Myelodysplastic Syndromes: A More Global 5-Hydroxymethylcytosine Deficiency Disorder Than Suggested By the Presence of TET2 Mutations

Abstract The high frequency of TET2 mutations in myelodysplastic syndromes (MDS) and the sole function of TET-dioxygenases as 5-hydroxymethylcytosine (5-hmC) hydroxylases emphasize the key role of this gene in disease pathogenesis. However, the broad down-regulation of 5-hmC argues for a role of DNA demethylation in MDS beyond TET2 lesions, which albeit the high frequency, do not convey any impact on survival outcomes. In fact, decrease in 5-hmC levels is by far more widely spread than TET2 lesions pointing towards other pathways affecting TET2 activity, thereby obscuring a precise determination of its mutational and clinical consequences. Herein, we investigated TETs expression to identify factors explaining the widespread deficiency of 5-hmC in MDS possibly determining clinical phenotypes and prognosis. An integrative data analysis of genomic studies (whole genome and deep targeted NGS), RNA-sequencing and 5-hmC quantification was performed on 1,665 patients with MDS and 91 healthy controls (HC). Meta-analytic studies of 5-hmC levels in myeloid neoplasia (n=598) and data of RNA-sequencing of fractionated CD34 (GSE63569) were also included as confirmatory cohorts. We started by analyzing the clinical impact of TET2 mutations carried by 23% of our study population. No impact on survival was found in carriers of TET2 lesions including those with biallelic, truncating or missense mutations compared to wild-type (WT) (Fig1A). By using 5-hmC levels as a functional readout of TET activity, we found a TET deficiency in about 70% of patients, a proportion higher than one would conclude by considering the mere presence of TET2 mutations (Fig1B). To explain the decrease in 5-hmC levels in WT cases, we next examined transcriptome modifications. Analysis of the expression of TET family of genes showed that MDS patients had lower TET2 mRNA levels in total and in CD34+ cells as compared to HC, irrespective of their TET2 status. Therefore, we reasoned that TET2 deficiency is more ubiquitously involved in MDS pathogenesis than what would be expected by the only estimation of mutant cases. Indeed, "low expressor" status (defined by TET2 expression < 25%ile of HC) was found in 74% of MDS. Along with variable 5-hmC levels, concomitant differences in TET1/TET3 expression were also investigated. While TET1 levels were too low to be evaluated, TET3 expression levels were markedly higher in all and in WT MDS compared to HC, possibly in an attempt to compensate TET2 dysfunction (Fig1C). In addition, TET3 expression did not correlate with TET2 mutational burden, confuting a compensatory feedback mechanism in TET2 mutant cases. Further uni- and multivariate analyses showed that elevated TET3 levels compensated TET2 deficiency in terms of clinical outcomes (Fig1D) and linear regression analyses confirmed that indeed lack of compensation by TET3 (low TET3 expression) was associated with high risk features. To explore whether other factors might be associated with low TET2 levels, we studied TET2 expression in WT cases as to the presence of other mutations. We found that TET2 expression was significantly lower in patients harboring DNMT3A (P< 0.0001), SF3B1 (P< 0.0001) and SRSF2 (P= 0.04) compared to HC. However, lack of correlation between levels of TET2 and mutational burden failed to prove a direct relationship of these mutations (Fig1E). Decreased hydroxylation of 5-mC may also be caused by endogenous L-2-hydroxyglutarate (L2HG) produced via malate shunt. Accordingly, L2HG dehydrogenase (L2HGDH) levels catabolizing L2HG and malate dehydrogenases (MDH1/2) supplying L2HG, would influence TET2 activity in a reciprocal fashion. Consistently we found that MDH1/2 levels were increased in MDS and that L2HGDH showed also a likely compensatory increase to handle elevated L2HG loads. Further, linear regression analyses revealed that L2HGDH levels were correlated inversely with TET2 and positively with TET3 expression in WT cases (Fig1F). In sum, MDS can be considered a wide-ranging 5-hmC deficiency disorder driven by direct or indirect loss of TET2functions by mutations or down-modulation due to a variety of mechanisms. Disease phenotypes and outcomes are both influenced by counteracting factors such as expression of TET3. Application of precision therapeutic approaches should be informed by the analyses of all these factors. Figure 1 Figure 1. Disclosures Carraway: Astex: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kim: Paladin: Consultancy, Honoraria, Research Funding; Bristol-Meier Squibb: Research Funding; Pfizer: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Minden: Astellas: Consultancy. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy.

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Immunogenetic Drivers in Post-Transplant Acute Myeloid Leukemia Recurrence: Genomic Dysfunction of Human Leukocyte Antigen Diversity

Blood ◽

10.1182/blood-2021-153577 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3921-3921

Author(s):

Simona Pagliuca ◽

Carmelo Gurnari ◽

Laila Terkawi ◽

Ishani Pandit ◽

Tariq Kewan ◽

...

