Clonal Abnormalities Research Articles

Porokeratosis: An enigma beginning to unravel.

Porokeratosis is a keratinization disorder with unclear etiopathogenesis, varied clinical presentation and characteristic histopathology, and is usually unresponsive to current therapeutic options. Until now, it was considered to be a clonal disorder with immunity, ultra violet radiation and other factors playing important roles in etiopathogenesis. It is now known that abnormalities in the mevalonate pathway are responsible for this clonal keratinization abnormality. New variants of porokeratosis like eruptive bullous, pruriginous, lichen planus like, follicular variants and porokeratoma have been described. While the cornoid lamella is the classical histopathologic feature, dermoscopy and reflectance confocal microscopy make the diagnosis clearer. Development of malignancy in a few variants is a concern. Linear, disseminated superficial actinic and giant lesions are most prone to developing malignancies. Bowen's disease, squamous cell carcinoma, basal cell carcinoma and even melanoma have been reported in cases of long-standing porokeratosis. Newer modalities of therapy such as photodynamic therapy, ingenol mebutate and HMGCoA inhibitors may play a role in the future.

Eight-Color Flow Cytometry Phenotypic Markers and Disease Progression in Monoclonal Gammopathy of Unknown Significance

Introduction: Flow cytometric immunophenotyping is considered an indispensable tool for the diagnosis, classification, and monitoring of plasma cell disorders. Herein, we seek to study the clinical significance of expression of phenotype markers in monoclonal gammopathy of unknown significance (MGUS).Methods: We identified a cohort of patients with a diagnosis of MGUS from the institutional myeloma database. Bone marrow (BM) aspirate assessment was performed using 8-color immunophenotypic next-generation flow cytometric (NGF) analysis with a minimum sensitivity of 10 -5 cells at the time of diagnosis or first visit to our institution. BM aspirate samples were immunophenotyped on a FACSCanto II flow cytometer using antibodies (BD) to delineate normal and abnormal plasma cells [CD138 (V-500), CD38 (FITC), CD19 (PE-Cy7), CD45 (V-450), CD27 (PercpCy5.5), CD81 (APC-H-7), CD56 (APC) and CD20 (PE)]. The sensitivity or the Limit of Detection (LOD) for this assay was validated to 20 cells in 2 ×10 6 events (0.001%), and the reproducibility or Lower Limit of Quantitation (LLOQ) is 50 cells in 2 ×10 6 events. Clinical and laboratory variables were also collected. Based on previously published data, expression (CD19, CD45, CD81), and lack of expression (CD56, CD27, CD20) of the above-mentioned surface antigens were analyzed. Additional variables such as IgA isotype, size of M-protein (≥15 g/L), and abnormal free light chain ratio(abnFLR) (defined as <0.1 or >10) were included in regression fitting models.Results: A total of 157 patients with MGUS were included in this analysis. The median age at diagnosis was 60 years (range 24- 84), 84 (53 %) patients were female and 25 (16%) were African American. Overall, IgG Kappa (75/148, 50%) was the most common isotype. Fluorescent-in-situ hybridization (FISH) data were available in 35 patients with t (4:14) and t (14;16) seen in 3 patients each. At a median follow-up of 18.15 years (quantiles 11.35, 33.62), 28 patients experienced disease progression (25 to MM, 2 to Waldenstrom macroglobulinemia, and 1 Smoldering myeloma). The median progression-free survival of this cohort was 17.3 years. Among these, occurrences of the bone lesion (8/28; 28.6%) were the most common pattern of disease progression to MM. This analysis showed lower odds of progression with the expression of CD27 (OR-0.39, 95% CI 0.15-0.99) (figure 1A). Disease progression was more common in patients with an abnormal plasma cell clone size ≥ of 3.1% at diagnosis (60% vs. 12.5%, p=0.0005). An abnormal plasma cell clone of ≥3.1% at diagnosis, was associated with increased odds of progression (OR-10.79, 95% CI 4.02-28.98) and a shorter PFS (12.5 years versus NR, p=0.01) (figure 1B). Serum M-spike ≥1.5 g/dL (OR-3.54;95% 1.30-9.62) and abnFLR (OR-2.30, 95% CI 1.00-5.32) were also associated with a higher odds of progression. However, in this population, the presence of IgA isotype did not increase the odds of MGUS progression. In a stepwise regression model, serum M-spike≥1.5 g/dL, abnFLR, and the lack of expression of CD27 were associated with the risk of disease progression.Conclusion: In addition to previously published risk factors, our cohort shows that the expression of CD27 antigen by eight-color flow cytometry confers a lower risk of disease progression of MGUS. This is consistent with our previous report that CD27 is progressively down-regulated in the transition from normal plasma cells (NPC) to MGUS to MM (Zhan et al, Blood 2006). Furthermore, we show that size of the myeloma clone (≥ 3.1% ) is a possible surrogate marker for disease progression in MGUS.Figure 1: 1A shows forest plot of odds ratios for flow cytometry markers, IgA isotype, size of M protein, abnFLR and plasma cell clone size. 1B shows the Kaplan Meier estimates of PFS for patients stratifies by plasma cell clone size. [Display omitted] DisclosuresMohan: Medical College of Wisconsin: Current Employment. Atrash: GSK: Research Funding; AMGEN: Research Funding; Jansen: Research Funding, Speakers Bureau.

A Multicenter, Prospective Study for Pediatric Chronic Myeloid Leukemia in Chronic Phase: The JPLSG CML-08 Study

Background: Tyrosine kinase inhibitor (TKI) has been used in pediatric chronic myeloid leukemia (CML) for more than 10 years, but only a few prospective clinical studies have been conducted in pediatric patients with CML due to their rarity. We conducted the JPLSG CML-08 study to determine the efficacy and tolerability of TKIs in children and adolescents with newly diagnosed CML in chronic phase (CML-CP).Methods: The JPLSG CML-08 study was a prospective multicenter observational study (UMIN000002581). Patients under 18 years of age with untreated BCR-ABL1-positive CML-CP were eligible and treated according to the modified ELN-2009 recommendation, and the efficacy and safety of TKIs were evaluated.Results: From October 2009 until September 2014, 79 patients were enrolled in 46 hospitals in Japan. A total of 78 patients (49 males and 29 females) were eligible for inclusion. Median age at diagnosis was 11 years (range, 1-17). Median observational period for survivors was 82 months (range, 48-118). Median WBC, Hb and platelet counts were 275x10 9/L (range, 8-765), 9.6g/dL (range, 5.8-14.6) and 560x10 9/L (range, 110-2875), respectively. Splenomegaly was found in 76%. High risk of Sokal, Hasford, EUTOS, and ELTS scores were observed in 21, 13, 27, and 9%, respectively. Clonal chromosome abnormalities in Ph-positive cells occurred in 1 patient at diagnosis. Imatinib, dasatinib, and, nilotinib were used as a first-line treatment in 69 (88%), 7 (9%), and 2 (3%) patients, respectively. The median initial dose of imatinib, dasatinib, and nilotinib was 276, 63, and 262mg/m2, respectively. 5y-PFS and OS was 96.2% (95%CI, 88.6 to 98.7%) and 97.4% (95%CI, 90.1 to 99.4%), respectively. Deaths were observed in 2 patients due to transplant complications. Hematopoietic cell transplantation was conducted in 14 patients (18%). Nine patients (12%) discontinued TKI with the aim of treatment-free remission (TFR), and five of them achieved TFR. In 69 patients with first-line imatinib, complete hematologic response was achieved in 95.7% at 3 months, complete cytogenetic response in 75.4% at 12 months, major molecular response (MMR) in 40.1% at 18 months, and MR4.0 in 52.8% at 60 months; If a transplant was performed, the follow-up period was censored at the date of transplant. Of the 69 patients, 52% changed treatment from imatinib to another TKI or transplant due to poor response, and 20% did due to intolerance. The most common cause of intolerance to imatinib was musculoskeletal events. BCR-ABL1 (IS) <10% at 3 months strongly correlated with higher achievement of MMR, MR4.0, and MR4.5. The EUTOS score was significantly associated with achievement of IS <10% at 3 months. Patients with a first-line second-generation TKI had a higher cumulative incidence of MR4.5 (P = 0.0191) than patients with a first-line imatinib. Second-generation TKI was used as first-line therapy only in patients older than 9 years, but other clinical characteristics, including risk scores, did not differ significantly between the two groups. The incidence of grade 3/4 adverse events (≥ 10%) included neutropenia (47%), anemia (39%), leukopenia (13%), arthralgia (13%), and myalgia (11%) for imatinib, neutropenia (21%), anemia (13%), and thrombocytopenia (11%) for dasatinib, and neutropenia (14%), elevated ALT (14%), hyperbilirubinemia (14%), skin rash (14%), and high CPK (14%) for nilotinib. Gastrointestinal bleeding was an adverse event specific to dasatinib (11% in all grades).Conclusion: This clinical study extends and confirms previous data showing that first-line treatment with imatinib is effective in children and adolescents, with response rates similar to those seen in adults. Although longer follow-up is needed to fully assess the long-term toxic effects, adverse events with imatinib, dasatinib, and nilotinib have been acceptable. As reported in adults, there was an advantage in selecting second-generation TKI over imatinib as first-line therapy to achieve deep molecular remission (DMR). Since discontinuation of TKI after achieving DMR is the preferred strategy, second-generation TKI is expected to become the standard therapy for children and adolescents. DisclosuresNo relevant conflicts of interest to declare.

