from 96 pts (77 HR+, 19 TN) was performed. Clinical benefit from A+P or P was defined as having PFS of at least 6 mos and lack of benefit as PFS less than 6 mos. Enrichment for cancer gene mutations was assessed using Fisher's exact test. Transcriptome data were evaluated for differential expression with DeSeq2 and gene set enrichment analysis (GSEA) and compared to cancer hallmark gene sets. Molecular features were compared between A+P responders and non-responders, P responders and non-responders, and A+P and P responders. P-values <0.05 were considered significant. Results: PIK3CA, TP53, and CDH1 were altered in 41%, 39%, and 13% of tumors, respectively, and these and other known cancer genes were not associated with A+P benefit. Alterations in AKT1, PIK3CA, ERBB2, CDH1, TP53, and KMT2C/ KMT2D, as well as amplifications of CCND1, MYB, and MDM2, did not preclude benefit from A+P. RNA expression of AURKA, or its upstream regulator, FOXM1, was not significantly different based on treatment group or benefit from A+P or P. Increased C-MYC RNA expression (p=0.008), and enrichment for MYC targets and UPR by GSEA were enriched in pts with lack of benefit from P compared to those with benefit from P (p<0.05), but not in pts with lack of benefit from A+P. Wnt/Beta-catenin signaling was enriched in pts with lack of benefit from P compared to those with benefit from P (p=0.03), and with lack of benefit from A+P compared to those with benefit from A+P (p=0.086). Benefit from A+P was associated with enrichment for MYC targets and UPR compared with benefit from P (p=0.024), suggesting activity of A+P, but not P, with high MYC activation and UPR. Conclusions: A added to P improved PFS in HR+ HER2- and TN MBC pts compared with P alone. Pts whose breast cancers had increased C-MYC expression, high MYC activation and increased UPR on RNA expression derived greater clinical benefit from A+P than from P alone. Clinical trial information: NCT02187991 .

e18738 Background: Advocacy programs in oncology has shown to improve outcomes through education, patient empowerment and civil society awareness, enabling better actions and efforts for patient care, especially in COVID-19 era. We present the results of a virtual survey conducted with the support of a Peruvian advocacy program for cancer patients in COVID-19 pandemic. Methods: In October 2020, a Peruvian Advocacy Program at Instituto Nacional de Enfermedades Neoplasicas (INEN) (“Club de la Mama”) formed by advocates and supported by a team of health professionals developed a virtual survey between October 2020 and July 2021 with the purpose of evaluating the impact of interruption of diagnosis or oncological treatment, detect a degree of anxiety/depression due to quarantine and provide information on the response of the oncological institution to the pandemic; from the patient’s perspective. This virtual survey includes: alternative treatment options, degree of anxiety/depression, early access to medical prescription, mode of appointment and quality of care. Patients answered the survey anonymously via web promoted on social networks (Facebook/ WhatsApp) and person-to-person diffusion. Results: A total of 244 patients were included, 78.7% females. Most of patients (86.5%) were from Lima and 86.9% had public insurance. 16.8% of patients had COVID-19 in the period of time evaluated. According to education level, 50.8% were superior, 37.7% high school, 10.7% elementary school and 0.8% illiterate. The most common cancer type was breast (36.1%), followed by hematologic (27.9%) and cervical (7.0%). The most common modality of care was in-person (73%), followed by telemedicine (15%) and only 2% not received medical consult. Regarding the degree of depression: 39% were mild, 14% moderate, 12% moderate-severe, 5% severe and 30% none. The most common degree of anxiety was 42% mild, 16% moderate, 12% severe, and 30% no degree. The most frequent reasons for not attending to hospital were unavailability of appointments (31%), followed by fear to COVID-19 (26%), geographic (10%) and difficulty in transportation (9.0%). The appointment cancellation/postponement time [reported in days] was: 18.0% not canceled, 16% [ < 15], 14% [15-30], 19% [30-60], 10% [60-90], 23% [ > 90]. Of the patients with surgical indication, 72% did not undergo surgery. Regarding prescription, 83% of patients received oncologic treatment, 10% did not received, and the remaining 7% answered that they did not need medication. Quality of care: 18% were considered excellent, 67% good, 10% fair, and 5% poor. Conclusions: Although the survey showed higher degrees of anxiety/depression in patients and longer cancellation/postponement/medication changes during COVID-19 pandemic, there was a significant adherence of medicine prescription, with no negative impact on the perception of quality of oncology care.