Keyword(s):

T Cell ◽

Human Leukocyte Antigen ◽

Board Of Directors ◽

Research Funding ◽

Human Leukocyte ◽

Review Committee ◽

Advisory Committees ◽

Leukocyte Antigen ◽

Mutational Burden ◽

Independent Review

Abstract Structural and functional variability of human leukocyte antigen (HLA) is the foundation for competent anti-tumor and infectious adaptive immune responses. HLA genomic heterogeneity enables the presentation of a broad immune-peptidome, sustaining an efficient diversification of T cell receptor repertoires (TCR). 1,2,3 Any perturbation impacting this diversity may be at the basis of pathological processes, hampering antigen presentation capabilities and T-cell reactivity. In allogeneic hematopoietic cell transplantation (allo-HCT) setting, the graft versus leukemia (GvL) effect should ensure disease control allowing the recognition of recipient neoantigen burden by donor T-cell effectors. However, the molecular dissection of graft versus host responses (GvH) remains elusive. Herein, by means of a broad immunogenetic study of a cohort of patients with myeloid malignancies who received a donor matched allo-HCT, we investigated how dysfunction of HLA variability could have an impact on alloreactive responses, ultimately hindering disease control. To that end, we combined NGS-based HLA genotyping and TCR-beta sequencing to molecularly characterize the HLA region in terms of locus-specific divergence and somatic mutational profile, and dissect features and clonotypic spectra of TCR repertoires. We first hypothesized that more diverse HLA genotypes could better present leukemic neoantigen burden than less diverse complexes, enhancing the GvL effect. Hence, we performed a matched-pair analysis between allo-HCT recipients relapsing after 3mo (median 6.2 mo. [IQR=4.6-12]), N=75) compared to patients without recurrence (N=193, matched for ethnicity, age, disease, graft source and conditioning regimens) and characterized the patterns of HLA evolutionary divergence (HED), 1 a metric recently conceived to quantitate the pair-wise distance (based on physiochemical composition) between the amino acids located within the peptide-binding groove of two homologous HLA alleles. Overall, the relapsed group was characterized by a lower global (class I/II) mean HED (p=.0029) compared to non-relapsed patients, with major differences seen for C (p=.0041), DQB1 (p=.0291), and DPB1 (p=.0396) loci. When studying the landscape of post-transplant TCR reconstitution (+3 months) in a subset of 25 patients, we observed an inverse correlation between TCR clonal expansion and global HED (AdjR 2=0.04, p=<2e-16), contributing to decrease the diversity of TCR repertoires in patients with lower HED. Although not different in number, the expansion of clonotypes with known anti-cancer specificity was higher in non-relapsing group (p=6.3e-08), possibly underlying a better tumor-surveillance. Next, we sought to investigate the patterns of somatic HLA dysfunction in relapsing patients (intended as allelic loss or mutations). Indeed, through a recently implemented HLA mutational calling algorithm, we observed somatic events encompassing both class I and II alleles in 23% (N=8/34 profiled patients). Interestingly, when analyzing patients with relapse who received a donor lymphocyte infusion-based treatment (DLI), none of the cases harboring mutational events (N=4/4) responded to this salvage strategy. It is noteworthy that in this last group, one patient relapsed with an extramedullary localization along with the acquisition of HLA mutations. HLA mutated group had a higher (although not significant) leukemia mutational burden compared to non-mutated group (mean number of leukemia-associated mutations: 3.6 vs 1.9/patient), underscoring the need for further driver mutational events compensating the possible lower immunogenic potential of HLA mutant clones. Despite a mild increase in mutational burden, driver hits (such as IDH1/2, FLT3, TP53, NPM1) were never present in patients carrying HLA aberrations, who instead harbored in a few cases mainly lesions in epigenetic regulators and chromatin modifiers (TET2, EP300, DNMT3A, EZH2). Altogether these findings pinpoint the role of the dysfunction of the structural variability of HLA complexes within both germline (HED) and somatic (HLA loss/mutations) scenarios as mechanisms hampering a successful neoantigen presentation and TCR recovery processes, possibly conveying a higher risk of disease relapse or treatment-resistance. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Carraway: Takeda: Other: Independent review committee; AbbVie: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee. Hamilton: Syndax: Membership on an entity's Board of Directors or advisory committees; Equilium: Membership on an entity's Board of Directors or advisory committees. Majhail: Anthem, Inc: Consultancy; Incyte Corporation: Consultancy. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Novartis: Consultancy.

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Deficiency of RPS14 Beyond the Haploinsufficient Loss in Del(5q)

Blood ◽

10.1182/blood-2021-151926 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2591-2591

Author(s):

Vera Adema ◽

Sunisa Kongkiatkamon ◽

Laura Palomo ◽

Wencke Walter ◽

Stephan Hutter ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Linear Model ◽

