Additional Cytogenetic Abnormalities Research Articles

Extragonadal germ cell tumors: A clinicopathologic study with emphasis on molecular features, clinical outcomes and associated secondary malignancies

Extragonadal germ cell tumors (EGCTs) are rare, representing <5% of all germ cell tumors (GCTs). Whilst EGCTs share morphological and immunohistochemical features with their gonadal counterparts, they tend to be more aggressive and are frequently associated with secondary somatic malignancies. The aim of our study was to evaluate the clinical, morphological and immunohistochemical features, and to analyze tumors for chromosomal abnormalities of 12p, in addition to any novel genetic alterations, in a series of EGCTs. Seventy-seven EGCTs were included. Anterior mediastinum was the most common anatomic site, followed by central nervous system, retroperitoneum, sacroccygeal area, and neck. Whole genome SNP array identified isochromosome 12p in 26% of tumors. Additional cytogenetic abnormalities included the presence of gain of chr 21 in 37% of tumors. Somatic-type malignancies were identified in 8% of patients. Disease progression (metastasis and/or recurrence) was documented in 8 patients, most of whom died from their relapse. Three patients who died of disease had somatic-type malignancies. Mediastinal seminomas had a significantly better overall survival when compared to mediastinal non-seminomatous GCTs. Our study demonstrates that EGCTs share similar histologic features, but diverse clinical outcomes compared to their gonadal counterparts. Outcomes vary according to anatomic location and histologic subtypes. Our data corroborate that somatic-type malignancies are frequently encountered in mediastinal EGCTs and that their presence portends a poorer prognosis.

Clinical implications of additional chromosomal abnormalities in adult acute myeloid leukemia with inv (16)/t(16;16)/CBFB::MYH11.

This study assesses the clinical significance of additional cytogenetic abnormalities (ACAs) and/or the deletion of 3'CBFB (3'CBFBdel) resulting in unbalanced CBFB::MYH11 fusion in acute myeloid leukemia (AML) with inv (16)/t(16;16)/CBFB::MYH11. We retrospectively evaluated the clinicopathologic features of 47 adult de novo AML with inv (16)/t(16;16)/CBFB::MYH11 fusion. There were 44 balanced and 3 unbalanced CBFB::MYH11 fusions. Given the low frequency of unbalanced cases, the latter group was combined with 19 published cases (N = 22) for statistic and meta-analysis. Both balanced and unbalanced cases were characterized by frequent ACAs (56.5% and 72.7%, respectively), with +8, +22, and del(7q) as the most frequent abnormalities. The unbalanced group tends to be younger individuals (p = .04) and is associated with a lower remission rate (p = .02), although the median overall survival (OS) was not statistically different (p = .2868). In the balanced group, "ACA" subgroup had higher mortality (p = .013) and shorter OS (p = .011), and patients with relapsed disease had a significantly shorter OS (p = .0011). Cox multivariate regression analysis confirmed that ACAs and history of disease relapse are independent risk factors, irrespective of disease relapse status. In the combined cohort, cases with ACAs had shorter OS than those with "Sole" abnormality (p = .0109). ACAs are independent high-risk factors in adult AML with inv (16)/t(16;16)/CBFB::MYH11 fusion and should be integrated for risk stratification in this disease. Larger studies are needed to assess the clinical significance of the unbalanced CBFB::MYH11 fusion resulting from the 3'CBFBdel.

Erythroid variant evolving from chronic myeloid leukemia resistant to multiple tyrosine kinase inhibitors: a rare case report

BackgroundChronic myeloid leukemia (CML) is characterized by the presence of BCR::ABL1 fusion gene resulting from a reciprocal translocation, t(9;22)(q34;q11.2), leading to prominent granulocytic proliferation. The majority of patients initially present in chronic phase (CP), which may progress to advanced CML with predominantly granulocytic phenotypes in the absence of proper treatment or response to tyrosine kinase inhibitors (TKIs). We present an exceptionally rare case in which an erythroid variant emerged from a CML patient resistant to multiple TKIs. This variant is characterized by the detection of t(9;22) BCR::ABL1 fusion in erythroid precursors at various maturation stages and the absence of granulocytic progenitor hyperplasia typically seen in classical CML.Case presentationA 33-year-old female with CP-CML had received multiple TKI therapies since her initial diagnosis in 2015. Due to intolerable side effects and inconsistent adherence, she exhibited an inadequate response and developed new-onset pancytopenia. Bone marrow (BM) biopsy specimen revealed a hypercellular marrow with significant erythroid hyperplasia (90% of marrow cellularity) and a reversed myeloid-to-erythroid (M: E) ratio of 1:10. Both erythroid and myeloid cells displayed progressive maturation without dysplasia or excess blasts. Chromosomal analysis identified t(9;22) (q34;q11.2) in 19 out of 20 metaphase cells. BCR::ABL1 fusion transcript (p210 isoform) was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Notably, no additional pathogenic cytogenetic abnormalities or ABL1 kinase domain mutations were detected. Here, we report the first published case of an erythroid variant emerging in a CML patient resistant to multiple TKIs—a distinct entity from the erythroid blast crisis evolving from CML.ConclusionThe erythroid variant of CML is distinguished by the presence of t(9;22) (q34;q11.2) BCR::ABL1 in predominant erythroid precursors at different stages of maturation. In a myeloid neoplasm showing predominant erythroid hyperplasia without typical CML features, it is vital to correlate morphology and t(9;22) BCR::ABL1 cytogenetic testing for accurate diagnosis, and to prevent confusion with PEL transformation in CML.

Long-Term Follow up for SWOG 1318: Combination of Dasatinib, Prednisone, and Blinatumomab for Older Patients with Philadelphia-Chromosome (Ph) Positive Acute Lymphoblastic Leukemia (ALL)

