Molecular and Expression Characterization of Monosomy 7 and Del(7q)

The loss of chromosome 7 (monosomy 7, -7) or its long arm [del(7q)] is a common karyotypic abnormality found in MDS and are associated with a poor prognosis. However recent studies may suggest a difference in survival outcomes. These lesions occur both independently and in a complex karyotype (CK). The leukemogenic features of -7/del7q can be explained through loss of heterozygosity or haploinsufficiency (HI) of key deleted genes. In addition, the phenotype of del(7q) and -7 might be further modified by the clonal architecture determining the mode of molecular evolution. Based on the hypothesis that a deletion will exert its biologic consequences in genes with HI expression and by co-associated mutations, a comprehensive analysis of genes on chr.7 was performed. Within a cohort of 8,161 patients with myeloid neoplasms (MN), we identified 511 with -7 (6%) and 143 with del(7q) (2%). In 29% of cases, del(7q) was found as an isolated alteration, while 90% of CK had del(7q); -7 showed similar percentages of isolated cases but was less common in CK (20%, 55%; respectively) cases. Del(7q) was significantly (P<.01) associated with MDS, while -7 was significantly (P=.0038) associated with sAML. When we focused on antecedent diagnosis, patients with -7 were more likely to have a previous diagnosis of aplastic anemia or chemotherapy treatment (60%; 41%, P<.01, for both). The most common co-associated molecular lesions with -7/del(7q) were: TP53 (31%), DNMT3A (13%), TET2 (13%), RUNX1 (12%), ASXL1 (11%), del(5q) (25%) and CK (54%). Del(7q) vs. -7 differed in the frequency of TP53 (39 vs. 29%; P=.03), DNMT3A (19 vs. 11%; P=.03), and IDH1/2 (13 vs. 52%; P<.01). Isolated -7/del(7q) was associated with mutations of TET2 (24% vs. 9%, P<.001), SRSF2 (12% vs. 3%, P<.001), EZH2 (10% vs. 3%, P<.001), and SETBP1 (8% vs. 2%; P<.001). Complex -7/del(7q) correlated with TP53 (39% vs. 4%; P<.01) and PTPN11 (9% vs. 6%; P=.02). When we focused on mutations on genes located in the 7q common deleted regions (CDR1, 7q22 and CDR2, 7q35-q36), EZH2 was the most represented 7q mutant gene (6 mutants in del(7q) and 24 mutants in -7) totaling 5% of all -7/del(7q) cases. Other mutated genes on 7q were: CUX1 in 1% del(7q) and 1% -7, CUL1 in 1% of del(7q), and LUC7L2 which was only found mutated in -7. Comparing the -7/del(7q) clone (calculated using allelic imbalance bio-analytic pipeline) to co-associated mutations allow us to identify the rank of -7/del(7q) in the clonal hierarchy: -7/del(7q) was an ancestral event in 58% and a secondary hit in 42% of the patients. When -7/del(7q) was the dominant hit, patients had fewer associated mutations, while in cases with secondary -7/del7q somatic mutations of TP53 (60%), IDH1/2 (30%), and DNMT3A (20%) were the ancestral hits suggesting a different clonal architecture according to -7/del(7q) clone size. Expression data was available for 49 of -7/del(7q) and 643 diploid cases for chr.7. Of the 694 informative genes on chr.7: 147 were deleted in all patients according to CNV analysis. We restricted the analysis to 15 uniformly HI genes selected because the expression inversely correlated with the level of 7q ploidy (ACTR3B, FASTK, GSTK1, IMPDH1, LRRC4, MEST, MGAM, NUP205, PRRT4, SLC37A3, SSBP1, TMEM209, TNPO3, ZC3HC1, ZNF277). Expression for these genes was then analyzed following normalization by -7/del7q clonal size. Unsupervised clustering grouped 72% of -7/del(7q) in one expression cluster. Classical -7/del(7q) signature genes fulfilled these criteria and with uniform HI in all cases. However, when we also included CUL1, CUX1, EZH2, KMT2C, and LUC7L2 to this analysis, the precision of -7/del(7q) clustering was 75% and defined a subcluster (#6). We were also able to identify cases previously misclassified (n=9) which were dispersed into 3 other clusters (14%, 8%, 3%) while 4% (n=23) of diploid cases were also misclassified and re-assigned into cluster 6. When we restricted the analysis to -7 cases only, a signature of 6 unique genes (C7orf43, CDK14, POLM, STAG3L5P, TRIM4) were found. Interestingly only POLM was on 7p, while the rest were on 7q22.1. In sum, we describe an integrated genomic and transcriptomic analysis of -7/del(7q) and we present data showing a complex heterogeneity of genes with HI expression which suggest that the clinical picture of patients carrying -7/del(7q) might be the result of a combination of clonality and HI. Disclosures Patel: Alexion: Other: educational speaker. Sekeres:Pfizer: Consultancy; Takeda/Millenium: Consultancy; BMS: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