Research Funding ◽

Review Committee ◽

Advisory Committees ◽

Tp53 Mutations ◽

Model Based ◽

Independent Review ◽

Cluster 2

Abstract The prevailing theory in del(5q) is that haploinsuffciency (HI) stemming from deletion and not simply LOH (loss of heterozygosity) is the culprit in clonal evolution. To date no haploinsufficient gene has been found to be the leukemogenic factor conveying growth advantage, but various other genes have been found to be important for phenotypic features or for propensity to acquire subsequent specific lesions. RPS14 is an example of such a gene, particularly in patients (pts) with isolated del(5q), responsible for macrocytic anemia and erythroid dysplasia and a propensity for acquisition of TP53 mutations. We hypothesized that RPS14 downmodulation and its consequences may be more common than del(5q) and it is frequent pathophysiologic feature in MDS. We first analyzed the genomic and expression profile of 170 pts with del(5q) and 825 diploid for 5q. We developed a new analytic pipeline to identify the most HI genes present in a large number of del(5q) pts. Genes within CDR (common deleted region) were classified as HI from the linear model fit if (i) clonality vs. gene expression slope from the isolated del(5q) was negative and FDR<.05; and (ii) effect of del(5q) at 50% clonality vs. other cases was negative and FDR<.05. A total of 62 genes met these criteria for linear-model based genes HI status, with a further 5 genes dropping due to low expression. Gene expression for these 57 HI genes among del(5q) samples was adjusted to 50%-clonality using the slopes from the estimated linear model to remove clonal heterogeneity. After applying model-based sparse clustering approach on all cohort, we obtained 7 clusters (Figure 1). As expected, del(5q) cases clustered together and showed consistent HI of 5q marker gene expression. Cluster-1 (n=146) included almost all del(5q) cases, except for 8 "mis-categorized" patients. It was characterized by low risk MDS (LR-MDS), presence of anemia/neutropenia and low mutational burden, with TP53 being the most commonly mutated gene and the only cluster with CSNK1A1 mutations. The remaining non-del(5q) patients were grouped in 6 clusters. Diploid cluster-2 (n=133) featured a normal karyotype, frequent ASXL1 and TET2 mutations, and profound down-modulation of RPS14 in all the patients included in the cluster (vs. other diploid pts). While the median RPS14 expression in cluster-1 (del(5q) cluster, with 50% adjusted clonality) was 7.29 (range 4.68-8.82 Log 2CPM), cluster-2 exhibited a median RPS14 expression of 6.12 Log 2CPM (range: 4.91-7.31 Log 2CPM). Clusters-3, -4, -5 (n=138, 90, 94, respectively) included most of the high risk MDS (HR-MDS). Cluster-3 was enriched for thrombocytopenia and SRSF2 mutations; cluster-4 for anemia, thrombocytopenia and ASXL1 and SRSF2 mutations. Cluster-5 was characterized by pancytopenia and frequent ASXL1 mutations and CK (complex karyotype). Cluster-6 (n=66) and -7 (n=233) contained the majority of non-del(5q) LR-MDS. When we analyzed the RPS14 expression in these clusters based on the RPS14 expression in cluster 2 we found 13% (n=18), 21% (n=19), 9% (n=8), 14% (n=9), 7% (n=16) of low RPS14 expressors in cluster-3, -4, -5, -6, -7, respectively. Cluster-2 showed a similar percentage of patients with anemia, and thrombocytopenia vs. Cluster-1 (69 vs. 50%, 23 vs. 30%; respectively). The mutational profile included a higher frequency of mutations for SRSF2 (29 vs. 0%), NRAS/KRAS (22% vs. 4%), ASXL1 (40 vs. 15%), TET2 (35 vs. 15%), and JAK2 (17 vs. 6%). These results indicate a more proliferative molecular spectrum of RPS14 downregulated cluster-2 than del(5q)-cluster-1, but RPS14 downmodulation did not lead to acquisition of TP53 mutations (4% vs. 76%). Considering all non-del(5q) RPS14 low expressors (n=186), only 3% of the cases had TP53 mutations. Since TP53 and CSNK1A1 mutations were characteristic of cluster-1 we studied interactions with HI RPS14. HI RPS14 in del(5q) and diploid low expressors showed a decreased expression of CDKN1A (P<.001) in comparison to the non-HI or low RPS14. We also found that CSNK1A1 mutations were not found outside of del(5q) pts, CSNK1A1 low expressors coincided with RPS14 low expressors. In conclusion, RPS14 expression defect is more widespread than del(5q) in MDS. However, only del(5q) RPS14 HI pts are prone to harbor TP53 and CSNK1A1 mutations; a group of diploid pts with low RPS14 and CSNK1A1 expressions might mimic some del5q features and could potentially respond to similar treatments. Figure 1 Figure 1. Disclosures Diez-Campelo: Takeda Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Carraway: AbbVie: Other: Independent review committee; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

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A Novel Machine Learning-Derived Molecular Classification Scheme with Prognostic Significance

Blood ◽

10.1182/blood-2021-152443 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3666-3666

Author(s):

Tariq Kewan ◽

Arda Durmaz ◽

Hassan Awada ◽

Carmelo Gurnari ◽

Waled Bahaj ◽

...

Keyword(s):