Outcomes in Patients with Poor-Risk Cytogenetics with or without TP53 Mutations Treated with Venetoclax Combined with Hypomethylating Agents

Outcomes in Patients with Poor-Risk Cytogenetics with or without TP53 Mutations Treated with Venetoclax Combined with Hypomethylating Agents

Automated Digital Morphometry of Peripheral Blood Smears Detects Both Infrequent Events and Cellular Population Patterns in Myelodysplastic Syndrome

Background: Complete Blood Count (CBC) analytical capacity is falling short of recognizing informative RBC morphology or WBC dysplastic morphological changes. Current morphologic peripheral blood smear (PBS) analysis is performed manually using a semi-quantitative scale on a limited number of cells introducing high degree of subjectivity and low sensitivity. The novel Full-Field Morphology (FFM) technology developed by Scopio Labs performs PBS analysis on a significantly larger scale of 1000 fields of 100X view in a routine manner, allowing a precise and highly sensitive automated quantification of cellular and sub-cellular morphological parameters. Current diagnosis of myelodysplastic syndrome (MDS) is based on invasive bone marrow aspirate, followed by subjective morphological analysis. In this study, we applied this digital morphometric approach to compare PBS morphology of MDS patients with age-matched controls.Methods: 32 MDS (average age 80+10, [range 41-97] y, F:M ratio 14:18) and 30 age-matched control (average age 79+9, [range 65-100] y, F:M ratio 13:17) PBS were scanned by the Scopio Labs system, and evaluated according to three distinct morphological features with known significance in MDS: blast percentage per 100 or 1000 WBC; neutrophil cytoplasmic granulation per 1000 neutrophils; RBC morphology of at least 150,000 RBC. Quantitative determination of neutrophils granulation, was measured by Granulation Index (GI, between 0-1) and GI Distribution Width (GIDW, between 0-1). RBC measurements included the quantitative measurements of RBC size, namely macro- and microcytosis, and RBC contour changes (deformation), i.e. the percent of RBC that deviate from normal RBC shape.Results: The mean GI of MDS samples was 0.36+0.15, [range 0.14-0.63] (Fig. 1A middle, Fig. 1E), significantly (p<10 -4) lower compared with the mean GI of age-matched control samples 0.53+0.10, [range 0.24-0.64] (Fig. 1A top, Fig. 1E). Mean GI were highly diverse among MDS samples compared with age-matched controls (Fig. 1E), but with no significant differences in GIDW (not shown). Interestingly, two sub-populations of neutrophils were detected in some of the MDS samples, differ in their mean GI (0.26 for one sub-population, 0.52 for the second one, Fig. 1A bottom). Such fingerprint, suggesting the presence of an abnormal and normal clones, was not detected in the control samples. Percentage of blasts was determined per 100 or 1000 WBC counts/sample (Fig. 1B). Blasts were detected in 13/32 (41%) of MDS samples compared with 1/30 (3%) of age-matched controls, when counts were performed per 100 WBC. However, when 1000 WBC were analysed, blasts were detected in 27/32 (84%) of MDS samples compared with 6/30 (20%) of age matched controls, a highly significant difference (p<10 -6). The percentage of blasts per 1000 WBC counts/sample of MDS samples was 0.92+1.35, [range 0-5] %, significantly (P<0.0008) higher compared with the percentage of blasts per 1000 WBC counts/sample of age-matched control samples 0.02+0.05, [range 0-0.2] % (Fig. 1E). RBC analysis revealed significant differences between MDS and age-matched samples (Fig. 1C). As expected, mean RBC size (49+4, [range 41-59] mm 2 MDS; 45+3, [range 40-51] mm 2 age matched) and % of macrocytosis (17+15, [range 2-60] % MDS; 3+4, [range 0-15] % age matched) were significantly (p<10 -5) higher in the MDS samples compared with the age matched controls (Fig. 1E). We found that MDS PBS contained significantly (p<10 -6) higher number of abnormally-shaped RBC (8+1, [range 5-12] %), compared with age-matched controls (6+1, [range 5-8] %) (Fig. 1E). Representative summaries of morphometric analyses of MDS and age-matched control are shown in figure 1D. Representative PBS scans of MDS and control samples are available in https://demo.scopiolabs.com/?_org=VCdaE756rjYwZW3Z#/scans.Conclusion:Our study demonstrates that FFM-based digital PBS analysis enables the detection and quantification of unique WBC and RBC morphologic alterations associated with MDS. The expanding therapeutic options for MDS, including for patients at early disease stages, makes the establishment of an accurate diagnosis of MDS, even at early stages, to be highly important. The proposed novel digital imaging technology opens the opportunity to screen patients, diagnose them early, based on peripheral blood morphology, and potentially, monitor their responsiveness to therapy. [Display omitted] DisclosuresKatz: Scopio Labs: Consultancy. Karni: Scopio Labs: Current Employment. Shimoni: Scopio Labs: Current Employment. Natan: Scopio Labs: Current Employment. Shaham: Scopio Labs: Current Employment. Pozdnyakova: Scopio Labs: Consultancy. Mittelman: Janssen · Roche · Novartis · Takeda · Medison / Amgen · Neopharm / Celgene / BMS · Abbvie · Gilead: Research Funding; Novartis · Takeda · Fibrogen · Celgene / BMS · Onconova · Geron: Other: Clini; Onconova · Novartis · Takeda · Silence: Membership on an entity's Board of Directors or advisory committees; MDS HUB: Consultancy; Celgene / BMS · Novartis: Speakers Bureau. Avivi: Novartis: Speakers Bureau; Kite, a Gilead Company: Speakers Bureau.