No Improvement in Survival for T-PLL Patients over the Last Two Decades

BACKGROUND: T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, post thymic lymphoid malignancy, with an incidence of approximately 0.1/100,000 people. T-PLL accounts for ~2% of all mature lymphocytic leukaemias in adults >30 years. Median overall survival (OS) ~20 months and long term remissions are infrequent. Intravenous alemtuzumab, a monoclonal antibody directed against CD52 remains the most effective treatment in T-PLL. At the Royal Marsden Hospital (RMH) we have been using alemtuzumab for T-PLL since the early 1990s. We describe our experience over the last 3 decades. METHODS: We included 174 T-PLL patients that were diagnosed or treated at RMH between 1989-2019. Immunophenotyping data was available through our hematological malignancy diagnostic service (HMDS) from 1998 onwards. Kaplan-Meier analysis was used for OS and disease free interval (DFI) RESULTS: There were 174 patients in total. Mean age at diagnosis was 61 years old (range 32-88) and M: F ratio was ~2:1 (113:61). 90 patients had reliable information on complete blood count (CBC) at diagnosis with median white blood cell count of 74 x 109/L (range 10-918), median hemoglobin 126 g/L (range 59-175) and median platelet count 116 x 109/L (range 7-513). Sufficient immunophenotyping data was available for analysis in 135/174 patients. The results showed predictably high expression of CD2, CD3, CD5, CD7, CD52 and TCRαβ. CD25 was positive in 67/135 (50%) of cases which is higher than the 18-35% seen in the current literature. CD4+/CD8- cases comprised 83/135 (61%) with CD4-/CD8+ 19/135 (14%), CD4+/CD8+ 32/135 (23%) and CD4-/CD8- 2/135 (2%). These results are summarized in table 1. Karyotyping/FISH (fluorescent in-situ hybridization) records showed a clonal result in 84 patients and of these 65/84 (77%) had an aberration of chromosome 14. These comprised 48/65 with inv(14), 6/65 t(14;14), 2/65 t(X;14), 1/65 TCL1 gene rearrangement by FISH and 8/65 with TRA/TRD involvement by FISH. Other frequent abnormalities seen were i(8)(q10) in 31/84 (39%) and complex karyotypic abnormality in 40/84 (48%). Lower frequency abnormalities seen were monosomy 11 in 8/84 (9%), monosomy 12 in 8/84 (9%), trisomy 8 in 6/84 (7%), 17p loss in 5/84 (6%) and mononsomy 13 in 4/84 (5%). Alemtuzumab was used in 116 patients, 69/116 (59%) receiving it as frontline treatment and 47/116 (41%) as salvage therapy. In 50/116 (43%) alemtuzumab was used as single agent, the remaining 66/116 (57%) receiving combination therapy, mostly pentostatin. Mean time from diagnosis to receiving alemtuzumab was 6.8 months (range 0-53 months). Overall response rate (ORR) to alemtuzumab was 94/116 (81%) with complete remission (CR) 69/116 (59%), partial remission (PR) 25/116 (22%) and no response (NR) 20/116 (17%). Alemtuzumab produced better response rates when used as frontline therapy. Table 2 summarizes these results and OS and DFI are shown in figures B and C. Allogeneic stem cell transplant (allo-SCT) was performed in 34 patients. Median OS post allo-SCT was 22 months and median DFI 31 months. Relapse rate post allo-SCT was 47%, non-relapse mortality 38% and transplant related mortality 29%. Figures D and E show OS and DFI post allo-SCT. Although relapse rates are high post allo-SCT there is a small cohort of patients who are achieving long term remission. We analysed OS by decade of diagnosis covering 3 decades 1990-1999, 2000-2009 and 2010-2019. The median OS for the whole cohort was 20.6 months with median OS of 21 months, 23 months and 19 months for each decade respectively. Results are shown in figure A. There was no statistically significant difference between the curves by log-rank analysis. DISCUSSION: Immunophenotyping results were similar to previous published data on T-PLL except for an increase in CD25 expression at 50%. Our data shows that outcomes in T-PLL have remained unchanged since the first decade of alemtuzumab usage, with no improvement in OS in the last 20 years. Despite improvements in diagnostic techniques and supportive care median OS is static at approximately 20 months. These results highlight the need for novel therapies in T-PLL. Given the rarity of T-PLL, international, multi-center, randomised trials are needed to improve outcomes. Disclosures Cross: Royal Marsden Cancer Charity: Other: MD Residency, Research Funding. Iyengar:Abbvie: Honoraria; Janssen: Honoraria. Dearden:Janssen: Honoraria; Genentech: Honoraria; Abbvie: Honoraria; Sanofi: Honoraria.