Machine Learning ◽

Board Of Directors ◽

Research Funding ◽

Prognostic Significance ◽

Molecular Classification ◽

Molecular Architecture ◽

Review Committee ◽

Advisory Committees ◽

Independent Review ◽

Normal Cytogenetics

Abstract The gold standard for the diagnosis of MDS relies on morphologic alterations hampered by a great deal of subjectivity. Cytogenetic and clinical features allow for clinical classifications predictive of survival. However, with a few exceptions (SF3B1MT, del5q, and certain balanced translocations), neither classic histo-morphology nor prognostic scoring systems (e.g., IPSS-R) are reflective of pathogenic underpinnings. To date supervised analyses of mutational data did not succeed to produce profiles specific or predictive of traditional disease sub-entities. Large cohorts with clinical annotation and a sufficient follow-up allow for innovative biostatistical approaches to subgroup patients according to molecular profiles. Objective operator-independent subcategorization may be congruent with common pathogenic links, rational applications of targeted therapeutics and better prognostications. We hypothesized that machine-learning (ML) strategies used for analysis of mutational/cytogenetic profiles will enable recognition of invariant disease subcategories according to their molecular configurations. Herein, we compiled a meta-analytic database (our cohorts and publicly available sources) of 3,011 MDS (median age 71yrs) and 6,788 pAML/sAML. Results of deep targeted sequencing of a panel of 55 myeloid mutations were collected together with cytogenetics. We then performed unsupervised analysis of MDS and AML patients using Bayesian Latent Class Analysis (BLCA). A consensus matrix was then clustered using Ward's criteria to generate final cluster assignment based on the highest silhouette value. To identify genomic signatures, we used Random Forest classification and extracted mutations with highest global importance indicated by mean decrease in accuracy. Using BLCA we identified 5 unique genomic clusters (GCs) with 3 distinct prognostic outcomes [low risk (LR), intermediate risk (Int), and high risk (HR)] that were validated by survival analysis (Fig.1A,B). The LR group included GC-1 and was characterized by the highest prevalence of normal cytogenetics (100%) and SF3B1 MT (25%) with co-occurring DNMT3A MT (14%), and absence of ASXL1 MT, ETV6 MT, STAG2 MT, TP53 MT, and complex/abnormal cytogenetics. Int group included GC-2 and GC-4. GC-2 was characterized by a higher percentage of abnormal cytogenetics cases than LR group and absence of STAG2 MT, SRSF2 MT, ASXL1 MT, TP53 MT, and normal/complex cytogenetics. GC-4 had the highest frequency of SRSF2 MT (52%) with co-occurring ASXL1 MT (59%), TET2 MT (40%), normal karyotype, and absence of complex/abnormal cytogenetics. Finally, HR included GC-3 and GC-5. GC-3 included ASXL1 MT (67%) with co-occurring SRSF2 MT (47%), TET2 MT (37%), STAG2 MT (22%), and absence of normal cytogenetics. GC- 5 had the highest frequency of -5/del(5q) (50%), -7/del(7q) (43%), -17/del(17p) (16%) and the highest odds of complex karyotype (92%) as well as TP53 MT (48%). Paralleling the genomic ML-based clustering, the clinical relevance of these subgroups was reflected in significantly different survivals [median (95% CI)]: i) GC-1 [69 (59-80)], ii) GC-2 [35 (29-41)], iii) GC-3 [12 (10-16)], GC-4 [27 (22-34)], and GC-5 [9 (7-11)] months (Fig.1C). We then classified the MDS cohort according to the recently published and validated AML GCs (Awada et al Blood 2021) to investigate overlapping genomic features. Overall, 90% of MDS GC-1 and 67% of MDS GC-2 had the same molecular architecture of AML GC-2 and 70% of MDS GC-5 had the same molecular features of AML GC-4. In addition, 98% of MDS GC-3 and 92% of MDS GC-4 had the same features of AML GC-3 (Fig.1D). In sum, we propose a novel objective molecular classification of MDS and related diseases that allows subgrouping of patients according to shared pathogenesis for a better prognostic resolution without errors derived from subjectivity. The model was then internally and externally validated using a cohort of 200 cases. Results of a validation cohort and online URL site of molecular clustering will be presented at the meeting. Figure 1 Figure 1. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Patel: Alexion: Consultancy, Other: educational talks, Speakers Bureau; Apellis: Consultancy, Other: educational talks, Speakers Bureau. Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astex: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

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Germline and Somatic Variants of CSF3R Define Distinct Myeloid Neoplasia Syndromes

Blood ◽

10.1182/blood-2021-152546 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3667-3667

Author(s):

Tariq Kewan ◽

Hrishikesh M Mehta ◽

Carmelo Gurnari ◽

Waled Bahaj ◽

Simona Pagliuca ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Extracellular Domain ◽