Metformin for Treatment of Cytopenias in Children and Young Adults with Fanconi Anemia

Fanconi anemia (FA), a genetic disorder affecting DNA repair, is characterized by bone marrow failure and cancer susceptibility. In FA mouse models, metformin (N,N-dimethylguanide) a biguanide metabolic agent, improves blood counts and delays tumor development. Given these findings, we conducted a single institution pilot study of metformin in non-diabetic patients with FA to assess feasibility, tolerability, and impact of metformin on hematologic response as defined by modified MDS International Working Group (IWG) criteria (Table 1).Fourteen of 15 patients with at least 1 cytopenia (hemoglobin <10g/dL, platelet count <100k/uL, or an ANC <1000 cells/uL) who signed treatment consent were eligible to receive metformin for 6 months; 1 patient was deemed ineligible given clonal disease progression on pretreatment bone marrow evaluation. Dosing of metformin was age based and equivalent to that used for type II diabetes mellitus. Of patients treated, the median patient age was 9.4 years (range 6.0-26.5), and 8/14 were male (57%). Median blood counts at study entry were hemoglobin of 11 g/dL (range 8.4 -14.2), platelet count of 54k/uL (range 16 -78), ANC of 1100 cells/uL (range 600 -1600). One patient was transfusion-dependent for red blood cells. Twelve of 14 patients (86%) completed 6 months of metformin treatment. One patient was lost to follow up after 2 weeks of metformin treatment and a second withdrew from the study after 3 months to proceed to hematopoietic stem cell transplant. Thirteen of 14 subjects (93%) tolerated maximal metformin daily dosing for age; one subject had a dose reduction for grade 2 gastrointestinal symptoms. No subjects developed hypoglycemia or metabolic acidosis. Three subjects had mildly elevated insulin levels consistent with mild insulin resistance prior to drug start; in 2/3 instances insulin levels normalized after metformin therapy. With metformin treatment, the most common adverse event observed was diarrhea (12/14, 85.7%); n=11 cases were grade 1 and n=1 was grade 2. Additional toxicities observed are outlined in Table 2. Hematologic response was observed in 4 of 13 evaluable patients (30.8%, 90% CI: [11.3, 57.3]) who received at least one month of treatment. The median time to response was 84.5 days (range 71-128 days). Responses were noted in neutrophils (n=3), platelets (n=1), and red blood cells (n=1). One individual with severe transfusion-dependent anemia became transfusion-independent after 1 month of treatment. The magnitude of hematologic improvement for the entire study cohort is outlined in Table 3. No subjects met criteria for disease progression or relapse during treatment. Bone marrow assessment of cellularity was not significantly different post therapy (median of change 0; interquartile range: -12.5- 30, p= 0.82) and clonal abnormalities noted at enrollment did not evolve with treatment. Interestingly, chromosomal breakage with MMC or DEB was observed to be lower following metformin in paired pre/post metformin samples from 8/10 and 7/10 individuals, respectively. Colony forming cell assays were performed pre/post metformin in 4 bone marrow samples derived from individuals who lacked protocol defined hematologic response. In 2 of 4 cases, increased myeloid and erythroid colony-forming cells were observed following metformin therapy. Exploratory proteomic assessment of plasma proteins pre and post metformin exposure identified downregulation of cytokine and cytokine receptor interaction pathways and inflammatory pathways and upregulation of insulin signaling pathways after completion of metformin. We conclude that metformin is safe and tolerable in non-diabetic patients with FA and may provide therapeutic benefit for a subset of patients with this marrow failure/cancer predisposition condition. Future efficacy endpoints should address the utility of metformin as both a prophylactic agent aimed at curtailing progression of bone marrow failure and as a preventive agent aimed at reducing cancer risk in FA. A phase II multicenter study is warranted to study whether the treatment is efficacious in a larger subset of patients and to further elucidate the signaling pathways and mechanism of action by which metformin may provide benefit in FA. [Display omitted] DisclosuresPollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees. Furutani: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Esrick: bluebird bio: Consultancy. Nakano: Novartis: Consultancy. Thompson: Celgene: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding; Biomarin: Research Funding; Vertex: Research Funding; Graphite Bio: Research Funding; Baxalta: Research Funding; CRISPR Therapeutics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Consultancy; Beam Therapeutics: Consultancy; Global Blood Therapeutics: Current equity holder in publicly-traded company. OffLabel Disclosure:metformin- use in Fanconi anemia in an effort to improve hematopoiesis

High-Throughput Characterization of Cytogenomic Heterogeneity of MDS Using High-Resolution Optical Genome Mapping

IntroductionIntroduction of next-generation sequencing has defined the somatic mutational landscape in MDS. Comprehensive high-throughput structural variant profiling (SVP) is as important as mutation profiling in characterizing MDS clonal architecture since these large genomic aberrations have already shown to be critical for diagnosis and risk-stratification of MDS. A subset (MECOM, KMT2A rearrangements) are therapeutic targets in clinical trials. At this time, technical advances in SVP for copy number alterations (CNAs) and fusions have not been congruent with mutation profiling due to the inability of short-read (150bp) NGS to detect SVs. Currently available long-read (10-20Kbp) and whole genome sequencing cannot detect all SVs due to the presence of repeat sequences. Hence, conventional karyotyping (CK) remains the gold standard. Optical genome mapping (OGM) is a novel single-platform technique that measures ultra-long-range sequence patterns (>300Kbp), thereby unaffected by repeat sequences, enabling unbiased evaluation of all types of SVs at a high resolution. Here, we performed comprehensive SVP and mutation profiling in a large well-characterized cohort of MDS.MethodsWe selected samples with available fresh/frozen BM cells from consecutive treatment-naïve MDS pts who also underwent standard-of-care tests (CK, FISH, targeted 81-gene NGS for mutations). For OGM, ultra-high-molecular-weight-DNA was extracted, followed by labeling, linearization and imaging of DNA (Saphyr, Bionano) [median coverage:>300X]. The results were analyzed using de novo (>500 bp), rare variant (>5000 bp) and copy number (>500,000 bp) pipelines. The data was compared against 200 healthy controls to exclude common germline SVs. Clinical significance of the SVs was determined based on the location/overlap with the coding region of myeloid malignancy associated genes. The detection sensitivity was 10%.ResultsThere were 76 treatment naïve MDS patients. Baseline characteristics, comprehensive cytogenetic scoring system (CCSS) and R-IPPS risk categories and somatic mutations are in Fig 1.OGM identified all clonal abnormalities detected by CK [CNAs, inversions, inter/intra-chromosomal translocations, dicentric, complex derivative chromosomes]. Precise mapping of SVs by OGM at gene-level allowed determining the status of clinically informative biomarkers such as TET2, MECOM, TP53 and KMT2A, without the need for confirmatory assays. Detailed gene-level characterization of different SVs included KMT2A-ELL [t(11;19)] in MDS with WT1 mut, t(9;11) with SYTL2 fusion (and not KMT2A), der(1;7) leading to del(7q) in MDS with GATA2 mut/IDH2 mutand t(1;3)(p36;q21) rearrangements with potential PRDM16 disruption in SF3B1 mut/RUNX1 mutMDS, among others. Using OGM, we mapped the sequence patterns in both samples with IM with high level of confidence.Additionally, OGM identified 23 cryptic, clinically significant SVs in 14 (18%) of 76 pts. These included deletions of TET2, KMT2A, and del(5q), KMT2A amplification in MDS with FLT3-ITD/DNMT3A mut/RAS mut, NUP98-PRRX2, MECOM rearrangement in TET mut mutated NK-MDS. In addition, there were SVs of uncertain significance: duplications of chr1 (PDE41P), deletions of chr21 (involving RUNX1), chr2 (DNMT3A, ASXL2), chr12 (ETV6) and chr22 (EP300) and der(16)t(12;16)(q21.1;q12.1). These cryptic SVs were noted across all R-IPSS risk categories (highest yield in very-low and low R-IPSS) and across all cytogenetic risk-groups (very-good to very-poor).In complex karyotype setting, OGM could resolve the markers and additional genetic material, and in most cases, showed a much higher the degree of complexity within the genome than was apparent by CK. Four pts showed SV patterns typical of chromothripsis/chromoplexy.The median number of mutations per pt was 1 (0-6). When compared to mutation subsets, cryptic SVs were only identified in pts with ≤3 mutations. Majority represented either MDS with TP53 mut (6, 29%) or SF3B1 mut/TET mut (deletions of TET2, KMT2A, NOTCH1 and EP300 genes).ConclusionsUnbiased, high-throughput whole genome SVP revealed cryptic, clinically significant SVs in ~18% of MDS pts. OGM is a single-platform cytogenomic tool that can facilitate SVP at a gene-level resolution. This study provides strong support for further validation in expanded cohorts to guide clinical implementation and integration of SVP for routine work-up. [Display omitted] DisclosuresWei: Daiichi Sanko: Research Funding. Kantarjian: Ipsen Pharmaceuticals: Honoraria; Amgen: Honoraria, Research Funding; Astellas Health: Honoraria; Astra Zeneca: Honoraria; AbbVie: Honoraria, Research Funding; KAHR Medical Ltd: Honoraria; NOVA Research: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Distinct Senescent Bone Marrow Microenvironment in Therapy-Related Myeloid Neoplasms