Turner's Syndrome: Approach to Diagnosis and Treatment

Turner's syndrome is the most common karyotypic abnormality causing gonadal failure and primary amenorrhea. It is characterized by short stature and absence of secondary sexual characteristics. It is diagnosed by increased plasma FSH and LH level with low level of estrogen i.e. hypergonadotrophic hypogonadism. Ultrasound abdomen reveals streak ovaries and atrophic uterus. Karyotype confirms the diagnosis of Turner's syndrome (45XO). We present here a 15 years girl who presented with primary amenorrhea with short stature with breast development corresponds to Tanner stage I. Her FSH was raised. Ultrasound abdomen showed uterine agenesis and streak ovaries. Karyotype showed 45XO which confirmed the diagnosis of Turner's syndrome. She is now on estrogen therapy and her height has increased and breast development corresponds to Tanner stage II. Keywords: hypergonadotrophic hypogonadism, primary amenorrhea, Turner's syndrome

Epidemiology of Mylofibrosis from Three Different French Area Covered By a Population-Based Registry

Introduction: Myelofibrosis (MF) is one of the myeloproliferative neoplasms BCR-ABL1-negative. It is a rare disease, difficult to diagnose particularly with changes of the diagnosis criteria in the last twenty years. It is also difficult to treat MF because of lack of specific dug and/or validated therapeutic scheme. Scarce epidemiological data were available on MF and we used databases from three specialized registries in France to evaluated incidence rate, survival and treatment strategy used in the general population. Methods: Cases diagnosed with Myelofibrosis (ICD-O-3: 9961/3) in Gironde, Côte d'Or and Basse-Normandie covering 3 530 000 inhabitants, between 01/01/20014 and 12/31/2014 were registered. Data were collected from laboratories and clinical charts. The vital status was updated at 01/01/2017. Incidence, clinical characteristics, survival and treatment were studied using the STATA-V13 software. Results: They were 288 cases, 68% being male with a median age of 73 y-o. 65% of the cases were died at 5 years. 12% of the cases occurred after a previous solid tumor and 5% were secondary to a previous hemopathy. 62% had comorbidities at diagnosis in which 50% were cardiac conditions. At diagnosis, 83% had a splenomegaly; blast and bone marrow cells were present in peripheral blood in respectively 30% and 73%. Jak2 V617F was found in 88%, CALR in 8% and MPL mutation in 4% of the cases. The most frequent karyotypic abnormality was the del 20q (20%). IPSS score was calculated in 34% of the cases and 20% of them had a low risk score. 