Review Committee ◽

Advisory Committees ◽

Intracellular Domain ◽

Secondary Aml ◽

Juxtamembrane Region ◽

Independent Review ◽

Myeloid Neoplasia

Abstract Somatic and germline (GL) variants of CSF3R are found in myeloid neoplasia (MN) and severe congenital neutropenia (SCN). In particular, somatic gain-of-function mutations in the juxtamembrane region of the receptor occur in chronic neutrophilic leukemia (CNL) or secondary AML. Another hotspot for somatic nonsense variants frequently mutated in these categories of pts involves the intracellular domain which regulates inhibitory growth pathways. We hypothesized that the somatic CSF3R variants could reveal previously unrecognized GL SCN mutations. When we studied a cohort of 2,610 pts with MN, we identified a total of 68 CSF3R variants (CSF3RMT). Using a bioanalytic pipeline, we assigned pathogenicity and type of origin (somatic vs. GL) to these variants, particularly those not previously described. In total, we found 32 GL (CSF3RGL) and 36 somatic (CSF3RS) mutations. Of the GL variants, 4 were previously described in pts with SCN consistent with heterozygous loss of function of the CSF3R gene. However, 15 additional alterations were located in similar regions and were predicted to be pathogenic while 13 variants were previously never described. Most of the CSF3RGL mutations were identified in pts with AML and MDS (88%). Interestingly, 2 (6%) pts had co-existing idiopathic neutropenia that progressed to secondary MDS. Another pt had aplastic anemia that eventually progressed to secondary AML. CSF3RGL were most often located in either the intracellular domain (44%) or the extracellular domain (34%) while none of the CSF3RGL mutations were found in the juxtamembrane region (Fig1). AML was detected in 21% of the pts with a CSF3RGL intracellular domain mutation and 18% of the pts with extracellular domain mutations. Of the germline missense variants, E808K (28%), R698C (9%), and E149D (9%) were the most frequently detected. Among the pts with E808K, 22% developed AML. The previously non-reported variants were detected in either the intracellular (50%) or the extracellular domain (50%). Missense variants were detected in 9/10 of the novel mutations in the following locations: L723V (20%), R428K (10%), G731R (10%), V406fs (10%), G687S (10%), P682H (10%), T154I (10%), and S413L (10%). One truncating mutation was found (c.1865-6delC) and it was located in intron 14 and has unknown impact on CSF3R function. Complex karyotype was noted in 19 % of the cases with CSF3RGL. DNMT3A (19%), NRAS (13%), FLT3 (9%), and BCOR (9%), were the most commonly found co-mutations. CSF3R S mutations were all heterozygous and found in 18 pts with AML and 18 pts with MDS and other MN. Overall, these lesions mapped within the intracellular proximal and distal domains (53%), the extracellular domain (14%) the juxtamembrane domain (25%), and the transmembrane domain (8%). Of note, MDS/MPN pts with CSF3RS mutations (11%) had lesions distributed between the intracellular, juxtamembrane and extracellular domains while none of the AML pts had mutations in the extracellular domain. Of all mutations, 36% were truncating events previously described in the context of post SCN AML while 61% were missense mutations. T618I was the most frequent CSF3RS detected (25%), followed by Q749X (11%), Q741X (9%), Q743X (6%). Juxtamembrane hits (CNL-like lesion) were all in the same canonical region (T618I). In contrast, somatic hits otherwise typical for post SCN AML were found in 33% of CSF3RS alterations and included the following: Q749X(4), Q741X (3), Q739X (2), S742X, Q743X, and E405K (not typical for post SCN AML). Taken together the combined allelic burden of these variants did not exceed that of general population (OR: 0.9503) suggesting that they are not significant risk alleles. Of note is that none of these variants were found to be in biallelic (somatic/GL) configurations. Complex karyotype was found in 19% of the pts with CSF3RS followed by del7q in 13% of cases. Importantly, an antecedent history of neutropenia was noted only in 14% of the pts carrying CSF3RS. Regarding associated mutations, ASXL1 (43%), RUNX1 (23%), SETBP1 (23%), TET2 (23%), DNMT3A (17%), SRSF2 (16%), EZH2 (14%), IDH2 (11%), and NRAS (11%) were the most common co-mutations. We have investigated CSF3RS mutations for the presence of GL alterations, but compound heterozygous configurations were not identified. We concluded that CSF3R mutations typically associated with SCN transformation to myeloid neoplasia can occur without GL variants associated with this defect. Figure 1 Figure 1. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Patel: Apellis: Consultancy, Other: educational talks, Speakers Bureau; Alexion: Consultancy, Other: educational talks, Speakers Bureau. Advani: Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Glycomimetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; OBI: Research Funding; Immunogen: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding. Carraway: AbbVie: Other: Independent review committee; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Astex: Other: Independent review committee; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Maciejewski: Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

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Genomic Landscape of PHD Finger Protein 6 (PHF6) Mutant Myeloid Neoplasia

Blood ◽

10.1182/blood-2021-153554 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1154-1154

Author(s):

Laila Terkawi ◽

Carmelo Gurnari ◽

Sunisa Kongkiatkamon ◽

Simona Pagliuca ◽

Minako Mori ◽

...

Keyword(s):