Background: Therapy-related myeloid neoplasms (tMN) is a second haematological malignancy associated with distinct molecular profile (Singhal et al Leukemia 2019) and dismal outcome. tMN is believed to arise from cytotoxic DNA damage to haematopoietic stem cells (HSC). Although, cytotoxic therapy (CT) can damage bone marrow (BM) microenvironment, its role in tMN pathogenesis remains unknown.Aim and Methods: We performed comprehensive multiomic profiling (transcriptomic, cytokine quantification, phenotype, DNA damage) of BM stromal cells (BMSC) from (i) tMN patients previously exposed to CT. Critically, we compared it with (ii) patients with MN and a history of another cancer without CT (pMN+Ca), (iii) primary MN (pMN) and (iv) age-matched controls (Healthy).Results: To decipher microenvironmental changes induced by CT from that of MN and age-related changes, whole transcriptome analysis was performed on BMSC isolated from tMN and compared with all control cohorts. Twenty-nine genes were differentially expressed in tMN compared to Healthy (FDR < 0.1, P < 0.05). Unexpectedly 146 genes were differentially expressed in tMN BMSC compared to other MN and interestingly, ~90% of differentially expressed genes were involved in senescence. Moreover, functional enrichment and GO analysis suggest DNA damage repair, cell cycle regulation, and senescence pathways were deregulated in tMN BMSC (Fig. 1A). Genes such as CDKN1A (a critical cyclin dependent kinase inhibitor orchestrating cell cycle arrest), TNFRSF10D (senescence associated), and FGF-2 (a key player in cell proliferation) were highly expressed (P < 0.001).These findings were validated by demonstrating other features of senescence in tMN BMSC: (i) enlarged/flattened cellular morphology, (ii) decreased cell proliferation and colony-forming potential, (iii) increased β-galactosidase expression, and (iv) defective DNA damage repair (Fig. 1Bi-iv). Interestingly, within the tMN cohort there was no correlation between latency period (the interval between completion of CT until tMN diagnosis) and senescence, indicating that higher senescence is persistent even after several years of CT.Senescence associated secretory phenotype (SASP), a mixture of inflammatory cytokines and chemokines such as IL-7, IL-1β, IL-13, and IL-6, were significantly higher in conditioned media of tMN BMSC (9/14, 64%) (Fig. 1 Bv).Despite reduced proliferation and senescence, transcriptome analysis showed enrichment of metabolic and energy production pathways in tMN BMSC compared to controls. TXNRD1, regulator of glucose and lipid metabolism, and BNIP3, a negative regulator of mitochondrial potential, were highly expressed in tMN BMSC (P < 0.001). These findings were further verified by Seahorse bioenergetic analyses. The overall energetic rate (as assessed by ATP production) was higher in tMN compared to Healthy BMSC (P = 0.002), with higher proportion of ATP generated by glycolysis (77% versus 35.5%) (Fig. 1C).Adipogenic differentiation potential of senescent BMSC is not well known. Transcriptome analysis showed reduced expression of genes involved in adipogenesis in tMN BMSC. This was further validated by two independent in vitro assays showing reduced adipogenesis (Fig. 1D). Interestingly, PNPLA2, a catalyst of the first lipolysis reaction, were significantly de-regulated in tMN BMSC (P < 0.001).The key difference in tMN and other MN is prior exposure to CT. Hence, we hypothesise that prior CT leads to long-term irreversible damage to BM microenvironment and induced senescence, which in turn propagate senescence in surrounding normal cells and promote clonal abnormalities in HSC. Other possibility is that tMN clone can induce these changes in BM microenvironment. To decipher it, we assessed serial BMSC. We observed aberrant stroma proliferation and bi-differentiation capacity, following CT, well before the diagnosis of tMN.Conclusions: By multiple orthogonal indices, our results show that tMN BMSC lie on an extreme trajectory away from normal and typical MN, with massive defect in senescence and distinct metabolic phenotype. Importantly, prior CT leads to long-term irreversible damage to the BM microenvironment which potentially contributes to tMN pathogenesis. Together, these data provide a valuable resource for future strategies to delay or prevent the onset of tMN and assist in marrow regeneration in patients undergoing CT. [Display omitted] DisclosuresHughes: BMS: Research Funding; Novartis: Honoraria, Research Funding; Takeda: Honoraria. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

MDS-Associated Splicing Factor Mutations Promote Alternative Splicing of a Differentiation-Impaired CSF3R Isoform