75% of cases were treated: 60% had hydroxycarbamide, 29% had ruxolutinib phosphate and 10% had thalidomide. Hydroxycarbamide was used in 48% of the cases in Basse Normandie, 68% in Côte d'Or and 64% in Gironde (p=0.001). EPO was used in 48% of cases and corticoids in a quarter of them. The transformation rate into acute myeloid leukemia was 25% in Côte d'Or, 12% in Gironde and 9% in Basse-Normandie (p=0.01). The 5 years observed survival was 25% and the 5-years net survival was 36%, without difference between male and female. It was 66% in cases under 55 y-o and was 25% in patients older than 75 y-o (p<0.001). The age-standardized 5-years survival was 50% in Basse Normandie, 42% in Côte d'Or and 37% in Gironde (ns). Conclusions: This study highlights biological and clinical characteristics of this rare disease in France. It also emphasizes some differences in the way of taking in charge these patients and in their survival rate. Even if it was not always statistically significant, patients from Basse Normandie appeared to receive less hydroxycarbazine, had a lower rate of AML transformation and had a slight better survival. This type of study should be extended to French cancer registries data for validation on a larger cohort and for guidance in patient management. Disclosures Troussard: Gilead: Other: scientific advisory board.

Effect of assisted reproductive technology on the molecular karyotype of missed abortion tissues

Missed abortion is one of the common complications of assisted reproductive technology (ART). Genetic abnormality is the most important factor. However, the effect of ART on the molecular karyotype of products of conception (POC) remains unknown. We explored the effect of ART on the molecular karyotype of POC in miscarriage. POC were obtained from women undergoing ART. Single nucleotide polymorphism (SNP) microarray was used to analyze the molecular karyotype. A total of 1493 POC were collected for SNP array analysis. The total rate of karyotypic abnormalities was 63.1% (943/1493). The proportion of karyotypic abnormalities was 70.4% (193/416) in >35-year-old group, which was significantly higher than that (60.6%) (343/566) in <30-year-old group and that (60%) (307/511) in the 30–35-year-old group. In natural conception (NC) group, the proportion of karyotypic abnormalities was 64.6% (201/311), whereas in ART group it was 62.7% (742/1182) and, there was no significant difference. The ratio between male and female fetuses was 1:1.13 (698/795). The rate of karyotypic abnormalities in male was 62.9% (439/698) and that in female was 63.4% (504/795), and these values did not differ significantly (P=0.84). Molecular karyotypic abnormality is the most important reason in miscarriage, and female age is a significant factor influencing the karyotypic abnormalities. Comparison with NC, ART, and gender of aborted embryos may not increase the rate of molecular karyotypic abnormality in miscarriage.