Few studies have evaluated the temporal variation of treatment patterns and survival of Latin American females with triple-negative breast cancer (TNBC). Despite increased uptake of neoadjuvant therapy in stage III females, we found a stage-specific worsening survival. Outcomes for other cancer stages remained unchanged since 20 0 0. Tailored regional clinical guidelines should be developed in the neoadjuvant setting.Background: Previous studies have reported a higher prevalence of triple-negative breast cancer (TNBC) in US Hispanic/Latina populations. However, survival outcomes and treatment approaches over time in Latin American females are scarcely reported. We aimed to evaluate the temporal variation in treatment patterns and overall survival (OS) outcomes of females with TNBC according to cancer stage. Materials and Methods: We performed a single-center retrospective cohort study on 1840 females from 2000 to 2014. Patients were classified in 3 calendar periods (20002004, 2005-2009, and 2010-2014). The Kaplan-Meier method and multivariable regression analyses were employed. Results: Stage III cancer was identified in half of the population. Five-year OS estimates for cancer stages I, II, and IV remained unchanged across all calendar periods. However, we found worsening 5-year OS estimates in stage III females (49% in 2000-2004 and 31% in 2010-2014; P < .001). Despite increased uptake of overall use of neoadjuvant therapy in stage III females, the time from diagnosis to treatment initiation ( P = .013) and time to complete the planned cycles ( P < .001) increased over time. Fifty-sex percent of stage IV patients were untreated. Females aged >= 70 years were less likely to receive treatment. Conclusions: Survival estimates were lower than those reported in high-income countries. Most females were diagnosed with advanced disease, and the OS for stage III females worsened over time. Our outcomes show difficulties in delivering timely neoadjuvant therapy in an overwhelmed healthcare system. Public health authorities should improve screening practices, develop regional clinical guidelines, and expand trial enrollment.

Consensus Matrix of the GSVA Enrichment Scores Computed for 50 Hallmark Gene Sets. xCell Immune Score Values by GSVA Cluster.