▪Background. Myelodysplastic syndromes (MDS) constitute the most common group of bone marrow failure disorders, characterized by ineffective hematopoiesis and a significant risk of transformation to AML. Efforts to understand the molecular basis of MDS led to the identification of acquired somatic mutations in the RNA splicing factors SF3B1, U2AF1, SRSF2, ZRSR2, and LUC7L2 in 45-85% of adult MDS patients. How they perturb hematopoiesis and promote expansion of abnormal clones remain unknown. Elevated levels of alternate isoforms of granulocyte colony stimulating factor receptor (CSF3R) patients with MDS and other myeloid neoplasias have been reported with a strong correlation reported with loss of chromosome 7. Constitutive splicing produces the canonical Class I CSF3R isoform that supports proliferation and differentiation. Alternative splicing promoting intron excision in CSF3R exon 17 results in a differentiation-defective Class IV CSF3R isoform. We hypothesized that aberrant splicing activity of MDS-associated splicing factor mutations promote Class IV CSF3R expression, resulting in dysgranulopoiesis. Methods. We constructed a CSF3R minigene that was transiently expressed in K562 cells along with eukaryotic expression vectors containing cDNAs for wild-type or mutant forms of SF3B1, U2AF1, or SRSF2. To determine role of LUC7L2, a splicing factor residing on chromosome 7, the CSF3R minigene was transiently expressed in K562 cells with knockout LUC7L2. Class IV or Class I was measure by qPCR using specific primers. Putative exonic splicing enhancer motifs (ESEs) within exon 17 were deleted in the minigene and transfected into K562 cells expressing either wildtype or mutant SRSF2. To determine impact of mutant SRSF2 on granulopoiesis, wildtype or mutant SRSF2 were overexpressed in GCSF-treated CD34+ cells and morphology was assessed on day 14. To determine impact of Class IV isoform on granulopoiesis, Lin-, Sca-1+, and c-Kit+ (LSK) cells from Csf3r null mice were transduced with Class I or Class IV and colony forming unit (CFU) assay for granulopoiesis was performed with Methocult 3534. Colony scores and cell morphology were assessed on day 8.Results. K562 cells with transient expression of mutant SRSF2 P95H and LUC7L2 knockout resulted in statistically significant increase in Class IV:I CSF3R mRNA ratios normalized to a minigene-only control. Interestingly, expression of mutant U2AF1 S34F significantly decreased Class IV:I while U2AF1 Q157P increased Class IV:I ratios. K562 cells expressing mutant SF3B1 K700E did not show significant differences in Class IV splicing. To further investigate how SRSF2 regulates CSF3R splicing, we deleted two putative SRSF2-binding ESE motifs (ESE1 and ESE2) within exon 17 of the CSF3R minigene and transfected them into K562 cells stably expressing either wildtype SRSF2 or mutant P95H, P95L, or P95R. Deletion of either ESE1 or ESE2 resulted in decreased Class IV:I in all SRSF2 P95 mutant cells compared to minigene-only and wildtype controls. Next, we assessed the effect of SRSF2 P95 mutations on neutrophil differentiation in CD34+ cells. Overexpression of SRSF2 P95H, P95L, or P95R led to increased neutrophilic precursors compared to untransduced CD34+ cells. To determine the effect of a single CSF3R isoform on granulopoiesis, we cultured LSK cells from Csf3r null mice transduced with either Class I or Class IV on MethoCult 3534 with addition of GCSF. On day 8, cells expressing Class I had more total colony numbers with significantly higher CFU-GM colonies than cells expressing Class IV compared to empty vector control.Conclusions. We demonstrated that mutated SRSF2, U2AF1, and LUC7L2 deficiency alters CSF3R splicing in its terminal exon. Interestingly, mutations on different residues of the same gene (U2AF1) had opposing effects on CSF3R splicing. Mutant SRSF2 resulted in increased intron excision to promote increased levels of Class IV isoform. Mutation of two putative SRSF2 binding sites within CSF3R exon 17 reversed the increased splicing promoted by mutant SRSF2, further support our observation that CSF3R is a target for mutant SRSF2. Our observation with overexpression of mutant SRSF2 P95 in CD34+ cells suggests that defective neutrophil differentiation is related to increased Class IV. Our findings shed insights into how aberrant splicing of CSF3R drives MDS progression and provides a new model of dysgranulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Complex Clonal Evolution Can Occur Following Transplantation for Transfusion Dependent Thalassaemia in the Context of Mixed Myeloid Chimerism and Reduced Conditioning Regimens

Haemopoietic stem cell transplantation (HSCT) is a well-established treatment modality for the cure of transfusion dependent thalassaemia (TDT) and sickle cell disease (SCD). Clonal evolution has recently been identified as a concerning event in the setting of mixed chimerism and/or ineffective haemopoiesis following conventional bone marrow transplantation and gene therapy for haemoglobinopathies. This has so far been restricted to SCD only, with the presumption that despite both conditions sharing an ineffective erythropoietic marrow compartment, there may be inflammatory and hypoxic differences enabling clonal evolution, in addition to the different exposure to hydroxycarbamide as a therapeutic agent. However, there is a need to investigate whether this may also be an occurrence in TDT.From 2011 to 2021, over a ten-year period, sixty-five consecutive paediatric patients received a sibling HSCT (n=55) or a haploidentical HSCT (n=10) for TDT in our institution. Conditioning intensity was minimised at the start of this cohort in order to limit toxicity and late effects, abandoning the use of Bu/Cy, which resulted in approximately 50% of the patients having stable mixed chimerism long-term. Sibling HSCT was conditioned with fludarabine 160 mg/m 2, treosulfan 42 g/m 2, thiotepa 10 mg/kg and ATG (Thymoglobulin) or alemtuzumab, and received GvHD prophylaxis with ciclosporin and MMF. Haploidentical HSCT was conditioned with fludarabine 150 mg/m 2, cyclophosphamide 30 mg/kg, TBI 2 or 4 Gy, and ATG 4.5 mg/kg (thiotepa 10 mg/kg added if TBI 2 Gy only) with GvHD prophylaxis provided by two doses of post-transplantation cyclophosphamide 50 mg/kg, sirolimus and MMF. All patients had pre-transplantation endogenous haemopoiesis was suppressed pre-transplantation with hypertransfusions for a minimum of 8 weeks, and/or the use of hydroxycarbamide and azathioprine. GvHD prophylaxis was provided with ciclosporin and MMF. All patients were Pesaro class I or II at the time of transplantation. The median age was 5 years (2 - 19). The median survival was 26.8 months (2.6-101.8). The OS was 93.8% and DFS was 89.2%.Three patients in this cohort developed clonal evolution in the context of myeloid mixed chimerism identified due to the development of cytopenias and transfusion dependence. All patients had a complex karyotype and it involved deletion of chromosome 7: •Patient 1 had a sibling BMT at 3 years and 2 months. Day +28 chimerism was >95% in whole blood and 95% in T cells. The myeloid fraction had a progressive reduction from day +60 onwards. At 15 months post-transplantation clonal evolution was identified [18% ring sideroblasts, 46;XY, del (7) (q22 q34) [5]/46;XY [5], chimerism 13% donor in whole marrow and 41% marrow T cells. Two months later he became red cell transfusion dependence. A second BMT with busulfan based conditioning resulted in long-term cure.•Patient 2 had a sibling BMT at 3 years and 8 months of age. Day +28 chimerism was 99% donor in whole blood and 99% donor in T cells. Day +161 post-transplantation he started requiring erythropoietin support to maintain him transfusion independent and on day +217 we first identified in the bone marrow the appearance of myeloid mixed chimerism (75% donor in whole marrow and 91% marrow T cells) and complex clonal evolution involving -7 and FISH identified deletion of one copy of KMT2E (7q22) and MET (7q31) detected [16/200]. The patient is under monitoring at present with a progressive reduction of the size of the abnormal clones.•Patient 3 had a haplo BMT at 5 years and 8 months. Day +28 chimerism was 97% donor in whole blood and 99% donor in T cells. She developed mixed myeloid chimerism following cessation of immunosuppression on day +206: 86% donor whole blood and 99% donor in T cells. At 22 months post-BMT she started to require transfusion and a complex clonal evolution involving deletion 7 in her bone marrow when the chimerism was 42% donor whole blood and 81% donor marrow T cells. FISH identified deletion of one copy of KMT2E(7q22) and MET(7q31) detected[22/100]. She is being prepared for a second bone marrow transplant.In conclusion, complex clonal evolution also occurs post HSCT in TDT, at least in the context of reduction of conditioning intensity and development of mixed myeloid chimerism. This finding warrants further investigation and may have significant implications for the design of both conventional HSCT and gene therapy strategies. DisclosuresNo relevant conflicts of interest to declare.