Transcriptional Regulation ◽

Board Of Directors ◽

Research Funding ◽

Binding Capacity ◽

Review Committee ◽

Advisory Committees ◽

Arginine Residues ◽

Independent Review ◽

Myeloid Neoplasia ◽

The Impact

Abstract Clinical impact and mechanistic contributions to leukemogenesis are difficult to assign to less common somatic mutations. However, the genetics of inherited syndromes can often be helpful in discerning the biological functions and mechanistic consequences of genes in other diseases. PHF6 (Xq26.2) encodes a protein consisting of two PHD-type zinc finger domains with activity in transcriptional regulation. PHF6 translocations were originally described in T-ALL and its mutations were later observed also in CML and adult AML. Germline (GL) PHF6MT cause Börjeson-Forssman-Lehmann syndrome (BFLS), an X-linked disorder characterized by intellectual disability and, to date, only a few BFLS cases were found to develop lymphoma or T-ALL. While regularly encountered in myeloid neoplasia (MN), the impact and functional meaning of PHF6 are not well established. To determine the incidence, distribution and molecular context of PHF6MT we studied a large cohort of patients with MN (n=8617) collected from our institution and public series. 1 Overall, 73% of patients were AML (pAML 69%; sAML 4%), MDS (22%) and MDS/MPN (5%) with a median age at diagnosis of 67 ys (18-100). We detected 149 patients (2%) carrying at least 1 PHF6MT with 11 harboring more than 1 hit. Four patients carried -X in addition to PHF6MT (2 males; 2 females). Majority of patients (68%) carried frameshift del/ins and nonsense. Mutations were scattered across all coding region with a slightly enrichment (47%) in the second PHD domain (239-330 aa) including the frequent R274Q/X (n=17). Common hits mainly affected arginine residues essential for DNA binding capacity (R129X n=9, R116X=7, R319X=5, R225X=3) followed by other hits (I314T=6, Y301X and C20fs=4 each). Of note, R116X, R225X, R274X, C280Y, H329R and Y303* lesions overlapped with the T-ALL PHF6MT spectrum while no overlap was found with GL mutations found in BFLS. Overall, 75% of all PHF6MT carriers were males and carried mostly (80%) truncating lesions. Compared to mutational frequencies observed in other X-linked genes, truncating PHF6MT behaved similarly to those in ZRSR2 (78%), STAG2 (73%) and BCOR (62%). Conversely, BCORL1MT, KDM6AMT and PIGAMT were evenly distributed between genders. When evaluating mutational characteristics in males and females, no differences were found in sex-adjusted median variant allelic burden of PHF6MT (54.8 vs 51%) nor its mRNA expression levels suggesting locus inactivation. PHF6MT tended to be older than PHF6WT patients (72 vs 68 ys; P= .05) and had mostly (63%) AML followed by MDS (23%) and MDS/MPN (14%). OS was similar between PHF6MT and PHF6WT patients (P= .16). Expression analyses showed that PHF6 loss leads to deregulation of chromatin and transcriptional factor genes. Indeed, in our cohort the most comutated genes were transcriptional factors and chromatin modifiers genes such as RUNX1 (26/149, 17%), ASXL1 (23/149, 15%) and TET2 (17/149, 11%). Of note, this group characterized by the triple ASXL1, RUNX1, TET2 mutational configuration clustered in one of the genomic groups previously identified (GC-3) 1 but the presence of these lesions did not worsen the OS as compared to PHF6MT without this mutational constellation. A low frequency of SF3B1MT (4%) was also noted confirming the enrichment of PHF6MT in AML rather than in low risk MDS. Further, 12% (14 males; 4 females) of PHF6MT patients had X-mutation mosaicism as shown by concomitant hits in BCOR (n=8), ZRSR2 (4), STAG2 (5), KDM6A (1). PHF6MT were equally founder lesions (30%; 44/149) and subclonal (34%; 50/149) whereas the rest was indistinguishable by VAF discrimination (co-dominant). The most common subclonal mutations were U2AF1 (14%, 6/44), IDH1/2 (9%, 4/44) and RUNX1 (7%, 3/44). When PHF6MT were subclonal, the founder hits were in TET2 (14%, 7/50), DNMT3A and RUNX1 (12%, each 6/50) genes. Given the high frequency of RUNX1MT in PHF6MT we investigated whether RUNX1 and PHF6 might be correlated. Transcriptomic analysis of 6246 patients (from 9 public studies) 2 showed a direct linear correlation (AdjR2= .03, P=5.55e-05) between the expression of the two genes. Our study is the largest to date to investigate the genetic landscape of PHF6MT in MN and highlights a strong connection of PHF6 with transcriptional regulation and chromatin genes. Ongoing scDNA-seq will clarify whether these mutations were acquired in distinct clones helping in dissecting the clonal hierarchy of PHF6MT cases. Disclosures Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Takeda: Other: Independent review committee; Astex: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Advani: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Glycomimetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Macrogenics: Research Funding; Immunogen: Research Funding; OBI: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding. Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

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The Drive to Acquire Biallelic Hits Inversely Correlates with the Functional Impact of the Primary TP53 Lesion: The Complexity of TP53 Role Assessment

Blood ◽

10.1182/blood-2021-152848 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3322-3322

Author(s):

Waled Bahaj ◽

Carmelo Gurnari ◽

Tariq Kewan ◽

Suresh Kumar Balasubramanian ◽

Simona Pagliuca ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Clinical Impact ◽

Dominant Negative ◽

Rapid Progression ◽

Compound Heterozygous ◽

Review Committee ◽

Advisory Committees ◽

Tp53 Mutations ◽

Independent Review

Abstract While the clinical impact and the mechanistic contribution of TP53 mutations have been a subject of intense research, many questions about its role in myeloid neoplasia (MN) remain unanswered. Previous molecular studies have confirmed the assertion that biallelic inactivation confers less favorable prognosis as opposed to monoallelic hits. These evidences agree with the observation that carriers of Li-Fraumeni syndrome do not always exhibit a complete penetrance of the recessive TP53 lesion. Thus, the presence of a residual function of TP53 appears to be somehow protective until it is offset by additional damage of contralateral allele or compound heterozygous hits in synergistic pro-leukemogenic pathways. TP53 can be affected by lesions of diverse configurations (e.g. biallelic, homo/hemizygous) targeting different locations [missense mutation (ms) in various hotspots vs truncations], and their assessment in terms of clinical consequences is complex. Only large cohorts of patients allow to discern the often discrete nuances of TP53 effects in individual inactivation patterns. We have compiled molecular and clinical data of a meta-analytic cohort (CCF and public datasets) of 1,011 patients with TP53 alterations, along with 3,419 cases found to be TP53 wild type (WT). A total of 1,258 TP53 mutations/deletions were found, 66% classified as biallelic and 37% as monoallelic hits (including single deletions). We investigated the closest hotspot ms mutations, hypothesizing that lesions mapping sequences in proximity will have the same phenotypic impact. Next, we arranged ms mutations into 6 main sites with each one containing lesions mapping within 5 amino acidic positions from the canonical hotspot location. These sites were mutated in 58% of patients with presence of truncating hits in 27% of cases. When ms mutation sites were compared to each other, a less dismal survival was observed for only the R175H hotspot (p .03). Most hotspots are known to exhibit dominant-negative effects (likely due to tetramer protein configurations) and thus, inhibit >50% of the TP53 activity as opposed to truncations which should inactivate ~50%. Consequently, one would expect that hotspot mutations produce a more aggressive phenotype. However, patients with ms hits had similar survival as those with truncating mutations (p=.6), likely because truncations were more often biallelic than ms mutation (81% vs 65%, p=.006). Indeed, we can stipulate that the strength (functional impairment) conveyed by a mutation will inversely correlate with the propensity to acquire biallelic hits. Therefore, we hypothesized that truncations (inactivating less TP53) would require an additional hit if compared to the stronger dominant-negative ms lesions. Notably, double hits were identified in 81% of cases carrying truncating mutations vs. 66% in those with ms canonical sites mutations (p<.009). Carriers of biallelic mutations had worse prognosis than those with monoallelic hits in adjusted multivariate analysis (HR 2.2 95% CI 1.8-2.7 p<.001). However, unlike in previous reports, in our large cohort containing several MN types, monoallelic hits were not survival neutral, but worsened the prognosis as compared to WT patients (p<.001). This finding implies a strong driver effect for TP53 lesions, which are characterized by a rapid progression even in the monoallelic configuration. Similarly, monoallelic hits were associated with a higher mutational burden compared to biallelic ones (1.22 vs 0.91 co-mutations/patient, p=.02), which likely compensated the need for further TP53 inactivation. When focusing on the accompanying genomic landscape of our cohort, we found that 45% of cases had TP53 mutations as the sole molecular lesion vs 55% of patients who also harbored co-occurring somatic events. In particular, complex karyotype was more frequent among patients without co-occurring mutations (79% vs 57%, p<.001). As of associations with disease subtypes, primary AML cases had a lower burden of co-mutations (p<.001) while the highest percentages were registered in LR-MDS (p=.005). In summary, our study demonstrates the complexity of assigning a correct clinical impact to TP53 mutations, which are characterized by a high degree of genomic heterogeneity. In addition to the genetic context, TP53 role may also vary in different subtypes of MN (e.g., AML vs MDS) shaping in a different fashion individual patients' trajectories. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Hamilton: Syndax: Membership on an entity's Board of Directors or advisory committees; Equilium: Membership on an entity's Board of Directors or advisory committees. Carraway: Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Takeda: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee. Maciejewski: Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

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Role of Remission Status and Prior Transplant in Optimizing Survival Outcomes Following Allogeneic Hematopoietic Stem Transplantation (HSCT) in Patients Who Received Inotuzumab Ozogamicin (INO) for Relapsed / Refractory (R/R) Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood.v130.suppl_1.886.886 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 886-886

Author(s):

Partow Kebriaei ◽

Matthias Stelljes ◽

Daniel J. DeAngelo ◽

Nicola Goekbuget ◽

Hagop M. Kantarjian ◽

...

Keyword(s):

Board Of Directors ◽

Survival Probability ◽

Research Funding ◽

Employment Equity ◽

Inotuzumab Ozogamicin ◽

Advisory Committees ◽

Allogeneic Hsct ◽

Post Transplant ◽

Equity Ownership

Abstract Introduction: Attaining complete remission (CR) prior to HSCT is associated with better outcomes post-HSCT. Inotuzumab ozogamicin (INO), an anti-CD22 antibody conjugated to calicheamicin, has shown significantly higher remission rates (CR/CRi and MRD negativity) compared with standard chemotherapy (SC) in patients (pts) with R/R ALL (Kantarjian et al. N Engl J Med. 2016). Pts treated with INO were more likely to proceed to HSCT than SC, which allowed for a higher 2-yr probability of overall survival (OS) than patients receiving SC (39% vs 29%). We investigated the role of prior transplant and proceeding directly to HSCT after attaining remission from INO administration as potential factors in determining post-HSCT survival to inform when best to use INO in R/R ALL patients. Methods: The analysis population consisted of R/R ALL pts who were enrolled and treated with INO and proceeded to allogeneic HSCT as part of two clinical trials: Study 1010 is a Phase 1/2 trial (NCT01363297), while Study 1022 is the pivotal randomized Phase 3 (NCT01564784) trial. Full details of methods for both studies have been previously published (DeAngelo et al. Blood Adv. 2017). All reference to OS pertains to post-HSCT survival defined as time from HSCT to death from any cause. Results: As of March 2016, out of 236 pts administered INO in the two studies (Study 1010, n=72; Study 1022, n=164), 101 (43%) proceeded to allogeneic HSCT and were included in this analysis. Median age was 37 y (range 20-71) with 55% males. The majority of pts received INO as first salvage treatment (62%) and 85% had no prior SCT. Most pts received matched HSCTs (related = 25%; unrelated = 45%) with peripheral blood as the predominant cell source (62%). The conditioning regimens were mainly myeloablative regimens (60%) and predominantly TBI-based (62%). Dual alkylators were used in 13% of pts, while thiotepa was used in 8%. The Figure shows post-transplant survival in the different INO populations: The median OS post-HSCT for all pts (n=101) who received INO and proceeded to HSCT was 9.2 mos with a 2-yr survival probability of 41% (95% confidence interval [CI] 31-51%). In patients with first HSCT (n=86) the median OS post-HSCT was 11.8 mos with a 2-yr survival probability of 46% (95% CI 35-56%). Of note, some patients lost CR while waiting for HSCT and had to receive additional treatments before proceeding to HSCT (n=28). Those pts who went directly to first HSCT after attaining remission with no intervening additional treatment (n=73) fared best, with median OS post-HSCT not reached with a 2-yr survival probability of 51% (95% CI 39-62%). In the latter group, 59/73 (80%) attained MRD negativity, and 49/73 (67%) were in first salvage therapy. Of note, the post-HSCT 100-day survival probability was similar among the 3 groups, as shown in the Table. Multivariate analyses using Cox regression modelling confirmed that MRD negativity during INO treatment and no prior HSCT were associated with lower risk of mortality post-HSCT. Other prognostic factors associated with worse OS included older age, higher baseline LDH, higher last bilirubin measurement prior to HSCT, and use of thiotepa. Veno-occlusive disease post-transplant was noted in 19 of the 101 pts who received INO. Conclusion: Administration of INO in R/R ALL pts followed with allogeneic HSCT provided the best long-term survival benefit among those who went directly to HSCT after attaining remission and had no prior HSCT. Disclosures DeAngelo: Glycomimetics: Research Funding; Incyte: Consultancy, Honoraria; Blueprint Medicines: Honoraria, Research Funding; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Shire: Honoraria; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; BMS: Consultancy; ARIAD: Consultancy, Research Funding; Immunogen: Honoraria, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding. Kantarjian: Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding. Advani: Takeda/ Millenium: Research Funding; Pfizer: Consultancy. Merchant: Pfizer: Consultancy, Research Funding. Stock: Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wang: Pfizer: Employment, Equity Ownership. Zhang: Pfizer: Employment, Equity Ownership. Loberiza: Pfizer: Employment, Equity Ownership. Vandendries: Pfizer: Employment, Equity Ownership. Marks: Pfizer: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau.

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Patterns of Clonal Evolution Assessed By Whole Exome Sequencing during Progression from MDS to AML Are Related to Therapy

Blood ◽

10.1182/blood.v128.22.4309.4309 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4309-4309

Author(s):

María Abáigar ◽

Jesús M Hernández-Sánchez ◽

David Tamborero ◽

Marta Martín-Izquierdo ◽

María Díez-Campelo ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Clonal Expansion ◽

Driver Mutations ◽

Advisory Committees ◽

Disease Evolution ◽

Mutational Burden ◽

Whole Exome ◽

Leukemic Phase

Abstract Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Impact of Interim FDG-PET (iPET) in the Outcomes of Patients with Aggressive B-Cell Lymphomas Receiving DA-EPOCH-R

Blood ◽

10.1182/blood-2019-122393 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2898-2898

Author(s):

Vania Phuoc ◽

Leidy Isenalumhe ◽

Hayder Saeed ◽

Celeste Bello ◽

Bijal Shah ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Fdg Pet ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Advisory Committees ◽

End Of Treatment

Introduction: 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) remains the standard of care for baseline and end of treatment scans for aggressive non-Hodgkin lymphomas (NHLs). However, the role of interim FDG-PET remains not as well defined across aggressive NHLs, especially in the era of high-intensity chemoimmunotherapy. Interim FDG-PET (iPET) can serve as an early prognostic tool, and prior studies evaluating the utility of iPET-guided treatment strategies primarily focused on diffuse large B-cell lymphomas (DLBCL) and frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Classification criteria systems assessing response also differ between studies with no clear consensus between use of Deauville criteria (DC), International Harmonization Project (IHP), and the ΔSUVmax method. Methods: This study evaluates our institutional experience with iPET during treatment with DA-EPOCH ± R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin with or without Rituximab) in aggressive NHLs. We retrospectively evaluated 70 patients at Moffitt Cancer Center who started on DA-EPOCH ± R between 1/1/2014 to 12/31/2018 for aggressive NHLs. Response on interim and end-of-treatment (EOT) scans were graded per DC, IHP, and ΔSUVmax methods, and progression free survival (PFS) probability estimates were calculated with chi-square testing and Kaplan Meier method. PFS outcomes were compared between interim negative and positive scans based on each scoring method. Outcomes were also compared between groups based on interim versus EOT positive or negative scans. Results: We identified 70 patients with aggressive NHLs who received DA-EPOCH ± R at our institute. The most common diagnoses were DLBCL (61%) followed by Burkitt's lymphoma (10%), primary mediastinal B-cell lymphoma (9%), plasmablastic lymphoma (7%), gray zone lymphoma (6%), primary cutaneous large B-cell lymphoma (1%), primary effusion lymphoma (1%), and other high-grade NHL not otherwise specified (3%). Of the 43 patients with DLBCL, 21/43 (49%) had double hit lymphoma (DHL) while 7/43 (16%) had triple hit lymphoma (THL), and 3/43 (7%) had MYC-rearranged DLBCL while 2/43 (5%) had double expressor DLBCL. Thirty nine out of 70 (56%) were female, and median age at diagnosis was 58.39 years (range 22.99 - 86.86 years). