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Abstract Background Clonal cytopenia of undetermined significance (CCUS) is a risk factor for development of myeloid neoplasms (MN) such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Therapy-related myeloid neoplasm (t-MN) is defined as the development of a MN in the context of prior DNA damaging therapies including chemotherapy, radiation, and immunosuppressive therapies. CCUS diagnosed in the setting of prior DNA damaging therapy (t-CCUS) and its outcomes are not known. The aim of this study was to study clinicopathological features and outcome of t-CCUS and compare it with t-MN. Methods Patients who had received DNA-damaging therapy and subsequently determined to have CCUS or t-MN were identified. The diagnosis of CCUS was made if the patient had non-diagnostic bone marrow biopsy evaluation combined with evidence of pathogenic myeloid somatic genetic alterations using conventional cytogenetics or next generation sequencing (NGS) panel. Pathogenic/likely pathogenic variants (PV) were stratified by DTA (DNMT3A, TET2, and ASXL1) genes vs. others. Event-free survival (EFS) was calculated using interval from t-CCUS to t-MN with death as a competing risk. Overall survival (OS) for t-CCUS and t-MN patients was calculated using interval from t-CCUS or t-MN diagnoses respectively to the last follow up. Statistical analysis was performed using JMP (v14.1, SAS Institute). Results We identified 30 patients with t-CCUS and 259 patients with t-MN. Eleven (37%) t-CCUS patients developed t-MN and were included in both cohorts (Fig. A). Clinical characteristics of t-CCUS patients are shown in Table 1. Briefly, age (median, interquartile range or IQR) at the diagnosis of primary malignancy and t-CCUS was not different between those who developed t-MN and those did not (P=0.09 and 0.378 respectively). There was no difference in hemoglobin, white blood cells, or ANC between the two groups. Patients who developed t-MN had a significantly lower platelet count (68 vs. 130, P<0.01) and higher bone marrow blast percent (2 vs. 0, P=0.04) compared to those who did not. At t-CCUS diagnosis, cytogenetics was abnormal in 11 (37%) patients and none had complex karyotype (CK). Paired cytogenetic analysis at t-CCUS and t-MN was available for 10 patients: 4 had unchanged karyotype at t-MN, whereas 6 had cytogenetic evolution. In contrast, 213 (83%) of 256 t-MN patients with available cytogenetics had abnormalities and 126/256 (49%) had CK. Thus, a significantly higher proportion of patients had CK t-MN compared to t-CCUS (χ 2 26.4, P<0.01). A 42-gene NGS panel identified 36 PV in 21 patients (median 1, range 1-4) at t-CCUS. The most common PV were in TET2 (37.1%), TP53 (11.4%), DNMT3A (8.6%), and ZRSR2 (8.6%) genes. Two patients had paired NGS available at t-CCUS and t-MN: one had BCOR and U2AF1 PV at t-CCUS and acquired an additional NRAS PV at t-MN. The second patient had 2 PV in TP53 at t-CCUS diagnosis, that remained the only PV identified at t-MN. In the t-MN cohort, median number of PV was 2 (range 1-9). The most common PV identified were TP53 (21.5%), TET2 (6.7%), DNMT3A (6.4%), and ASXL1 (6.4%), whereas 4.8% patients had no PV. Three of 22 patients in the t-CCUS cohort had TP53mut, compared to 55 of 157 in the t-MN cohort (χ 2 4, P=0.05). Management strategies for t-CCUS included observation (20), growth factors (4), treatment of primary malignancy (2), immunosuppressives (1), hypomethylating agent followed by allogeneic SCT (1), and others (2). Following t-CCUS, 9 patients progressed to MDS and 2 to AML with a median of 3.5 months (IQR 1.7-43.4). EFS was 43.4 months (IQR 6.8-80.9). The presence of abnormal cytogenetics (Fig. B) and the absence of DTA-mutations (Fig. C) predicted shorter EFS. At last follow up, 12 (40%) deaths were noted. Four (33%) patients died without developing t-MN. Causes of death in the t-CCUS cohort were: t-MN (3), primary malignancy (1), progressive cytopenia without MN (1), infection (1), graft-vs.-host-disease (1), and undetermined (5). t-MN patients had a significantly shorter survival compared to t-CCUS (Fig. D). Conclusion More than 10% of t-MN patients had a precursor t-CCUS. On the other hand, t-CCUS patients are at a high risk of progression to MN. t-MN is characterized by the acquisition of TP53mut, genomic instability, and a significantly shorter survival compared to t-CCUS. The presence of abnormal cytogenetics and the absence of DTA mutations at t-CCUS predict shorter EFS. Figure 1 Figure 1. Disclosures Litzow: Omeros: Other: Advisory Board; AbbVie: Research Funding; Jazz: Other: Advisory Board; Amgen: Research Funding; Astellas: Research Funding; Pluristem: Research Funding; Actinium: Research Funding; Biosight: Other: Data monitoring committee. Patnaik: Kura Oncology: Research Funding; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees. Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution.