DIAGNOSTIC APPLICATION AND CLINICAL SIGNIFICANCE OF FCM WELLS SCORING SYSTEM IN MYELODYSPLASTIC SYNDROMES

Myelodysplastic syndromes (MDS) are group of clonal diseases of the hematopoietic system characterized by ineffective hematopoiesis, dysmyelopoiesis, a high frequency karyotype abnormalities and the risk of transformation into acute leukemias. Cytopenic and dysplastic changes are not pathognomonic for MDS, and there are many diseases that can imitate MDS. According to various sources, clonal karyotypic abnormalities are present only in 20-60% of MDS. The diagnosis of MDS is not difficult if blasts or sideroblasts are present in the bone marrow, or there are chromosomal aberrations as evidence of clonal hematopoiesis. The diagnostic problem arises in cases of MDS without sideroblasts, with normal karyotype and/or bone marrow hypoplasia. Since 2012, the ELNet Working Group has proposed and subsequently supplemented guidelines for Flow Cytometry as a complementary diagnostic tool. The aim of the study was to compare the results of the FCM Wells score MDS with the results of the İPSS-R score MDS The study included 30 patients initially diagnosed with MDS . The classification was carried out according to the WHO Classification of MDS 2016: MDS SLD-6 (20%), MDS-MLD-5 (16.7%), MDS RS-MLD-2 (6.7%), MDS-EB1-9 (30%), MDS EB2-8(27%). According to the IPPS-R, patients were scored based on blasts, cytogenetic examination, hemoglobin/platelet/absolute neutrophil count and scored as very-low, low, intermediate, high, very-high. Using the Wells evaluation criteria, which takes into account cytometric analysis of the cells of the main myelopoiesis lines, changes were found in the compartment of granulocytes in 93%, monocytes in 40% and erythrocytes in 73% of cases. High scores on the Wells scale (> 4) were obtained in 89% of (8/9) MDS-EB1, 100%(8/8) MDS-EB2, 80% (4/5) MDS MLD patients, 17% (1/6) MDS –SLD, 50%(1/2) MDS RS-MLD. According to IPPS-R, MDS patients received a score <1.5 very low risk group include 50%(3/6) MDS –SLD, 20%(1/5) MDS-MLD, score > 1.5-3 - Low risk group include MDS –SLD 50%(3/ 6), MDS-MLD-80% (4/5), MDS RS-MLD 50% (1/2), MDS-EB1-78%(7/ 9), score > 3- 4.5-intermediate risk qroup got MDS-EB1 22%(2/ 9), MDS EB2- 25% (2/8), MDS RS-MLD- 50%(1/2), Score > 4.5 respectively high risk group got patients MDS –SLD- 17%(1/6), MDS EB2- 50%(4/8), Score > 6 very high risk group got MDS EB2- 25%(2/8). The Pearson's correlation coefficient (PCC) showed high correlation between İPSS-R and FCM Wells score was 0.83, p<0.002. In our study, the FCM score had a positive correlation with the IPSS-R prediction. Expanded analysis of the main compartments of the bone marrow (early precursors of myelopoiesis, the population of granulocytes and monocytes, erythrocytes) using the Wells scale as an additional tool improves the diagnosis and distinguish low-grade MDS from non-clonal cytopenias.

Clonal cytogenetic abnormalities in donor-derived cells after sex mismatched allogeneic stem cell transplantation

Clonal cytogenetic abnormalities (CCA) in donor-derived cells after stem cell transplant (SCT) are typically reported in donor-derived cell neoplasms, but CCA also may reflect a constitutional abnormality in the donor or may be present in a recipient without overt hematological malignancy. We reviewed 8515 tests on 2035 patients, who had allogenic sex mismatched SCT and underwent serial cytogenetic analysis between 2006 and 2020 in our institution. A constitutional CCA was observed in 3 patients: inv(10), t(1;5), and t(13;14). A somatic CCA without overt neoplasia was detected in 12 patients: del(7q) (n = 6), del(20q) (n = 3), der(11)t(11;11) (n = 1), t(1;9) (n = 1), dup(6p)(n = 1). In this group, four patients with cytopenia had del(7q), and an association between del(7q) and an adverse overall survival (OS) was observed [HR:5.99; 95%CI 1.23–29.92). Four patients had a donor-derived cell neoplasm: myelodysplastic syndrome (n = 3) and acute myeloid leukemia (n = 1), and all four neoplasms had loss of 7q. In our cohort, ∼1% of the patients (19/2,035) had CCA in donor-derived cells. Balanced constitutional CCA can pose a reproductive risk to donor. Loss of 7q is the most common somatic CCA, in donor-derived cells.

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

NS-62: The Importance of regular disease monitoring for patients with low or low intermediate risk Monoclonal Gammopathy of Undetermined Significance (MGUS)

<h3>Background</h3> Monoclonal gammopathies encompass a number of disorders characterized by a clone of a normal protein. Although biologically heterogeneous, monoclonal gammopathy of undetermined significance (MGUS) represents the most common of these disorders with the production of a monoclonal protein (Mprotein). This abnormal clone of plasma or other lymphoid cells carries a variable risk of progression to a plasma cell disorders (PCDs) requiring treatment. Few studies report real-world incidence of progression from low risk MGUS to PCDs requiring therapy and with newer risk models. Purpose: To describe characteristics of patients who progress from MGUS to PCD requiring plasma cell directed therapy. <h3>Methods</h3> A list of all patients with MGUS seen at the Cleveland Clinic from 1/1998 to 6/2021 was obtained from an internal IRB approved data base. Demographic information, the date of antecedent MGUS diagnosis, and disease characteristics were confirmed. Patients were categorized as having low, low-intermediate, high intermediate, or high risk MGUS by assigning a point system as follows: Mprotein with IgA isotype (1point), Mprotein concentration of 1.5g/dL or more (1point), serum FLC ratio less than 0.1 or more than 10 (1point), and immunoparesis (≥1 uninvolved immunoglobulins below lower level of normal; 1-2 points).In the scoring system for light-chain MGUS, serum FLC ratio less than 0.1 or more than 10 (1 point) and immunoparesis (1, 2, or 3 points) were identified as risk factors. Patients were excluded if MGUS was diagnosed prior to 2004, missing sFLC, or MGUS <1 year. <h3>Results</h3> Included in this study were 3,188 charts of patients with confirmed MGUS. Of these, 209 were excluded as did not meet criteria for analysis. An additional 102 patient charts were excluded due to incomplete baseline disease evaluation, or a diagnosis of MGUS prior to 2004 (when light chain assay was commercially available). A total of 2,853 patients remained without evidence of disease progression according to 2014 IMWG diagnostic criteria. 23 patients (12M;11F) progressed from MGUS:14 progressed to MM, 6 progressed to amyloidosis, 2 progressed to WM and 1 progressed to MGRS. The median days from MGUS diagnosis to PCD diagnosis requiring therapy was 2,018 days (5.53y). Interestingly, 5 patients had 0 risk factors for progression but still progressed (3 MM, 2 Amyloidosis); 11 had 1 risk factor/low intermediate risk (7 MM,4 Amyloidosis); 6 had 2 risk factors (4 MM, 2 WM) while 1 had 3 risk factors (to MM). <h3>Conclusions</h3> Despite this small sample from a single institution, our findings are consistent with other studies in that the number of patients who progress from MGUS to other PCDs remains low. Routine monitoring can improve the time to diagnosis and minimize the risk of organ damage. Advanced practice providers and nurses are well-suited to monitor and mangage patients with MGUS.These findings support regular blood and urine testing for MGUS and ongoing risk assessment.

Therapy-related myeloid neoplasms in 109 patients after radiation monotherapy

AbstractTherapy-related myeloid neoplasms (t-MNs) are a late complication of cytotoxic therapy and are defined as a distinct entity by the World Health Organization. Although the link between chemotherapy exposure and risk of subsequent t-MN is well described, the association between radiation monotherapy (RT) and t-MN risk is less definitive. We analyzed 109 consecutive patients who developed t-MNs after RT and describe latencies, cytogenetic profile, mutation analyses, and clinical outcomes. The most common cytogenetic abnormality was a clonal abnormality in chromosome 5 and/or 7, which was present in 45% of patients. The median latency from RT to t-MN diagnosis was 6.5 years, with the shortest latency in patients with balanced translocations. One-year overall survival (OS) was 52% and 5-year OS was 22% for the entire cohort. Patients with chromosome 5 and/or 7 abnormalities experienced worse 1-year OS (37%) and 5-year OS (2%) compared with other cytogenetic groups (P < .0001). Sixteen patients underwent net-generation sequencing; ASXL1 and TET2 were the most commonly mutated genes (n = 4). In addition, 17 patients underwent germline variant testing and 3 carried pathogenic or likely pathogenic germline variants. In conclusion, patients with t-MN after RT monotherapy have increased frequencies of chromosome 5 and/or 7 abnormalities, which are associated with poor OS. In addition, pathogenic germline variants may be common in patients with t-MN after RT monotherapy.

Plasma-Cell Myeloma with Double M-Bands on Serum Protein Electrophoresis

Plasma-cell myeloma (PCM) is a monoclonal gammopathy (MGM) characterized by proliferation of abnormal clone of plasma cells infiltrating the bone marrow with consequent end-organ damage. The clonal plasma cells secrete a single clone of immunoglobulins (Igs) leading to presence of M-protein in the serum and/or urine. The M-protein is appreciated as a discrete band on serum protein electrophoresis (SPE) in the gamma globulin region, also called the M-band. Biclonal gammopathy (BGM) occurs due to neoplastic transformation of a plasma-cell clone undergoing Ig class switching or due to an independent neoplastic transformation event yielding proliferation of unrelated plasma-cell clones, therefore resulting in two distinct M-bands on SPE. It is, however, vital to distinguish a true BGM from an apparent one (MGM presenting with two distinct bands on SPE) so as to make an accurate diagnosis. Hereby, we report a case of a 61-year-old man, diagnosed with PCM and presenting with two discrete bands on SPE (simulating a BGM) which turned out to be monoclonal in nature.

Recurrent Episodes of Angioedema as Presenting Feature of JAK2-Positive Myeloproliferative Disorder Consistent with Polycythaemia Vera

Polycythaemia vera (PV) is one of the chronic myeloproliferative neoplasms, which are collectively characterised by clonal proliferation of myeloid cells with variable morphologic maturity and haematopoietic efficiency. PV is distinguished clinically from other myeloproliferative neoplasms by the presence of an elevated red blood cell mass because of uncontrolled red blood cell production. This is accompanied by increased white blood cells and platelet production, which is because of abnormal clone of haematopoietic stem cells with increased sensitivity to the different growth factors for maturation. PV can present with variable symptoms because of impaired oxygen delivery caused by slugging of blood, such as headaches, dizziness, vertigo, tinnitus, visual disturbances, and angina pectoris. Some patients present with bleeding complications (1%); another 1% of patients present with thrombotic complications. It is uncommon for patients with myeloproliferative disorders to present with features of angioedema. There are many reported cases in the literature that describe the relationship between the occurrence of angioedema and lymphoproliferative diseases; however, there are no reported cases describing instances of myeloproliferative neoplasm with angioedema. In this article, the authors have studied a case of a 53-year-old male who presented with recurrent episodes of features that are suggestive of angioedema. He was diagnosed with JAK2-positive myeloproliferative disorder consistent with PV; this is the first reported case in Jordan.

Cytogenetic findings of ectopic endometriotic tissue in women with endometriosis and review of the literature

ObjectiveTo determine chromosome and gene alterations in ectopic endometrial (EM) tissue which may be implicated in the clinical course or the progression of endometriosis and to review the literature concerning the cytogenetic findings of women with endometriosis. Study design15 women who underwent laparoscopic endometriosis surgery at the Athens Genesis Clinic were enrolled in the study. Ectopic endometrial tissue was surgically removed and further analyzed by conventional and molecular cytogenetic techniques. Fluoresent in situ hibridization (FISH) with probes for p53, ATM, MYC, MLL1 and IGH genes, the centromeres of chromosomes 7 and 8 and 7q22/7q31 chromosomal regions was carried out. ResultsKaryotypic analysis revealed no clonal chromosomal abnormalities. However, an increased frequency of polyploidy (55.6%) and sporadic chromosomal abnormalities (40.0%) concerning chromosomes 9, 11, 17 and X were noticed involving mainly deletions, trisomies or monosomies. FISH analysis showed IGH gene rearrangements in 54% of the EM cases and MLL gene rearrangements in 73% of the examined samples. Normal hybridization patterns were observed for p53, ATM and MYC. The increased frequency of polyploidy shown by conventional karyotyping was also confirmed by FISH. ConclusionPolyploidy, sporadic chromosomal abnormalities, as well as IGH and MLL gene rearrangements, may provoke genetic instability and play a potential role in the development of endometriosis. IGH and MLL gene rearrangements indicate a genetic relation between endometriosis and carcinogenesis. Confirmation of the above gene rearrangements in a large series of women may allow the determination of their possible involvement in the pathogenesis of this complex disease and their possible contribution in the early identification of women in danger for malignant transformation.

Clinical and Molecular Characterization of Clonal Chromosomal Abnormalities Appearing in Philadelphia-Negative Metaphases of Chronic Phase CML

▪BackgroundClonal chromosomal abnormalities in Philadelphia chromosome-negative metaphases (CCA/Ph-) emerge as patients (pts) with chronic phase chronic myeloid leukemia (CP-CML) are treated with tyrosine kinase inhibitors (TKI). The clinical significance of CCA/Ph- remains unclear specifically among pts treated with second generation TKI. Very little is known about the molecular abnormalities associated with them.MethodsWe conducted a retrospective analysis of pts enrolled on prospective, single-institution frontline trials for CML-CP. Of the 598 pts enrolled, 279 pts (46%) were treated with imatinib, 126 (21%) with nilotinib, 143 (23%) with dasatinib and 50 (8%) with ponatinib. CCA/Ph- were defined as abnormalities present in ≥2/20 Philadelphia-negative (Ph-) metaphases or if present in one metaphase in ≥2 cytogenetic assessments. The clinical outcomes of pts with CCA/Ph- were compared to a control group without additional chromosomal abnormalities (ACAs). We performed targeted deep sequencing (median depth 490x) on a pilot study from 10 pts with various CCA/Ph- (panel of 295 genes recurrently mutated in hematological malignancies). Single nucleotide variants (SNV) and small insertions and deletions (indels) were called against virtual normal sequence developed in-house. An automatic pipeline was used to filter and annotate raw variants, and then select non-polymorphism, protein-coding changing mutation, thus identifying high confidence driver mutations.ResultsCCA/Ph- occurred in 58 pts (10%) and the most common were - Y in 25 (43%) and trisomy 8 in 7 pts (12%). Most CCA/Ph- appeared within the first year of the start of the TKI (median 6 months, range 3-78 months). CCA/Ph- were transient and were detected in 2 or less time points in 36/58 cases (62%). The median number of Ph-negative metaphases containing CCA/Ph- per analysis was 5/20 (range 1-20). We further categorized CCA/Ph- into those where - Y was the only clonal abnormality, and all others. Pts who developed CCA/Ph- were significantly older than those without ACAs (median age 59 vs 47; P<0.001). Specifically, pts with -Y CCA/Ph- were the oldest (median age 64; P<0.001), followed by those with non-Y CCA/Ph- (median age 52; P=0.003) when compared to those without ACAs (median age 47). Four pts with ACAs in Ph+ metaphases who subsequently developed CCA/Ph- were excluded from response and survival analyses. Response to TKI therapy was similar for pts with CCA/Ph- and those without ACAs. With a median follow-up for survivors of 7.6 years (range 0.2-14.7 years), we found that pts with non -Y CCA/Ph- had worse failure-free survival (FFS), event-free survival (EFS), transformation-free survival (TFS) and overall survival (OS) compared to those without ACAs with the following 5-year rates: FFS (52% vs 70%, P = 0.02), EFS (68% vs 86%, P = 0.02), TFS (76% vs 94%, P < 0.01) and OS (79% vs 94%, P = 0.03). In a multivariate analysis, non -Y CCA/Ph- increased the risk of transformation or death when baseline characteristics were considered with a hazard ratio (HR) of 2.81 (95% CI 1.15-6.89, P = 0.02). However, this prognostic impact was not statistically significant when achieving BCR-ABL < 10% at 3 months was included in the analysis (Table 1).A total of 46 high-confidence driver mutations in 18 genes in 10/10 (100%) pts with CCA/Ph- were identified (Figure 1). Median number of driver mutations was 2.5 (range 1-9). ASXL1 frameshift mutation was detected in all examined pts (10/10 pts) mostly at low variant allelic frequency (VAF) with a median VAF of 1.6% (range 0.7% - 4.2%). Mutations were also commonly detected in PTPN11 (7/10 pts). Mutations in PTPN11 have been reported in about 4% of pts with acute myeloid leukemia (Papaemmanuil et al. NEJM 2016). Mutations in DNMT3A (R635Q) and ABL (Y253H, associated with resistance to imatinib) were observed in the same case in addition to ASXL1. In this cohort, the observed mutation rate is higher than what is expected in CP-CML when comparing to published literature (Kim et al. Blood 2017). Sequencing of a control cohort from pts with deep molecular response without ACAs is ongoing.ConclusionIn conclusion, non -Y CCA/Ph- are associated with decreased survival when emerging in pts with chronic phase CML across various TKIs. Preliminary results from deep sequencing indicate a higher than expected rate of mutations in CP-CML pts with CCA/Ph- compared to those without ACAs, mostly detected at low VAF. [Display omitted] DisclosuresKantarjian:Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding. Takahashi:Symbio Pharmaceuticals: Consultancy. Jabbour:Bristol-Myers Squibb: Consultancy. Cortes:ARIAD: Consultancy, Research Funding; Sun Pharma: Research Funding; BMS: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

MDS and AML in Shwachman-Diamond Syndrome: Clinical Features and Outcomes

Genetic predisposition to MDS and AML is recognized in a growing subset of patients, but data to inform medical management and outcomes are sparse. Shwachman-Diamond syndrome (SDS) is an inherited marrow failure syndrome associated with increased risk of myelodysplasia (MDS) and acute myeloid leukemia (AML). The aim of this multi-institutional retrospective study was to investigate clinical features, treatment, and outcomes of 37 patients with SDS who developed MDS or AML. Diagnosis of SDS was established by SBDS genetic testing (n=30), exocrine pancreatic dysfunction (n=5), or data were unavailable (n=2). Nine individuals presented with AML (4 male, 5 female), 26 with MDS (13 male and 13 female), one male with refractory cytopenia (RC), and one male with isolated persistent somatic TP53 mutation. Eight of 26 MDS patients, had MDS-EB1 or EB2 at diagnosis, of whom two progressed to AML. Three patients initially presenting with early MDS progressed to MDS-EB1, MDS-EB2, or AML. Median age (years) at diagnosis of early MDS was 13.8 (range 0.5-28.8), for MDS-EB1/2 was 16 (range 9-30) and for AML was 33 (range 5.5-47). Nine patients with MDS and 1 patient with AML were receiving G-CSF chronically prior to diagnosis of MDS/AML.Complex cytogenetics, (>3 cytogenetic abnormalities per clone), were noted in 8/9 AML cases. One AML patient presented with normal cytogenetics. Complex clonal cytogenetic abnormalities were noted in 5 of 6 patients with MDS-EB1/EB2 for whom reports were available. Cytogenetic data were available for 16 MDS cases and clonal abnormalities were noted in 10. Aberrations involving chromosome 7 were most common. The patient with the somatic TP53 mutation had normal cytogenetics. Centralized bone marrow pathology review is currently ongoing.Follow up data were available for 8 patients with AML; 6 are deceased. Six received chemotherapy with intent to proceed to hematopoietic stem cell transplant (HSCT), of whom 5 failed to achieve remission and died with disease without proceeding to transplant. One AML patient proceeded to HSCT with progressive disease post-chemotherapy, but died with relapsed disease. One patient proceeded to HSCT without prior chemotherapy and was in remission at day 30 post-HSCT. The sole long-term surviving AML patient achieved remission with chemotherapy and underwent HSCTs with 3 separate stem cell infusions due to primary graft failure after the initial two infusions. He remains alive in remission more than 4 years after diagnosis. Notably this surviving AML patient was the only AML patient with normal cytogenetics at diagnosis.Follow up data are available for 6 of 8 patients presenting with MDS-EB1/EB2, 4 of whom are deceased. One patient who presented with MDS-EB1 is alive with relapsed disease after HSCT and one patient is in remission 100 days after HSCT.Follow up data are available for 17 of 18 early MDS patients, 7 of whom are deceased including three who had progressed to MDS-EB1, MDS-EB2, or AML. Four of the 7 deceased patients had received a HSCT. One individual with MDS died of hepatic failure unrelated to MDS. Seven MDS patients are alive in remission after HSCT without prior chemotherapy. Treatment was not reported for an additional 2 surviving patients. One patient receiving a hypomethylating agent is alive with stable disease 4.8 years from diagnosis. The patients with RC and with a TP53 clonal abnormality are alive at 6.5 years and 1.7 years from diagnosis respectively.In summary,prognosis is poor for patients with SDS who develop AML or MDS-EB1/2, with few long-term survivors due to resistant disease and treatment-related complications. Outcomes are better if transplanted for MDS prior to acquisition of increased blasts, with 10 of 17 (59%) subjects surviving (median follow up 6 years, range 1.5-11.2). AML often presents in adulthood highlighting the need for continued surveillance of SDS patients with bone marrow examinations including cytogenetics and FISH. These data demonstrate the importance of early detection of clonal evolution in this leukemia predisposition syndrome. Better markers for risk stratification are needed to identify patients who would benefit from early transplant prior to advanced clonal disease. Novel therapeutic strategies, such as targeted therapies, are urgently needed to improve outcomes of SDS patients with advanced MDS or AML. DisclosuresDiNardo:Daiichi-Sankyo: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Norkin:Celgene: Honoraria, Research Funding. Shimamura:TransCellular Therapeutics: Other: Spouse is majority shareholder .