INTRODUCTION: Peripheral T-cell lymphomas (PTCL) represent a rare and heterogenous group of mature T-cell lymphomas often characterized by aggressive behavior. Previous studies evaluating the distribution of PTCL subtypes across Latin America (LATAM) were limited in their representation of most countries in the region. Additionally, a lack of standardized management for several subtypes and the absence of comprehensive lymphoma registries in LATAM suggests exploring real-world treatment patterns and clinical outcomes. We conducted an international pooled analysis to assess the distribution of PTCL across LATAM countries and report treatment outcomes. METHODS: We compiled data from patients aged ≥18 years with newly diagnosed PTCL from the retrospective registry of the Grupo de Estudio Latinoamericano de Linfoproliferativos (GELL, n=988, 1975-2023), the Brazilian T-cell Project (Brazilian TCP, 2015-2017, 168 cases retrospective; 2017-2023, 425 cases prospective) and the prospective registry of the International T cell Project (ITCP, n=529, 2006-2023). Data were abstracted from medical records in a standardized form. Survival data was only available from the GELL and Brazilian TCP. Overall survival (OS) was estimated from diagnosis to death from any cause, while progression-free survival (PFS) was defined from diagnosis to relapse, progression, or death from any cause. We used the Kaplan-Meier method and Log-rank test to estimate and compare survival probabilities. RESULTS: We enrolled 2110 patients from 11 LATAM countries. Overall, the median age at diagnosis was 54 years (range 18-95 years), most were male (59%), present with advanced stage disease (Ann Arbor III-IV, 67%), and had good performance status (ECOG ≤1, 71%) (Table 1). After PTCL not otherwise specified (NOS, 39%), adult T-cell leukemia/lymphoma (ATL, 18%) and extranodal NK/T cell lymphoma (ENKTL, 16%) were the most frequently diagnosed PTCL subtypes with varying distribution across LATAM countries. Peru had a higher prevalence of ATL (39%) and ENKTL was frequently diagnosed in Central America (43%). In contrast, ALK-negative (ALK-) anaplastic large T-cell lymphoma (ALCL) was the second more frequent subtype in Brazil (18%), Chile (16%), and Argentina (9%). The percentage of mature T-cell NOS was 27% in Argentina and 28% in Chile. A total of 1620 received chemotherapy. First-line chemotherapy varied across subtypes. Patients with ENKTL were frequently treated with asparaginase/platinum-based therapy (62%), while CHOP was more commonly used for ATL or PTCL NOS (46% for both). Chemotherapy with CHOEP/EPOCH was frequent for patients with ALK- ALCL (45%), ALK+ (ALCL 47%), or AITL (48%). With a median follow-up of 33 months, the 3-year OS and PFS for the overall cohort were 40% and 30%, respectively. ALK+ ALCL had superior survival estimates, with a 3-year OS of 77% and a 3-year PFS of 73%. The 3-year OS for patients with ENKTL was 48% and the PFS was 45%. Patients with ATL experienced the lowest survival rates (OS and PFS of 23% and 16% at 3 years, respectively). The use of asparaginase/platinum or CHOP-based therapy was associated with superior 3-year OS (61% and 52%, respectively; p=0.011) and PFS (57% and 49%, respectively; p=0.017) among patients with ENKTL. For ATL, the use of CHOEP/EPOCH was associated with improved 3-year OS (21%, p=0.009) and PFS (15%, p=0.024), but outcomes remained dismal. CONCLUSION: To our knowledge, we report the largest pooled cohort of PTCL subtypes across LATAM by leveraging the retrospective registry of the GELL consortium and data from the Brazilian TCP and ITCP. Our findings suggest a distinct distribution of PTCL subtypes across LATAM countries, with a higher prevalence of ATL and ENKTL compared to the epidemiological patterns in Western countries. This distribution underscores a unique opportunity to increase trial enrollment and accrual of rarer PTCL subtypes. The relatively high percentage of mature T-cell NOS suggests difficulties in providing specific lymphoma diagnoses in the region. The low survival rates for some subtypes indicate the need to develop novel therapies to improve patient outcomes. A larger prospective assessment of PTCL epidemiology and treatment outcomes is being planned to expand the ascertainment of cases, improve pathological classification of the different PTCL subtypes, and validate our results in the LATAM region.

Additional Longitudinal Analysis of Relapsed and Refractory Childhood Solid Tumors.