Abstract Purpose: Multiple myeloma (MM) is a plasma cell (PC) malignancy with an increasing incidence in the United States. Fluorescence in situ hybridization (FISH) is currently the gold-standard diagnostic assay to detect recurrent genomic abnormalities of prognostic and therapeutic significance in MM. Hyperdiploid MM, a standard risk abnormality, is characterized by gains of odd-numbered chromosomes including chromosome 15. While gains of chromosome 15 have been reported in approximately 53% of standard risk MM patients (Chretien, Blood, 2015), losses of chromosome 15 are rarely reported. Enumeration of chromosome 15 centromere is detected using the alpha satellite FISH probe. We previously discovered ~9% of cases had false-negative chromosome 15 FISH results when compared to Mate Pair Sequencing (MPseq) (Smadbeck, BCJ, 2019). To our knowledge, no previous studies have reported a reduced performance of the alpha satellite D15Z4 FISH probe in PC FISH studies. A false-negative result for D15Z4 could result in an underreporting of trisomy 15 and overreporting of monosomy 15. Here, we investigated the incidence of false-negative D15Z4 FISH results by examining the presence of monosomy 15 from patients with a monoclonal gammopathy. Methods: We identified 1231 samples from patients with a monoclonal gammopathy who had undergone uniform testing to identify MM-specific cytogenetic abnormalities. DNA from bone marrow samples was genotyped on the Precision Medicine Research Array and biogeographical ancestry was assessed using the Geographic Population Structure Origins tool (Elhaik, Nat Commun, 2014; Home DNA 2016). FISH of immunoglobulin (cIg)-stained PCs was performed as described (Baughn, BCJ, 2018). Chromosome 15 centromere probe (Vysis CEP 15 D15Z4, Abbott Molecular, Abbott Park, IL) was used for chromosome 15 enumeration. Samples were divided into two groups: Cases with or without evidence of monosomy 15 by FISH. Mann-Whitney U test was applied to compare difference between groups for data deviating from normal distribution. A Chi-squared test evaluating the differences across these two groups was used. Results: We identified monosomy 15 in 31 cases (2.5% of cohort). Monosomy 15 was observed in nearly 100% of both the PC (cIg+) and non-PC (cIg-) populations, by contrast to the other recurrent PC-specific genomic abnormalities which were restricted to only the PC population. All samples with a banded analysis revealed most metaphases had two normal chromosome 15s demonstrating that the D15Z4 FISH probe failed to correctly enumerate the number of chromosome 15 centromere signals. Of the samples with a monosomy 15 result, the median African ancestry was 76.2% (95% percentiles: 77.5%-89.0%), while the median African ancestry of the non-monosomy 15 cohort was 2.3% (95% percentiles: 0%-87.7%) (). Although not all patients with >50% African ancestry had evidence of monosomy 15, 26/31 (83.9%) of patients with monosomy 15 had >50% African ancestry. Accordingly, patients with African ancestry (similarity ≥ 0.70) had 8.02-fold (95% confidence interval: 3.73-17.25, P = 9.92 X10 -8) increased possibility to have monosomy 15. Of cases with monosomy 15, 22 (71.0%) had an IGH rearrangement with the most prevalent IGH partner being CCND1. The incidence of t(11;14) was greater in the monosomy 15 group (13/31:41.9%), in contrast to the non-monosomy 15 group (330/1200=27.5%) (P = 0.12). Conclusions: We report a polymorphic variant of the chromosome 15 centromere resulting in a false-negative FISH result using the D15Z4 FISH probe. Surprisingly, the false-negative FISH result was not uniformly distributed among patients and was highly enriched in individuals of African ancestry. This