Tyrosine kinase inhibitors (TKIs) have improved the outcomes of patients with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Many older patients (pts) are not good candidates for intensive chemotherapy and are treated with TKIs plus corticosteroids or low-intensity chemotherapy. Although the remission rates with this approach have been high, the median disease-free survival (DFS) has been short. Therefore, novel treatment strategies are needed. This trial evaluated the feasibility of combining the TKI dasatinib with prednisone and blinatumomab in older pts with Ph+ ALL. Here we present updated results (median follow-up, 4.3 years). Methods: This trial was activated through the NCTN in January 2015 and closed to accrual in April 2021. Pt eligibility included: age ≥ 65 years; Ph+ or Ph-like ALL (with dasatinib-sensitive fusions or mutations); newly diagnosed or relapsed/ refractory; no evidence of central nervous system (CNS) disease; and adequate organ function. Treatment: For induction, pts received dasatinib 140 mg/d orally (PO) Days 1-56 along with prednisone 60 mg/m 2/d PO Days 1-24. Pts achieving complete remission (CR) or CR with incomplete count recovery (CRi) (Day 28 or Day 56) continued dasatinib until Day 84, followed by 3 cycles of post-remission therapy (PRT) with blinatumomab/ dasatinib. Pts not achieving CR or CRi by Day 56 received re-induction with blinatumomab. The response rate was assessed on Day 35 of blinatumomab. Pts not achieving CR/ CRi could receive a second cycle of blinatumomab. This was followed by 3 cycles of PRT with blinatumomab/ dasatinib. Maintenance therapy consisted of prednisone 60 mg/m 2/d x 5 days every 28 days for a total of 18 cycles, along with dasatinib 140 mg po qd indefinitely. CNS prophylaxis included intrathecal (IT) methotrexate every 4-6 weeks x 8 doses. IT methotrexate was given at least 2 days apart from blinatumomab. Response was assessed at Days 28, 56, and 84, and additional time points were dependent on response. Minimal residual disease (MRD) was assessed centrally by multi-color flow cytometry on Day 28. Statistics: A total of 24 eligible patients were accrued. The primary objective was the feasibility of the combination therapy. The combination was to be deemed feasible if the dose-limiting toxicity (DLT) was &amp;lt; 33%. DLT included: &amp;gt; Grade 3 non-hematologic toxicities (excluding nausea, vomiting, diarrhea) and Grade 4 neutropenia lasting &amp;gt; 42 days. The primary results were reported in 2022 with a median of 2.7 years of follow-up. Herein, we report long-term outcomes for these patients. Results: The median age was 73 years (range 65-87). All pts had newly diagnosed Ph+ ALL; 79% of Ph+ pts had additional cytogenetic abnormalities. During induction, 2 pts experienced treatment-related non-hematologic Grade 4 toxicities. No Grade 4 or higher treatment-related non-hematologic toxicities occurred during post-remission therapy or maintenance. The toxicity of the combination was deemed acceptable. Twenty-two of 24 pts (92%) achieved a CR during dasatinib and prednisone induction therapy. Four did not receive PRT (2 due to adverse events, 1 to receive a transplant, and 1 because of insurance issues). Sixteen pts who achieved CR had MRD data. Six of 16 pts (38%) were MRD undetectable by multi-color flow cytometry on Day 28. Most sites also monitored patients' blood or bone marrow with real-time quantitative PCR to quantify the BCR-ABL1 transcript for molecular response. Of 19 patients analyzed, 17 (89%) were in a major or complete molecular remission at some time point after treatment, with at least 12 of these patients achieving complete molecular remission. Four patients remain on maintenance. The median follow- up for pts who are alive is 4.3 years (range 2.6-6.5 years). The median overall survival (OS) is 6.5 years (95% CI 3-NA) and the median DFS has not been reached as of June 29, 2023. Kaplan-Meier 3-year estimates of OS and DFS are 75% (95% CI 52%-88%) and 72% (95% CI 49%-87%), respectively (Figure). Of the 8 relapses, 4 patients were CD19+ and 3 had T315I mutations. Conclusions: Median DFS and OS with dasatinib and blinatumomab in this older group of Ph+ ALL patients remain excellent despite only 1 patient proceeding to an allogeneic transplant. Further correlative studies (single cell transcriptomics and DNA methylation sequencing) are being planned to evaluate predictors of relapse and will be presented at the meeting.

Prospective Assessment of Co-Morbidities and Framingham Risk Score in Newly Diagnosed Chronic Myeloid Leukemia (CML) Patients and Its Impact on Clinical Outcomes Following Frontline TKI Therapy: Toronto CML Genomic Alliance in Greater Toronto Area & Ontario (TCGA-GTA)

Introduction Front-line (1L) tyrosine kinase inhibitor (TKI) drug selection is a pearl of clinical practice in CML. It requires consideration of multiple aspects including the goal of therapy, potency of the drug as well as comorbidity of the patient (pt). Contemporary CML practice emphasizes the importance of comorbidity for TKI selection. However, such data is scarce particularly the prevalence of comorbidity and its impact on 1L drug selection. We are developing a shared care model with community hematologists in Greater Toronto Area (TCGA-GTA). Newly diagnosed CML pts in the community will be referred to the study core center for prospective enrolment into the CML registry, informed consenting for genetic testing/future research, and evaluation of baseline comorbidity. Pt will return to referring hematologists for management of CML, including 1L TKI treatment as per standard of care, and is scheduled to visit the study center at 6 and 12 months, then annually for 10 years. It gives us a unique opportunity to evaluate the impact of comorbidity on 1L TKI drug selection. Patients and method Adult pts with newly diagnosed CML of any phase within 2 months of diagnosis are eligible for the study unless they are not able to visit study core center. Comorbidities and Framingham risk score (FRS) were assessed at the time of initial evaluation. Mutation profile testing is done by a bar-coded error-corrected sequencing platform for 40 genes developed at the Ontario Institute for Cancer Research. Cumulative incidence of molecular response with 2 log (MR2), 3 log (MR3), 4 log reduction or deeper (MR4), and treatment failure (TF) were calculated using cumulative incidence method considering competing risk. Event-free survival (EFS) was defined as time from start of TKI until TKI stop/switch, TF or death. Failure-free survival (FFS) was defined as time from start of TKI to TF [primary resistance, loss of complete cytogenetic response, new additional cytogenetic abnormalities (ACAs), and progression to accelerated or blasts phase (AP/BP)] or death. Results From November 2020 until June 31, 2023 (data cutoff), a total of 77 pts were enrolled in the study. The median follow-up time was 12 months. The frequency of 1L TKI selection was as follows: IMATINIB (IM) (n=23, 30%), DASATINIB (n=25, 32%), NILOTINIB (n=22, 29%), BOSUTINIB (n=1, 1%), ASCIMINIB (n=6, 8%). Majority of the pts were male (n=51, 66%). Ninety-three percent of pts were in chronic phase at diagnosis, while 4% and 3% were in AP and BP, respectively. Presence of ACAs was detected in 18 pts (23%), 10 of which were high risk, while 6% (n=5) were not evaluable. The baseline clinical and disease characteristics of pts on 1L IM vs other TKIs (2G-TKI) are summarised in Table 1. Of note, pts on IM were significantly older with higher FRS and comorbidities, including coronary artery disease, diabetes mellitus, and hypertension. The OS of all pts at 1 year was 98.5% (95% confidence interval 90%-99.8%). At 1 year, pts with intermediate to high risk FRS had lower FFS (73.2% vs 86.5%, p=0.037) while history of hyperlipidemia was associated with lower EFS (59.3% vs 81.5%, p=0.023) and 6 times more likely to stop or switch therapy due to intolerance or resistance (HR 5.9, p=0.002). Baseline comorbidities did not demonstrate signifincant impact on MR2 and MR3 at 1 year but pts with low FRS were more likely to achieve MR4 within 1 year (22.3% vs 0%, p=0.023). Age did not affect any of the clinical and molecular endpoints. Compared to 2G-TKI, IM had worse EFS (HR 3.93, p=0.003), FFS (HR 5.66, p=0.011), and MR2 (47.3% vs 84.4%, p=0.003) at 1 year. Further, IM pts experienced higher rate of any side effects compared to those on 2G-TKI (69.6% vs 31.5%, p=0.003). Conclusion The TCGA-GTA study provides valuable insights into the impact of comorbidity on selection of 1L TKI and its outcome in a real-world setting. Pts who are older and with higher FRS and comorbidities tend to receive 1L IM. However, pts on 1L 2G TKIs are more likely to reach treatment goals faster with lower probability of TF. The present study did not reach a clear conclusion whether the worse outcomes in the pts receiving IM are from IM treatment or concurrent risk factors such as age and comorbidities, thus further study with an expanded cohort and longer follow-up is required. Elderly pts or those with comorbidities would need alternative treatment with better efficacy and tolerability, such as Asciminib, that can overcome comorbidity issues in the future.