RECENT ADVANCES IN THE 5Q- SYNDROME

The 5q- syndrome is the most distinct of the myelodysplastic syndromes (MDS) and patients with this disorder have a deletion of chromosome 5q [del(5q)] as the sole karyotypic abnormality. Several genes mapping to the commonly deleted region of the 5q- syndrome have been implicated in disease pathogenesis in recent years. Haploinsufficiency of the ribosomal gene RPS14 has been shown to cause the erythroid defect in the 5q- syndrome. Loss of the microRNA genes miR-145 and miR-146a has been associated with the thrombocytosis observed in 5q- syndrome patients. Haploinsufficiency of CSNK1A1 leads to hematopoietic stem cell expansion in mice and may play a role in the initial clonal expansion in patients with 5q- syndrome. Moreover, a subset of patients harbor mutation of the remaining CSNK1A1 allele. Mouse models of the 5q- syndrome, which recapitulate the key features of the human disease, indicate that a p53-dependent mechanism underlies the pathophysiology of this disorder. Importantly, activation of p53 has been demonstrated in the human 5q- syndrome. Recurrent TP53 mutations have been associated with an increased risk of disease evolution and with decreased response to the drug lenalidomide in del(5q) MDS patients. Potential new therapeutic agents for del(5q) MDS include the translation enhancer L-leucine.

Hypoplastic Myelodysplastic Syndrome (h-MDS) Is a Distinctive Clinical Entity with Poorer Prognosis and Frequent Karyotypic and FISH Abnormalities Compared to Aplastic Anemia (AA).

Abstract 3199 Poster Board III-136 Differences between hypoplastic MDS (h-MDS) and aplastic anemia (AA) are not defined. Role of karyotype and fluorescent in situ hybridization (FISH) in these diseases is not established. Medical record review at Seoul National University Hospital between 1990 and 2008 was performed. Patients diagnosed as either h-MDS or AA based on morphology was reviewed. We assessed overall survival (OS) and leukemic conversion. 369 AA and 40 h-MDS patients (median age 39 years, range 15-82) were analyzed. 235 and 165 patients underwent karyotyping and FISH at diagnosis respectively. Compared to AA, karyotypic abnormality, 5q deletion, trisomy 8 and trisomy 1q FISH abnormalities were more frequently found in h-MDS. Median OS of h-MDS was shorter than AA (60 vs. 219 months, p<0.001) with prognosis of h-MDS falling between severe and very severe AA. Patients with h-MDS had more frequent leukemic conversion (p<0.001) than AA patients. Karyotypic abnormality was not prognostic in AA (p=0.225). For h-MDS, complex karyotype and trisomy 1q FISH abnormality predicted poor prognosis. The prognosis of h-MDS falls between severe and very severe AA. h-MDS accompanies frequent karyotypic and FISH abnormality and is prone to leukemic conversion. Complex karyotype and trisomy 1q FISH abnormality may have a prognostic role in h-MDS. Table 1 Characteristics of patients according morphologic classification in 409 patients Characteristics Aplastic anemia (N=369) Hypoplastic MDS (N=40) p-value Age (median) 38.0 49.5 0.005 Gender 0.846 Male 197 22 Female 172 18 White blood cell (mean) (/μL) 2724 3042 0.368 Reticulocyte count (mean) (%) 1.1 1.8 0.007 Hemoglobin (mean) (g/dL) 8.3 7.9 0.357 Platelet (mean) (/μL) 44130 109000 0.008 Severity of cytopenia* 0.007 Moderate 244 36 Severe 96 4 Very severe 28 0 PNH component NA Present 15 NA Absent 351 NA IPSS risk category NA Intermediate-1 NA 25 Intermediate-2 NA 4 High NA 1 Oxymetholone administration# 0.005 Yes 167 9 No 196 30 Immunosuppressive treatment# 0.005 Yes 114 4 No 249 35 Stem cell transplantation# 1.000 Yes 49 5 No 314 34 Figure 1 OS of patients according to disease subtype h-MDS had inferior OS compared to AA in general (A), and its prognosis falls between SAA and VSAA (B). h-MDS had shorter OS compared to AA even stratified by the degree of cytopenia. IPSS score had a prognostic impact on h-MDS (D). Figure 1. OS of patients according to disease subtype h-MDS had inferior OS compared to AA in general (A), and its prognosis falls between SAA and VSAA (B). h-MDS had shorter OS compared to AA even stratified by the degree of cytopenia. IPSS score had a prognostic impact on h-MDS (D). Disclosures No relevant conflicts of interest to declare.