may be due to a variation of the chromosome 15 alpha satellite in some individuals that prevents the D15Z4 FISH probe from sufficiently binding (O'Keefe, Genome Research, 2000). Although this study focused on identification of monosomy 15, the incidence of a false-negative result is likely greater than we have reported in this study since we did not evaluate cases that had a true trisomy 15 that was reported as normal chromosome 15 enumeration by FISH. This study highlights a critical need to ensure diagnostic reagents have equal performance for all patients independent of their ancestry. Evaluation of genomic events using unbiased approaches such as whole genome sequencing may circumvent some of these limitations. Disclosures Elhaik: DDC: Consultancy. Baird: DDC: Current Employment. Kumar: Antengene: Consultancy, Honoraria; Bluebird Bio: Consultancy; Beigene: Consultancy; BMS: Consultancy, Research Funding; Tenebio: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Carsgen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Abstract KMT2A gene rearrangements are a major oncogenic driver in multiple hematologic neoplasms. Apart from t(9;11)(p21;q23) (KMT2A/MLLT3) in acute myeloid leukemia (AML), KMT2A gene rearrangements are considered to convey high risk and poor overall survival. Herein, we report a case of a 7 year old boy with newly diagnosed AML and a cryptic KMT2A/AFDN gene fusion resulting from a 5′KMT2A insertional event. The results of conventional chromosome studies revealed trisomy 8 in all 20 metaphases, with normal-appearing chromosomes 6 and 11. A KMT2A break-apart FISH probe identified 2 intact copies of the KMT2A gene region and an extra 5′KMT2A signal in 85% of interphase nuclei. Subsequent FISH studies using a KMT2A/AFDN dual-color dual-fusion FISH probe revealed positive results for a single fusion in 82% of interphase nuclei, indicating a KMT2A/AFDN gene fusion. Subsequently, metaphase FISH confirmed the location of the KMT2A/AFDN fusion at 6q27. To our knowledge, this represents only the second time in the literature that a cryptic KMT2A/AFDN gene fusion resulting from a 5′KMT2A insertional event was reported.

AbstractGain of 1q22 at diagnosis portends poorer outcomes in multiple myeloma (MM), but the prognostic significance of acquired 1q22 gain is unknown. We identified 63 MM patients seen at Mayo Clinic from 1/2004 to 12/2019 without 1q22 gain at diagnosis who acquired it during follow up and compared them to 63 control patients who did not acquire 1q22 gain with similar follow up. We also compared outcomes in the acquired 1q22 gain group with outcomes in 126 patients with 1q22 gain present at diagnosis. The incidence of acquired 1q22 gain was 6.1% (median follow‐up 6.8 years); median time to acquisition was 5.0 years (range: 0.7–11.5 years). Abnormalities on baseline fluorescence in situ hybridization (FISH) included trisomies (54%) and monosomy 13 (39%); 16 (25%) had high‐risk (HR) translocations or del(17p). Median progression‐free survival with front line therapy was 29.5 months in patients with acquired 1q22 gain, versus 31.4 months in control patients (p = .34) and 31.2 months in patients with de novo 1q22 gain (p = .04). Median overall survival (OS) from diagnosis was 10.9 years in patients with acquired 1q22 gain, versus 13.0 years in control patients (p = .03) and 6.3 years in patients with de novo 1q22 gain (p = .01). Presence of HR FISH at baseline increased risk of 1q22 gain acquisition. We demonstrate that acquisition of 1q22 gain is a significant molecular event in MM, associated with reduced OS. Among HR patients for whom this clonal evolution is determined, a risk‐adapted approach and/or clinical trial should be considered.

SummaryWe report a comparative analysis of patients with therapy‐related acute lymphoblastic leukaemia (tr‐ALL) vs de novo ALL. We identified 331 patients with B‐ALL; 69 (21%) were classified as tr‐ALL. The most common prior malignancies were breast (23·2%) and plasma cell disorders (20·3%). Patients with tr‐ALL were older (median 63·2 vs. 46·2 years, P < 0.001), more often female (66·7% vs. 43·5%, P < 0·001), and more likely to have hypodiploid cytogenetics (18·8% vs. 5·0%, P < 0·001). In multivariable analysis, patients with tr‐ALL were less likely to achieve complete remission [odds ratio (OR) = 0·16, P < 0·001] and more likely to be minimal residual disease‐positive (OR = 4·86, P = 0·01) but had similar OS after diagnosis and allo‐haematopoietic cell transplantation.

Abstract The detection of recurrent genetic abnormalities in acute myeloid leukemia (AML), including RUNX1T1/RUNX1 gene fusion, is critical for optimal medical management. Herein, we report a 45 year old woman with newly diagnosed AML and conventional chromosome studies that revealed an apparently balanced t(8;20)(q22;p13) in all 20 metaphases analyzed. A RUNX1T1/RUNX1 dual-color dual-fusion fluorescence in situ hybridization (FISH) probe set was subsequently performed and revealed a RUNX1T1/RUNX1 gene fusion. Metaphase FISH studies performed on abnormal metaphases revealed a cryptic, complex translocation resulting in RUNX1T1/RUNX1 fusion, t(8;20;21)(q22;p13;q22). This case study shows the importance of performing FISH studies or other high-resolution genetic testing concurrently with conventional chromosome studies for the detection of cryptic recurrent gene fusions in AML, particularly a focused genetic evaluation such as RUNX1T1/RUNX1 gene fusion, when specific abnormalities involving 8q22 are identified.

Abstract DDX41 mutations (germline and somatic) are associated with late onset myelodysplastic syndromes/acute myeloid leukemia (MDS/AML). Myeloid neoplasms (MN) with germline predisposition was identified as a distinct category in the 2016 WHO classification revision, including MN with germline DDX41 mutation. We retrospectively analyzed the molecular findings and clinical characteristics of thirty-three DDX41-mutated (mDDX41) patients at our institution. We identified 14 distinct pathogenic DDX41 variants in 32 patients and 8 DDX41 variants of unknown significance (VUS) in 9 patients. Five (16%) patients had a second DDX41 somatic mutation p.R525H and 13 (40%) had at least one additional oncogenic co-mutation in other genes. The median age at the time of diagnosis was 66 years, with male predominance (72%) and the majority of patients had normal cytogenetics (91%). Two-year overall survival (OS) was 86% and 6 (21%) MDS/AML patients with relatively preserved hematopoietic function were observed without further intervention. In comparison to AML patients with prognostically more favorable subtypes [t(8;21), n=27 and inv(16), n=40], mDDX41 patients in our cohort showed similarly favorable OS. Our study highlights that mDDX41-MN patients often have an indolent course and mDDX41-AML has comparable OS to favorable-risk AML.

Context.— Comprehensive genomic profiling has demonstrated that approximately 20% of pancreatic carcinomas with acinar differentiation harbor potentially targetable BRAF fusions that activate the MAPK pathway. Objectives.— To validate the above finding by BRAF break-apart fluorescence in situ hybridization (FISH) in a large series of pure acinar cell carcinomas (ACCs), evaluate tumors for the presence of BRAF V600E mutations, and compare clinicopathologic features of tumors with BRAF rearrangements with those without. Design.— Thirty cases of pure ACC and 6 cases of mixed acinar-neuroendocrine carcinoma (ACC-NEC) were retrieved. A break-apart FISH probe was used to detect BRAF rearrangements. Immunohistochemistry for BRAF V600E was performed. Results.— BRAF rearrangements by FISH were found in 6 of 36 cases (17%), 5 of which were pure ACC and 1 was a mixed ACC-NEC. Follow-up was available in 29 of 36 cases (81%). The median survival was 22 months for BRAF-rearranged cases and 16 months for BRAF-intact cases; the 2-year overall survival was 50% for BRAF-rearranged cases and 35% for BRAF-intact cases. No significant clinicopathologic differences were identified in cases with BRAF rearrangement compared with those without BRAF rearrangement. BRAF V600E mutation was identified in 2 of 34 cases (6%), both of which were pure ACC and were BRAF-intact by FISH. Conclusions.— This study supports the finding that BRAF rearrangements are present in approximately 20% of cases and identified BRAF V600E mutations in approximately 5% of cases. These cases may benefit from targeted therapy.

SummaryPatients with asymptomatic/smouldering Waldenström macroglobulinaemia (SWM) have a variable risk of progression to active WM. Our study evaluated 143 patients with SWM consecutively seen between January 1996 and December 2013. With a median [95% confidence interval (CI)] follow‐up of 9·5 [8·1–11·5] years, the cumulative rate of progression was 11% at 1 year, 38% at 3 years and 55% at 5 years. On multivariate analysis, haemoglobin (Hb) ≤123 g/l [risk ratio (RR) 2·08; P = 0·009] and β2‐microglobulin (β2M) ≥2·7 µg/ml (RR 2·0; P = 0·01) were independent predictors of a shorter time‐to‐progression (TTP) to active WM. Patients with myeloid differentiation factor 88 wild type (MYD88WT) genotype (n = 11) demonstrated a trend toward shorter TTP [median (95% CI) 1·7 (0·7–8·7) vs. 4·7 (2·4–7·7) years for the MYD88L265P cohort, n = 42; P = 0·11]. The presence of C‐X‐C chemokine receptor type 4 (CXCR4) mutation (n = 29) did not impact the TTP (median: 3 years for CXCR4WT vs. 5·6 years for CXCR4MUT, P = 0·34). The overall survival (OS) for patients with SWM (median: 18·1 years) was comparable to an age‐, sex‐ and calendar year‐matched USA population (median: 20·3 years, P = 0·502). In conclusion, Hb and β2M at diagnosis represent independent predictors of progression to active WM. Comparable survival of SWM and a matched USA population argues against pre‐emptive intervention in this patient population.

Abstract Purpose: Structural variants (SV) of the MYC gene region are common in multiple myeloma and influence disease progression. However, the prognostic significance of different MYC SVs in multiple myeloma has not been clearly established. Experimental Design: We conducted a retrospective study of multiple myeloma comparing MYC SV subtypes identified by next-generation sequencing (NGS) and FISH to MYC expression and disease survival using 140 cases from Mayo Clinic and 658 cases from the MMRF CoMMpass study. Results: MYC SVs were found in 41% of cases and were classified into nine subtypes. A correlation between the presence of a MYC SV and increased MYC expression was identified. Among the nine MYC subtypes, the non-immunoglobulin (non-Ig) insertion subtype was independently associated with improved outcomes, while the Ig insertion subtype, specifically involving the IgL gene partner, was independently associated with poorer outcomes compared with other MYC SV subtypes. Although the FISH methodology failed to detect approximately 70% of all MYC SVs, those detected by FISH were associated with elevated MYC gene expression and poor outcomes suggesting a different pathogenic role for FISH-detected MYC subtypes compared with other MYC subtypes. Conclusions: Understanding the impact of different MYC SVs on disease outcome is necessary for the reliable interpretation of MYC SVs in multiple myeloma. NGS approaches should be considered as a replacement technique for a more comprehensive evaluation of the multiple myeloma clone.