A Predictive Scoring System for Therapy Failure of Tyrosine-Kinase Inhibitors in Patients with Chronic-Phase Chronic Myeloid Leukemia

Background In the latest version of WHO criteria (5th edition, 2022), accelerated-phase chronic myeloid leukemia (CML-AP) was re-defined as “high-risk” chronic-phase chronic myeloid leukemia (CML-CP). However, there is no robust predictive scoring system for therapy failure of tyrosine-kinase inhibitors (TKIs) in these newly-defined CML-CP patients. Objectives To develop and validate a predictive scoring system for TKI-therapy failure in CML-CP patients. Methods Data from 2,038 consecutive CML-CP patients according to the WHO 2022 criteria receiving initially TKI-therapy at one centre were interrogated as the training dataset to develop a predictive scoring system which was subsequently-validated in 4,640 patients from 76 other centres (validation dataset). TKI-therapy failure was defined by the 2020 European LeukemiaNet recommendation. Results In the training dataset, 1,591 patients (78%) initially received imatinib; 326 (16%), nilotinib; 85 (4%), dasatinib; and 36 (2%), flumatinib (a novel 2G-TKI made in China). Median TKI-therapy duration was 58 months (IQR, 33-90 months). 515 patients (27%) experienced therapy failure at a median of 9 months (IQR 3-12 months) on TKI-therapy. Multi-variable analysis indicated that older age, male sex, lower hemoglobin concentration, higher proportion of blood blasts and basophils, larger spleen size below the costal margin and high-risk additional cytogenetic abnormalities (ACAs) in Ph + cells were significantly-associated with higher cumulative incidence of therapy failure. Based on the optimal Fine-Gray regression model, a formula to calculate the therapy-failure risk score was established: “ Initial TKI-therapy failure risk score = 1.9321 × (Age/100) + 0.2950 × Sex (male = 1, female = 0) - 0.8589 × (HGB/100) 2 + 0.0979 × Blood blasts + 0.023× Blood basophils + 0.0521 × Spleen size below the costal margin + 0.5075 × high-risk ACAs (existing = 1, no = 0)” which divided patients into low- (score ≤ 0.0545; n = 908; 47%), intermediate- (0.0545 &amp;lt; score &amp;lt; 1.1662; n = 791; 41%) and high-risk (score ≥ 1.1662; n = 216; 12%) cohorts with 7-year cumulative incidences of therapy failure of 8% (95%CI, 4, 12%), 39% (35, 43%) and 80% (76, 84%; p &amp;lt; 0.001; Figure 1A). Sub-distribution hazard ratios (sHRs) for therapy failure (low-risk cohort as reference) were 3.2 (2.6, 4.1, p &amp;lt; 0.001) and 9.5 (7.4, 12.2; p &amp;lt; 0.001) for the intermediate- and high-risk cohorts. In the validation dataset, 4,640 patients were classified into the low- (n = 2,168; 47%), intermediate- (n = 2,093; 45%) and high-risk (n = 379; 8%) cohorts using the scoring system. 7-year cumulative incidences of therapy-failure were 10% (7, 13%), 36% (33, 39%) and 70% (66, 74%; p &amp;lt; 0.001; Figure 1B). sHRs (low-risk cohort as reference) were 3.4 (3.0, 3.9; p &amp;lt; 0.001) and 8.8 (7.5, 10.3; p &amp;lt; 0.001) for the intermediate- and high-risk cohorts. Time-dependent AUROC values for therapy failure using the predictive scoring system were 0.84-0.92 and 0.79-0.86 in the training and validation datasets. Moreover, patients identified as intermediate- or high-risk cohorts by the predictive scoring system receiving initial 2G-TKI-therapy had significantly-lower therapy failure rate than those receiving imatinib-therapy ( p-values = 0.002 and 0.017) both in the training and validation datasets. Conclusions We developed and validated a robust predictive scoring system for TKI-therapy failure in newly-defined CML-CP patients according to the WHO criteria (5th edition, 2022), which might help physicians decide appropriate therapy strategy.

Achievement of Undetectable BCR::ABL1 (uBCR::ABL1) Is Predictive of Improved Survival in Philadelphia Chromosome Positive (Ph+ve) Acute Lymphoblastic Leukemia (ALL) Patients Not Receiving Allogeneic Stem Cell Transplantation

Background With the advent of tyrosine kinase inhibitor (TKI)-based therapy the outcome of Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukemia (ALL) has significantly improved. Moreover, patients (pts) who achieved undetectable (u) BCR::ABL1 at 3 months (mo) from the time of initiating therapy, tend to have improved outcomes (Short et al. Blood 2016; 128 [4];504-507). We conducted a retrospective multicenter study to analyze the predictors of achieving uBCR::ABL1 in adult (&amp;gt; 18 years) Ph + ALL pts. Methods A total of 458 adult Ph + ALL pts from 10 US academic institutions, who were diagnosed between May 2003 and April 2023, were evaluated to assess the rates of uBCR::ABL1 at 3 mo after initiating therapy and factors that predict the achievement of molecular response. Continuous variables were summarized as median (range), while categorical variables were reported as frequency (percentage). Unadjusted comparisons of patient/ treatment related characteristics and molecular response were made using a non-parametric test (continuous variables) or Fisher's exact test (categorical variables). Survival curves were estimated using the Kaplan-Meier method and compared between molecular responses via the log-rank test. Results Baseline characteristics The median age of the pts was 53 years (range [R] 19-85), median white blood cell count (WBC) was 17.6 (10 9/L) (R, 1.2-571), 249 (55%) pts had additional cytogenetic (CG) abnormalities, 98 (22%) pts had p210 fusion protein, and 35 (8%) pts had CNS disease at diagnosis. A higher proportion of pts received TKI in combination with intensive chemotherapy (IC) (69%), than TKI + steroids (15%), TKI + low intensity chemotherapy (LC; combination of vincristine, steroids +/- rituximab) (9%), TKI + blinatumomab (3%) and IC without TKI (4%) during induction. During induction, the highest proportion of pts received 2 nd generation TKI combinations (67%; majority dasatinib 98%), compared to imatinib (19%) or ponatinib (10%): 4% of pts did not receive TKI with induction. The complete remission (CR) rate was 94%; measurable residual disease (MRD) negative by flow cytometry 51% (181/352 evaluable pts) and 58.5% (209/354 evaluable pts) of pts had uBCR::ABL1 by PCR at 3 mo from induction. A total of 212 (46%) pts received allogeneic stem cell transplantation (allo-HCT) in CR1, among them 61% of pts received TKI maintenance post allo-HCT. Predictors of uBCR::ABL1 at 3 months Predictors for uBCR::ABL1 at 3 mo are summarized in Table 1. The proportion of pts with WBC &amp;gt; 100 (45% vs 60%, p= 0.04), p210 fusion protein (35% vs 85%, p= &amp;lt;0.001), additional CG abnormality with monosomy 7 (40% vs 61%, p= 0.03), additional somatic myeloid co-mutation (39% vs 61%, p= 0.09; 94 pts evaluable), received induction with steroid plus TKI (41% vs 63%, p= 0.002) or received imatinib based induction (47% vs 61%,p= 0.07) had inferior uBCR::ABL1 rates at 3 mo. Conversely pts who received ponatinib based induction had significantly better uBCR::ABL1 rates (79% vs 57%, p= 0.009). Survival The median relapse free survival (RFS) was 72.3 mo (95% CI; 50.1-94.4), RFS was numerically higher among pts who achieved uBCR::ABL1 at 3 mo but not statistically significant (84.2 mo vs 62.9 mo, p= 0.20). The median overall survival (OS) was 129.2 mo (95% CI; 81.2-177.2), OS was not significantly different among pts who achieved uBCR::ABL1 at 3 mo (129.2) versus no uBCR::ABL1 (not reached; 50% alive at 94 mo), p= 0.47. In sub-set analysis, among non-transplanted pts, RFS was not statistically significant between those who achieved uBCR::ABL1 vs those who did not (38.9 vs 22.0 mo, respectively p= 0.24). However, the median OS was significantly higher in pts who achieved uBCR::ABL1 at 3 mo compared to pts who did not achieve uBCR::ABL1 at 3 mo: 149.3 vs 77.5 mo, p= 0.04 ( Figure 1). Conclusion In this large multicenter study, we observed significantly inferior uBCR::ABL1 rates among pts with WBC &amp;gt; 100, p210 fusion protein, monosomy 7 CG abnormality, pts receiving induction with steroid plus TKI or imatinib based induction. Conversely, uBCR::ABL1 rates were significantly better in pts receiving ponatinib based induction therapy. Overall, we did not see significant impact of uBCR::ABL1 at 3 mo, on RFS or OS. It is likely subsequent therapies and allo-HCT abrogates the negative impact of not achieving uBCR::ABL1 at 3 mo. However, among non-transplanted pts achievement of uBCR::ABL1 at 3 mo showed a significantly improved OS.

LAMP5 Is an AML-Restricted Immunotherapeutic Target Enriched in High-Risk Acute Myeloid Leukemia

LAMP5, Lysosomal Associated Membrane Protein Family Member 5, a neuronal protein that plays a role in synaptic plasticity is normally found primarily in the brain, however limited studies have identified it to be expressed in leukemia, particularly KMT2A-rearranged leukemias. Importantly, there is low expression of LAMP5 in normal hematopoiesis making it an ideal potential immunotherapeutic target for both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). As a newly identified aberrant protein in leukemias, there is little data available on the prevalence or clinical implications of LAMP5 expression. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric AML patients to define the prevalence, co-occurring cytomolecular aberrations and clinical outcomes associated with LAMP5 expression. Ribo-depleted RNA seq data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years from BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients, LAMP5 was expressed (positive defined as mRNA transcripts per million (TPM) &amp;gt;2) in 396 patients (28%) with mRNA TPM ranging from 0-884 with similar prevalence found in adult AML (28%; BEAT AML N=126/452, SWOG N= 59/199 TCGA N=50/179), but with significantly higher TPM ranging from 0-3,200. Of the 396 pediatric patients with LAMP5 expression, there was a notable enrichment in KMT2A-r AML (N=203, 51%), which accounts for 59% of the total KMT2A-r cohort. Within the KMT2A-r cohort, there is enrichment in the MLLT1 (N=17/18, 94%), MLLT3 (N=61/93, 66%) and MLLT10 (N=58/69, 84%) fusion groups. Additionally, LAMP5 expression was seen in other high risk fusion subtypes including 100% of patients with KAT6A-CREBBP (N=12/12) and 29% of NUP98 fusions (N=45/154). There is absent LAMP5 expression in normal lymphoid and myeloid cells, but there is moderate expression seen in plasmacytoid dendritic cells, which could provide a therapeutic target for Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN). Given the association with high-risk AML fusion subtypes, the impact of LAMP5 on clinical outcome in AML pediatric patients was evaluated. High rates of relapse were seen in patients with LAMP5 expression, with a relapse rate of 45% compared to 36% in LAMP5-negative patients. 5 year event-free survival (EFS) was 36% for LAMP5-positive patients compared 45% for LAMP5-negative patients (p=0.0005). Similar poor outcomes were seen with overall survival 54% for LAMP5-positive patients vs. 62% for LAMP5-negative patients (p=0.001). In an analysis of a large cohort of AML patients, we found that LAMP5 expression is highly enriched in many high-risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and NUP98 fusions. Patients with LAMP5 expression have high rates of relapse with significantly worse outcomes compared to patients without LAMP5. Given low levels of LAMP5 in normal hematopoiesis, LAMP5 is an ideal AML-restricted immunotherapy target with broad applicability for many high-risk fusion subtypes of AML. Current efforts to generate a LAMP5 chimeric antigen receptor (CAR) T cell are underway which will be used to evaluate preclinical efficacy in AML via cell- and patient-derived xenograft models with the goal to translate this novel therapy into clinical use to improve the current dismal outcomes for these high-risk patients.

CLEC2A Is a Novel AML-Restricted Immunotherapeutic Target Enriched in KMT2A-Rearranged Acute Myeloid Leukemia

KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment related AML in adults leading to a highly refractory and deadly subtype of AML regardless of age. Targeted therapies for KMT2A-r AML have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML is in its infancy in part due to significant overlap of antigens expressed in AML and normal hematopoietic cells, which would limit efficacy and increase hematopoietic toxicity. We interrogated the AML transcriptome compared to normal hematopoiesis to identify optimal targets that are silent in normal hematopoieisis and are highly expressed in AML. Here we describe a novel immunotherapeutic target, CLEC2A, a member of the C-type lectin family, that is highly enriched in high-risk AML, particularly KMT2A-r AML and absent in normal hematopoeisis. We evaluated the association between KMT2A fusions and CLEC2A expression identifying CLEC2A as a putative oncoprotein and the potential for CLEC2A to serve as an immunotherapeutic target in high-risk AML via antibody-mediated cytotoxicity. Diagnostic specimens from 1,864 patients with AML (Pediatric 0-29 years from COG Phase III trials N=1486; Adult 18-88 years SWOG N=199 and TCGA-LAML N=179) were interrogated using contemporary NGS to identify genetic aberrations. Conventional karyotyping and mutational analysis were used to determine additional cytogenetic and molecular abnormalities. As CLEC2A was entirely absent in normal bone marrow (TPM 0-0.15), positive CLEC2A expression was defined as mRNA transcripts per million (TPM) &amp;gt;1. Although, CLEC2A was detected in only 18% of AML patients (252/1486; 17% pediatric and 90/378; 24% adult patients), it was highly enriched in those with KMT2A-r AML with 77% (N=193) of CLEC2A positive patients having KMT2A-r, accounting for 48% of the total KMT2A-r cohort, Fig 1A. CLEC2A expression was enriched in specific fusion subgroups: MLLT10 (80%), MLLT4 (71%), and MLLT3 (48%), Fig 1B. CLEC2A expression was low to absent in a majority of other patients, though was seen in other high-risk groups including non-KMT2A MLLT10 fusions, NUP98 fusions, CBFA2T3-GLIS2 fusions, and monosomy 7. CLEC2A is also expressed in a subtype of T-ALL (HOXA activated), which is a fusion driven T-ALL. We further evaluated a potential causal link between KMT2A fusions and CLEC2A expression and inquired whether KMT2A fusion proteins might directly bind to the CLEC2A promoter and induce CLEC2A expression. We used AutoCUT&amp;RUN profiling of genome-wide occupancy of KMT2A oncoproteins to evaluate direct binding of the KMT2A fusion protein to the CLEC2A promoter in primary AML patient samples with three different KMT2A fusions (MLLT10, MLLT4 and MLLT3). We found direct binding of KMT2A fusion proteins to the CLEC2A promoter region, indicating a causal relationship between KMT2A fusions and CLEC2A expression. We then evaluated the potential of therapeutic targeting of CLEC2A by an antibody-drug conjugate using a HumZap cytotoxicy assay. CLEC2A antibody conjugated to saporin (a toxin with cytolytic activity when internalized) was incubated with CLEC2A+ and CLEC2A- cells with target-specific cytotoxicity evaluated by colorimetric assay. CLEC2A+ OCI-AML2 cells had significantly higher rate of cytotoxicity compared to CLEC2A- control cells at 10nM Ab-toxin conjugate. Clinical outcomes for patients with and without CLEC2A expression was evaluated. Patients with CLEC2A had significantly worse outcomes (EFS 23% vs. 45.5%, p&amp;lt;0.0001 and OS 38% vs. 63%, p&amp;lt;0.0001 for patients with and without CLEC2A expression, respectively). Given high rates of co-occurring KMT2A-r AML, particularly high-risk fusions (MLLT10 and MLLT4), we evaluated clinical outcomes within the KMT2A-r cohort as these patients have poor EFS and OS. KMT2A-r patients with CLEC2A had significantly worse EFS (24% vs. 39%, p=0.0063) and OS (38% vs. 60%, p=0.0002). Within the KMT2A-r cohort, high rates of relapse were seen for patients with CLEC2A expression compared to those without CLEC2A (72% vs. 55%, p=0.0039). Here we describe CLEC2A, a novel AML-restricted cell surface target that is an ideal immunotherapeutic target. CLEC2A is highly expressed in KMT2A-r AML, entirely absent in normal hematopoietic cells, directly and causally linked to the KMT2A fusion, and can be used for target-directed cytotoxicity.

Outcomes of Patients with Multiple Myeloma and 1q Gain/Amplification Receiving Autologous Hematopoietic Stem Cell Transplant: The MD Anderson Cancer Center Experience

Background: There is contradictory data regarding the prognostic impact of additional copy numbers of chromosome 1q (1q+) on the outcomes of newly diagnosed multiple myeloma (NDMM) patients, and there is scarce data in the context of autologous stem cell transplantation (autoSCT). In this report, we studied outcomes of NDMM patients with 1q+ who received induction with contemporary anti-myeloma agents, followed by autoSCT and post-transplant maintenance therapy. Methods: We conducted a retrospective single-center chart review analysis of adult NDMM patients with 1q21 gain or amplification (3 or ≥4 copies of 1q, respectively; 1q+) detected by fluorescence in situ hybridization (FISH) that received autoSCT between 2008-2018. Progression-free survival (PFS) and overall survival (OS) were the primary endpoints. Results: 213 NDMM patients with 1q+ were included in the analysis, with a median age of 62.5 years and 53% were male. Overall, 169 (79%) patients had 1q gain, while 44 (21%) patients had 1q amplification. The most commonly used induction and conditioning regimens were bortezomib, lenalidomide, and dexamethasone (VRD) (41%) and melphalan (77%), respectively (Table 1). At day 100 after autoSCT and at best post-transplant response, 78% and 87% of patients achieved ≥VGPR, 34% and 56% achieved CR, 38% and 50% achieved MRD negative ≥VGPR, respectively. The median PFS and OS for the entire cohort were 35.5 months and 81.4 months, respectively. 1q amplification was associated with inferior PFS compared to 1q gain (HR=2.03, 95% CI 1.36-3.03, p&amp;lt;0.001; Figure 1). On multivariable assessment (MVA) for PFS, MRD negative &amp;gt;VGPR before autoSCT and at day 100 post-transplant [(HR=0.56, 95% CI 0.36-0.86, p=0.009) and (HR=0.64, 95% CI 0.44-0.94, p=0.022), respectively] were associated with better PFS, whereas 1q amplification was associated with inferior PFS (HR=1.94, 95% CI 1.29-2.92, p=0.001). On MVA for OS, R-ISS stage III (HR=4.08, 95% CI 1.07-15.50, p=0.039) was associated with inferior OS, whereas achieving MRD negative &amp;gt;VGPR at best post-transplant response was associated with superior OS (HR=0.45, 95% CI 0.24-0.85, p=0.014). Notably, the percentage of cells with 1q+ was not associated with PFS nor with OS, both when evaluated as a continuous variable and when evaluated as a categorical variable using thresholds of either 30% or 50%. The presence of additional high-risk cytogenetic abnormalities did not adversely affect survival outcomes. Conclusions: Patients with NDMM and 1q+, especially 1q amplification, have poor survival outcomes, despite the use of contemporary induction regimens, upfront autoSCT and post-transplant maintenance. These patients may benefit from novel treatment modalities, such as CAR-T and bispecific antibodies, earlier in their disease course.

Real-World Validation of the European Leukemia Network 2022 Risk Stratification in Acute Myelogenous Leukemia

Background: The 2017 European LeukemiaNet (ELN 2017) guidelines have become foundational for the risk stratification, prognostication, and treatment of acute myelogenous leukemia (AML). The updated 2022 European LeukemiaNet (ELN 2022) guidelines include additional cytogenetic abnormalities and genetic mutations for risk group assignment. It remains unknown how these updated guidelines compare for risk stratification and prognosis. Methods: We studied (N=287) adults with newly diagnosed AML from MedStar Georgetown University, MedStar Washington Hospital Center, and Baylor University Medical Center from 2012 to June 2023. Patients were stratified as favorable (fav), intermediate (int), or adverse (adv) risk according to ELN 2017 and ELN 2022 criteria. Overall survival (OS) was compared by age, race, and sex. Statistical analysis performed with SAS. Results: Median age was 65+16 years with a predominance of males (N=168, 59%). African-Americans (AA) composed 15% (N=44) of the cohort. Myelodysplastic-related mutations were present in 33 patients (15%), whereas the prevalence of epigenetic mutations was 25% (N=55). By ELN 2017 criteria, 19 (7%) patients were characterized as fav, 64 (23%) as int, and 198 (70%) as adv risk. Using ELN 2022 criteria, 18 (6%) patients were categorized as fav, 52 (19%) as int, and 211 (75%) as adv risk. Kaplan-Meier (KM) curves revealed a median overall survival of 200 days (d), 400 d, and 1532 d in the adv, int, and fav risk groups, respectively (log-rank p&amp;lt;0.001) by ELN 2017 criteria. Median OS was 379 d, 527 d, and 1552 d for the adv, int, and fav groups, respectively (log-rank p&amp;lt;0.001) in the ELN 2022 cohort. Stratifying by age &amp;lt;60 years, median OS was 390 d in the adv group and 832 d in the int group (log-rank p=.021)by ELN 2017 criteria,as compared to 350 d in the adv group and 757 d in the int group (log-rank p=.0012) by ELN 2022 criteria.Among those &amp;gt;60 years of age, median OS was 244 d and 349 d in the adv and int groups, respectively by ELN 2017 criteria (Anova p&amp;lt;0.25). In the ELN 2022 cohort, median OS among those &amp;gt;60 years of age was 284 d for the adv group and 209 days in the int group (Anova p&amp;lt;0.62). Among females, median OS in the adv group was 224 d and 342 d in the int group by ELN 2017 criteria (Anova p=0.4). In the ELN 2022 cohort, median OS among females was 214 d in the adv group and 421 d in the int group (Anova p=.13). Median OS among male patients in the ELN 2017 cohort was 324 d for those of adv risk and 623 d for those of int risk (Anova p&amp;lt;0.004). In the ELN 2022 cohort, median OS in males was 352 d and 563 d in the adv and int groups, respectively (Anova p&amp;lt;0.07) . In the ELN 2017 cohort, median OS was 259 d in AA patients as compared to 719 d in non-AA patients; this was not statistically significant (Anova p=0.16). Comparatively, in the ELN 2022 cohort, median OS for the AA patients was 153 d as compared to 212 d for the non-AA patient population (Anova p=0.86) .Conclusions: In this real-world dataset, the updated ELN 2022 guidelines reproduce differential survival for proposed favorable, intermediate, and adverse risk categories. When considering time to endpoint survival [Kaplan Meier curves] in our study, ELN 2022 seems to lack the ability to clearly discriminate among the adverse and intermediate risk subgroups. It is possible that patients seen in academic institutions not participating in FLT3 directed therapy trials retained inferior survival whereas similar patients fared better through early enrollment in trials using molecular targeted therapy. Finally, neither ELN 2017 nor ELN 2022 discriminate survival in AA populations for either adverse or intermediate subgroups. Efforts should be directed to develop novel risk stratification schemas to better encapsulate this vulnerable population.

Encouraging Outcome of AML with t(8;16) Following Allogeneic Stem Cell Transplantation in First Remission - a Retrospective Analysis on Behalf of the Acute Leukemia Working Party of the EBMT

In the 2022 European Leukemia Net (ELN) risk classification, acute myeloid leukemia (AML) with translocation t(8;16) was newly classified as AML defined by specific cytogenetic abnormalities, associated with unfavorable prognosis. Translocation (8;16)(p11;p13) is a rare abnormality in AML with female preponderance and occurs more frequently in secondary AML (sAML), especially therapy-related, than in de novo disease. Clinically, AML with t(8;16) is associated with high risk of bleeding, occurrence of extramedullary manifestations, myelomonocytic differentiation, and signs of hemophagocytosis in the bone marrow. The translocation leads to the fusion of acetyltransferase KAT6A (also known as MOZ or MYST3) and CREB binding protein ( CREBBP) genes, both of them involved in hematopoiesis. ELN risk stratification of AML with t(8;16) was based on studies that included relatively low numbers of patients receiving allogeneic stem cell transplantation (alloSCT). In the largest report by Kayser et al. ( Br J Haematol 2021) 21 patients (15 transplanted in first complete remission [CR1], median follow-up 5.5 years) were analyzed. A 5-year overall survival (OS) of 38% was achieved after alloSCT compared to 11% in patients who received chemotherapy only. The aim of our study was to evaluate the outcome of patients with AML harboring t(8;16) after alloSCT in a larger cohort from the EBMT registry. All types of donor, conditioning regimen and graft were included. Sixty patients were identified, of whom 44 had been transplanted in CR1. Median follow-up from alloSCT was 72.3 months, median age was 46.3 years, and 73.3% were female. Karnofsky performance status was &amp;gt;80% in 59 patients, one had missing data. At 2 years after alloSCT, OS and leukemia free survival (LFS) of the entire cohort were 42.7% and 42.2%, respectively, (28.6% and 14.3% in patients transplanted in advanced disease). Cumulative incidence of relapse (RI) and non-relapse mortality (NRM) at two years was 36.9% and 15.3%, respectively, in patients transplanted in CR1, and 78.6% and 7.1%, respectively, in patients transplanted in advanced disease. The 44 patients transplanted in CR1 were analyzed in detail. Basic characteristics were comparable to the entire cohort as described above. Twenty-two of the patients had de novo, 22 had sAML. According to the ELN 2017 classification (not yet including t(8;16) as a factor for poor prognosis), 54.5% had an intermediate and 45.5% an adverse cytogenetic risk. Furthermore, 38.8% had a complex karyotype. Donors were matched related in 34%, matched unrelated in 54%, haploidentical in 5% and cord blood in 7%. Conditioning was myeloablative in 52.3%, whereas 47.7% had received reduced intensity conditioning. Among patients transplanted in CR1, 2- and 5-year OS from alloSCT was 48% and 39.9%, the respective LFS was 46.8% and 35.5%. On multivariate analysis (MVA), harboring t(8;16) within a complex karyotype was the major risk factor for outcome, being associated with higher RI (hazard ratio [HR] 3.38, p=0.01), lower LFS (HR 4.17, p=0.016), and lower OS (HR 3.08, p=0.017). In contrast, patients with t(8;16) outside of a complex karyotype achieved excellent results (Figure 1). Besides a complex karyotype, MVA revealed sAML as an additional, independent risk factor with a higher RI (HR 3.73, p=0.026). Age was the major factor for NRM, reaching 5% vs. 26.5% in patients below or above the median (p=0.02). In conclusion, according to the largest series analyzed in this setting so far, overall results after alloSCT in AML with t(8;16) transplanted in CR1 were favorable, especially in patients without multiple additional cytogenetic abnormalities, achieving a low risk of relapse and a 5-year OS &amp;gt;60%. Hence, alloSCT in CR1 appears to abrogate the unfavorable prognostic value of this translocation. Whether or not t(8;16) does have any additional influence on the outcome of patients with a complex karyotype remains to be evaluated. Figure Legend Figure 1: Outcome of AML with t(8;16) with and without complex karyotype

TP53 Variant Allele Frequency and Therapy-Related Setting Independently Predict Survival in Myelodysplastic Syndromes with Del(5q)

Background: According to the international consensus classification (ICC), diagnosis of myelodysplastic syndromes (MDS) with del(5q) requires presence of i) del5q, alone or associated with no more than one other cytogenetic abnormality, exclusive of -7/del(7q), ii) &amp;lt;5% bone marrow (BM) and &amp;lt;2% circulating blasts, and iii) the absence of “multi-hit” TP53 ( Arber et al. Blood 2022;140: 1200). We have recently reported on TP53 variant allele frequency (VAF) ≥22% being the sole genetic predictor of inferior survival in MDS-del(5q) (Fleti. et al. AJH 2023;98:E76). In the current study, we examined the additional prognostic impact of therapy-related setting, in the context of TP53 VAF and other risk factors. Methods : The current study included patients with MDS-del(5q) who were seen at the Mayo Clinic or Moffitt Cancer Center, USA. Diagnosis of MDS-del(5q) and therapy-related qualification was according to ICC criteria ( Arber et al. Blood 2022;140: 1200), with the exception that patients with biallelic or multi-hit TP53 were included to allow unbiased analysis of impact on prognosis. Patient selection was based on availability of mutation information from next-generation sequencing (NGS). The latter was assessed either at diagnosis or at different time points during the disease course, but at least 3 months before leukemic transformation. Otherwise, all analyses, including estimation of overall and leukemia-free survival, were based on variables collected at time of initial diagnosis of MDS-del(5q). TP53 mutation VAF threshold of 22% was considered based on our previously published report cited above. Age cutoff at 67 years was determined by ROC analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: A total of 210 patients (median age 72 years; 65% females), meeting the above-stipulated criteria for MDS-del(5q), were considered: 172 primary and 38 therapy-related. Table 1 outlines clinical and laboratory characteristics obtained at the time of diagnosis: 42% were transfusion-dependent, 69% had hemoglobin level &amp;lt;10 g/dL, 30% platelet count &amp;lt;150 x 10(9)/L, and 15% carried additional cytogenetic abnormality. NGS data performed either at diagnosis or at least 3 months prior to leukemic transformation was available in 146 patients; the most frequent mutations were TP53 (21%), SF3B1 (16%), DNMT3A (12%), TET2 (12%), ASXL1 (10%), and JAK2 (7%) (Table 1). At a median follow-up of 3.9 years (range 0.002-18.5) for living patients, 95 (45%) deaths and 34 (16%) leukemic transformations were recorded; treatment undertaken included lenalidomide in 162 (77%) cases. NGS data-exclusive multivariable analysis that included all 210 patients identified therapy-related setting (p&amp;lt;0.01; HR 2.9; Figure 1a), age &amp;gt;67 years (p=0.01; HR 1.8), transfusion-dependency (p=0.04; HR 1.5), and platelet count &amp;lt;150 x 10(9)/L (p=0.03; HR 1.6), as independent predictors of inferior survival. The significant effect of age and platelet count was sustained when analysis was limited to patients with primary MDS-del(5q) but lost when applied to therapy-related disease. NGS data-inclusive multivariable analysis, which was applicable to the subset of 146 patients, identified, as risk factors, therapy-related setting (p&amp;lt;0.01; HR 2.3) and TP53 VAF ≥22% (p&amp;lt;0.01; HR 2.4; Figure 1b) while age &amp;gt;67 years sustained borderline significance (p=0.07; HR 1.8). Figure 1c depicts survival data based on the presence or absence of TP53 VAF ≥22% and therapy-related qualification; low-risk with no risk factors (N=112; median survival 11.7 years; 5/10-year survival 73%/52%) and high-risk with one or both risk factors (N=34; median survival 4 years; 5/10-year survival 42%/14%); leukemia-free survival was affected by TP53 VAF ≥22% (p&amp;lt;0.01) but not therapy-related setting (p=0.13). Conclusions : The current study highlights TP53 VAF and therapy-related setting as the most prominent risk factors for survival in MDS-del(5q) and proposes a simple risk model that distinguishes patients with a projected 10-year survival that exceeds 50%, in the absence of both risk factors, and plummets to 14%, in the presence of one of the two risk factors. Accordingly, allogeneic stem cell transplant might be deferred in the former instance while it should be pursued sooner than later, in the latter instance.

The Co-Occurrence of bZIP in-Frame CEBPA-Mutation with Unfavorable Genetic Abnormalities Is Associated with a Poor Prognosis in Acute Myeloid Leukemia

In a previous study (SY Ahn et al, Cancer Res Treat 2023), we reported that the presence of only the basic leucine zipper in-frame mutation (bZIP in-f) CEBPA is associated with favorable outcomes in patients with CEBPAdouble mutations. However, when bZIP in-fCEBPAmutations were accompanied by certain mutations in chromatic/DNA modifiers, cohesion complex, and splicing genes, patients exhibited inferior overall survival (OS). FLT3-ITD or additional cytogenetic abnormalities is known to rarely co-occur with CEBPA mutation in patients with AML. In cases where both bZIP in-fCEBPA and FLT3-ITD coexist, there are conflicting aspects in risk stratification, and research on its prognostic significance is limited. Therefore, we aimed to evaluate the prognostic significances of the co-occurrence of bZIP in-fCEBPAmutation with unfavorable genetic abnormalities in acute myeloid leukemia. In 832 patients who were diagnosed with AML excluding acute promyelocytic leukemia and underwent intensive induction therapy, 98 (11.8%) had bZIP in-fCEBPA. Among 98 patients with bZIP in-fCEBPA, eight cases had FLT3-ITD (5 with low allelic ratio, 3 with high allelic ratio), 19 patients had unfavorable genetic mutations according to the 2022 European LeukemiaNet guidelines, and 10 patients had non-favorable cytogenetic abnormalities as detected in diagnostic samples. We observed no clinical differences in features such as age, sex, WBC count, bone marrow blast percentage at diagnosis based on the co-occurrence unfavorable genetic abnormalities (unfavorable-gene). Among the 98 patients with bZIP in-f CEBPA, 30 out of 37 patients with unfavorable-gene achieved complete remission (CR) after induction therapy, and 55 out of 61 without unfavorable genetic abnormalities showed CR (p=0.20). Allogeneic hematopoietic cell transplantation was proceeded at first CR in 21 out of 37 unfavorable-gene group, and 35 out of 61 bZIP in-fCEBPAonly group ( p=1.00). In the survival analysis, patients with FLT3-ITD, unfavorable genetic co-mutated, or non-favorable cytogenetic abnormalities with bZIP in-fCEBPA showed inferior OS (p=0.036) (Fig. A). Due to the limited number of patients for analysis, we classified them into two group: the unfavourable-gene group and bZIP in-fCEBPAonly group. We observed that the unfavourable-gene group had a poor OS compared to the bZIP in-fCEBPAonly group (HR 2.71, 95 CI; 1.32-5.55, p=0.007) (Fig.B). On multivariate analysis, unfavorable-gene group was only poorrisk factor for OS. Despite the inclusion of a relatively small number of patients, our study demonstrates that when co-occurrence of bZIP in-fCEBPA withunfavourable genetic abnormalities, it results in a poor prognosis. To confirm its clinical significance, further validation with a larger patient population is necessary.

Characterization of MDS and CMML Patients Harboring Trisomy 8 Abnormality: Clinical, Autoimmune, and Mutational Features

Introduction Trisomy 8, seen in 5-7% of MDS and CMML patients, is regarded as an intermediate-risk cytogenetic abnormality in IPSS-R but not an MDS-defining cytogenetic abnormality. Recent studies have demonstrated an association between systemic autoimmune disease and trisomy 8, attributed to CD8 positive T-cell expansion and WT1 over-expression resulting in inflammation and suppression of hematopoiesis. Further, MDS and CMML with trisomy 8 respond favorably to immunosuppressive therapy. In this study, we investigated the clinical (including autoimmune manifestations), molecular and outcome characteristics of MDS/CMML with trisomy 8 in comparison to MDS with normal karyotype. Methods We retrieved all patients with trisomy 8 by conventional cytogenetic studies from our cytogenetic files between 2015 and 2023 who underwent bone marrow examination and met the WHO/ICC criteria for MDS/CMML diagnosis. In some cases, FISH testing was performed for confirmation. Majority of patients underwent amplicon-based targeted next-generation sequencing with a panel of 81 genes associated with myeloid malignancies (2500X coverage; LOD: 2% VAF). Clinicopathologic features were collected from medical records. For comparison, we retrieved a control cohort of all MDS patients diagnosed within the same period with normal karyotype (MDS-NK) at our institution. Results From a total of 362 patients with trisomy 8 (215 treatment naïve) detected at any time point during the disease course, we identified 165 MDS/CMML patients with trisomy 8 at baseline. These included 57 (35%) with isolated trisomy 8, 21 (13%) with 1 additional and 78 (53%) with ≥2 additional cytogenetic abnormalities (meeting the criteria for complex karyotype). Recurrent additional cytogenetic abnormalities included del(13)(q11q22) (n=3) and trisomy 21, del(5q), del(11q) involving KMT2A, and loss of Y, all seen in 2 patients each. We focused our subsequent analysis on 57 treatment naïve MDS/CMML patients with isolated trisomy 8 and compared them to 162 treatment naïve MDS/CMML patients with diploid karyotype (MDS-NK) at baseline. The median age was 74 years (range, 30-90) and 34 (60%) were men, with baseline characteristics shown in Table 1. There were no significant differences in age, gender distribution, median hemoglobin, ANC, MCV or platelet counts when comparing the two populations. Trisomy 8 MDS/CMML had a higher median baseline BM blast percentage [5% (1-18%) vs. 3% (0-17%); p=0.034] compared to MDS-NK. Distribution of IPSS-R subgroups in trisomy 8 MDS/CMML revealed a significantly lower representation of “Very Low” [0% vs. 13%; p=0.0027] and “High” [18% vs. 49%; p=0.0001], and higher representation of “Very High” [16% vs. 3%; p =0.0004] categories. NGS data was available in 36 (63%) of treatment naïve patients with isolated trisomy 8. The most frequent mutation was seen in ASXL1 (n=20, 56%), followed by TET2 (n=12, 33%), RUNX1 (n=12, 33%), SRSF2 (n=8, 25), KRAS/NRAS (n=8, 22%), and EZH2 (n=7, 19%). Compared to MDS-NK, the following mutations were significantly enriched in trisomy 8: ASXL1, EZH2, STAG2, RUNX1, KRAS/NRAS, IDH2 and PTPN11. Over a median follow-up time of 19 months, 28 (49%) with trisomy 8 and 19 (12%) MDS-NK patients died. MDS/CMML with trisomy 8 had a significantly lower median overall survival (OS) compared to MDS-NK [23.7% vs. not reached; p-value: &amp;lt;0.0001]. A total of 27 (7.6%) MDS/CMML patients with trisomy 8 (21 with trisomy 8 at baseline) were found to have a bona fide autoimmune disease, including autoimmune uveitis/scleritis (n=3), (rheumatoid and psoriatic), polyarteritis nodosa, giant cell arteritis with polymyalgia rheumatica, autoimmune sclerosing pancreatitis, immune hemolytic anemia, synovitis, and Grave's disease. Conclusions MDS/CMML with trisomy 8 showed unique clinicopathologic characteristics compared to MDS-NK: higher baseline BM blasts, higher representation of “Very High” IPSS-R risk categories with characteristic mutational signatures and a variety of underlying autoimmune diseases.

33. Optical Genome Mapping identifies additional cytogenetic abnormalities in patients with hematologic malignancies

33. Optical Genome Mapping identifies additional cytogenetic abnormalities in patients with hematologic malignancies

A rare FBXO25–SEPT14 fusion in a patient with chronic myeloid leukemia treatment to tyrosine kinase inhibitors: a case report

Abstract Objectives Exploring the pathogenesis of resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia. Case presentation This case report describes a rare fusion of FBXO25 and SEPT14 genes in a 58-year-old male patient with chronic myeloid leukemia. The patient had been treated with tyrosine kinase inhibitors for one year. After 6 months of imatinib treatment, the patient's symptoms improved significantly and the complete blood count returned to normal, but the optimal ratio of BCR::ABL transcripts to ABL transcripts is greater than 10 % indicating treatment failure. Then we switched to a second generation TKIs to continue treatment, During the Flumatinib treatment period, the patient developed severe bone marrow suppression and exhibited additional cytogenetic abnormalities involving chromosome aberration: 47, XY,+8[5]/47, idem, inv(Y)(p11.2q11.23)[15]. By adjusting the drug dose and elevating blood cells, the patient’s BCR::ABL P210/ABL was 2.56 % after six months of Flumatinib treatment. The patient’s BCR::ABL P210/ABL consistently remained above 1 % throughout the treatment, and additional cytogenetic abnormalities were present. Next-generation sequencing revealed the recombination of exon 4 of the FBXO25 and exon 10 of the SEPT14, and this mutation has not been previously reported. Conclusions Our findings suggest that the FBXO25-SEPT14 fusion may be associated with tyrosine kinase inhibitors resistance in chronic myeloid leukemia.

Additional cytogenetic abnormalities in patients with newly diagnosed acute promyelocytic leukemia predict inferior event-free survival.

The innovative combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has established a new chapter of curative approach in acute promyelocytic leukemia (APL). The disease characteristics and prognostic influence of additional cytogenetic abnormalities (ACA) in APL with modern therapeutic strategy need to be elucidated. In the present study, we retrospectively investigated disease features and prognostic power of ACA in 171 APL patients treated with ATRA-ATO-containing regimens. Patients with ACA had markedly decreased hemoglobin levels than that without ACA (p = 0.021). Risk stratification in the ACA group was significantly worse than that in the non-ACA group (p = 0.032). With a median follow-up period of 62.0 months, worse event-free survival (EFS) was demonstrated in patients harboring ACA. Multivariate analysis showed that ACA was an independent adverse factor for EFS (p = 0.033). By further subgroup analysis, in CD34 and CD56 negative APL, patients harboring ACA had inferior EFS (p = 0.017; p = 0.037). To sum up, ACA remains the independent prognostic value for EFS, we should build risk-adapted therapeutic strategies in the long-term management of APL when such abnormalities are detected.

Chronic myeloid leukaemia in blast phase with t(9;11)(p21;q23), KMT2A::MLLT3 gene fusion

Case: A 51-year-old man presented with lymphadenopathy and increasing fatigue. He had been diagnosed with chronic myeloid leukaemia (CML) seven months earlier and had a suboptimal response to first line nilotinib, failing to achieve MR1 with BCR-ABL1/ABL persistently >10% (IS). A repeat bone marrow examination showed 90% blasts, consistent with CML in blast phase (CML-BP). The blasts showed monoblastic morphology and immunophenotype. Conventional karyotype showed both t(9;22)(q34;q11.2), involving the long arm of chromosome 9, and t(9;11)(p21;q23), involving the short arm of the same derivative chromosome 9. FISH confirmed KMT2A breakapart. A core biopsy of cervical lymph node demonstrated a monoblastic leukaemic infiltrate and BCR-ABL1 mutational analysis did not identify any resistance mutations. Discussion: Progression of CML to accelerated or blast phase is usually accompanied by a series of genomic events including BCR-ABL1 resistance mutations, other somatic mutations and additional cytogenetic abnormalities (ACAs). Common ACAs include extra Philadelphia chromosome, trisomy 8, trisomy 9 and isochromosome 17q. KMT2A rearrangement involving the 11q23 locus are common in de novo or therapy related acute leukaemia (both myeloid and lymphoid), but rarely reported in CML blast phase (<1%). The few reported cases show an aggressive clinical presentation, poor outcomes and poor response to tyrosine kinase inhibitors (TKI).

Autologous Stem Cell Transplantation for Patients with Multiple Myeloma with Translocation (4;14): The MD Anderson Cancer Center Experience

Translocation between chromosomes 4 and 14, t(4;14), has been reported in 15% of patients with multiple myeloma (MM) and is considered a high-risk cytogenetic abnormality associated with inferior outcomes. Autologous hematopoietic stem cell transplantation (auto-HCT) is standard of care for patients with high-risk MM, yet there are scarce data on post-transplantation outcomes of patients with t(4;14) MM. The aim of the present study was to evaluate outcomes of MM patients with t(4;14) who underwent auto-HCT and received contemporary anti-myeloma agents for induction and post-transplantation maintenance. We conducted a retrospective analysis of MM patients with t(4;14), detected by fluorescence in situ hybridization (FISH), who underwent auto-HCT between 2008 and 2018 at MD Anderson Cancer Center. Primary endpoints were progression-free survival (PFS) and overall survival (OS), and secondary endpoints were hematologic response and minimal residual disease (MRD) status after auto-HCT. MRD status in the bone marrow biopsy was evaluated using 8-color next-generation flow cytometry with a sensitivity of 1/10-5 cells. Seventy-nine patients were included (52% male), with a median age of 60 years (range, 32 to 78 years). Forty-four patients (56%) had an additional high-risk cytogenetic abnormality. Fifty patients (63%) achieved at least a very good partial response (≥VGPR) prior to auto-HCT and 20 (25%) had MRD-negative ≥VGPR. At the best post-transplantation evaluation, 90% had ≥VGPR and 63% had MRD-negative ≥VGPR. The median follow-up for survivors was 35.7 months (range, 7.7 to 111.6 months). For the entire cohort, median PFS and OS were 22.9 months and 60.4 months, respectively. Patients with MRD-negative ≥VGPR prior to transplantation had improved PFS and OS on both univariate analysis (UVA) and multivariate analysis (MVA) (hazard ratio [HR], .35 [95% confidence interval (CI), .16 to .76; P=.008] and .12 [95% CI, .03 to .44; P=.002], respectively). The presence of additional high-risk cytogenetic abnormalities was not associated with inferior PFS (P=.57) or OS (P=.70). Post-transplantation lenalidomide-based combinations were associated with improved OS in both UVA and MVA (HR, .14; 95% CI, .04 to .45; P=.001), while their impact on PFS was not statistically significant (P=.37). Our results consolidate t(4;14) as a high-risk abnormality associated with poor outcomes despite novel agent induction, auto-HCT, and post-transplantation maintenance. Despite some inherent study design limitations, including a relatively small cohort and heterogeneity in treatment, we observed that deeper pretransplantation response and post-transplantation maintenance with lenalidomide-based combination were associated with improved outcomes. Novel immune and cellular therapies are needed to improve the outcomes in patients with t(4;14).