of EMD cells to date combining the whole exome sequencing (WES), bulk RNA sequencing (RNA-seq) and single cell RNA sequencing (scRNA-seq) data from the largest cohort of EMD samples ever sequenced (N=15) collected between 2017-2023.Methods: WES was performed for 15 EMD samples and 8 paired NDMM samples using Twist Comprehensive Human Exome.RNAseq was performed for 15 EMD, 8 paired NDMM, 7 unpaired NDMM and 15 unrelated RRMM samples using SMARTer Stranded Total RNA-Seq Kit v2.scRNAseq was performed for 5 EMD samples using Cedhromium 3' library kit.All sequencing was performed on Illumina platform.Only FACS/MACS sorted aberrant PCs were used in case bulk DNA/RNA sequencing.Chromosomal aberrations were evaluated using the combination of FISH and WES.Results: We show that combination of genomic lesions 1q21 gain/amplification and at least one mutated gene in MAPK signaling are the most frequent mutations in EMD, present in 14/15 (93%) and 13/15 (87%) EMD samples, respectively.Analyses of bulk RNAseq data showed up-regulation of pathways connected to cell proliferation and upregulation of potent growth factor IL6. Interestingly, CXCR4 gene, important for a homing of plasma cells in bone marrow, was significantly downregulated as well as production of immunoglobulins, with IGL being more prevalent than IGH.We focused on expression of molecular targets for modern immunotherapy, which could be effective in EMD.However, we observed decrease in expression of CD38, GPRC5D, SLAMF7 in EMD cells and lower expression of HLA molecules, which are important for action of immunotherapeutics.Intriguingly, our data suggested novel EMD targets with elevated expression of genes EZH2 and CD70, that are already in use in non-myeloma oncology.In addition, we identified CD8+ T and NK cells as the most abundant immune subsets in EMD microenvironment.Conclusions: We performed the largest genomic study of EMD tumors to date and revealed that combination of 1q21 gain/amp and mutations in MAPK pathway are presumably defining genomic features of EMD.Next, transcriptomic profiling of EMD cells suggested high proliferative potential, decreased BM homing, decreased IG production, autocrine growth regulation and decreased expression of some key therapeutically relevant molecules.Finally, for the first time, we dissected the microenvironment of EMD tumors.Overall, our findings represent a significant contribution to understanding of biology and resistance of EMD.

Introduction:Recent reports have highlighted a number of genetic resistance events in patients who have progressed on BCMA and GPRC5D targeted immunotherapies.We have leveraged the multi-dimensional data in the MMRF CoMMpass study to define the incidence of target allele loss in patients.Moreover, the usage of multiple advanced mutation callers identified recurrent internal tandem duplications (ITD) in TNFRSF17/BCMA reminiscent of the FLT3 ITD seen in AML.Methods: Whole genome, exome, and RNA sequencing results from IA22 of the MMRF CoMMpass study were processed through an updated pipeline with alignment to the GRCh38 reference genome.Small variant calling was performed with 5 independent variant callers; Mutect2, Strelka2, Octopus, VarDict, and Lancet.Copy number was determined using GATK CNV with an optimized diploid centering model, structural events were identified using Manta and gene expression estimates were determined using Salmon.Results: Integration of the available gene expression, copy number and mutational data facilitated the comprehensive evaluation of the current antibody, bispecific or CAR-T target antigens.At diagnosis all patients expressed the target genes and none had bi-allelic loss of a target loci, however, single copy loss was detected for FCRL5 (0.8%), CD38 (5.6%), GPRC5D (13.2%), and BCMA (3.4%).Four hyperdiploid patients had very high expression of BCMA, which were associated with high level amplifications (5, 12, or 30 copies) or an IgH translocation.High confidence mutation calls (>=3 callers) identified eight patients with SNV or INDEL mutations in BCMA.Two patients had inactivating nonsense or frameshift mutations while the remaining five had large 37-58 nucleotide ITD.Since large insertions are one of the most difficult events to identify we curated the single variant caller results and identified three additional patients with ITD of 46-73 nucleotides.Interestingly, all of these patients have a hyperdiploid karyotype and the ITD occur within the transmembrane domain with a minimal duplication of amino acids 61-66.The ITD are predicted to be gain-of-function events, as each patient was characterized by a high NF-kB gene expression index, exceeding the 90th percentile observed in the cohort in all cases.Conclusions: As we begin to prioritize the use of different immune-based therapies it will be important to consider the relative risk each patient has to develop resistance based on the preexisting loss of one target gene copy, as some targets are frequently lost (GPRC5D) while others are rarely lost (FCRL5).Some hyperdiploid patients have hyperactivation of NF-kB signaling as a result of BCMA overexpression by high level copy number gains and immunoglobulin translocations or ITD.These patients are likely dependent on BCMA mediated signaling and would likely be exquisitely sensitive to BCMA targeted therapies.It will be essential to determine if current BCMA directed therapies can still bind these BCMA ITD isoforms.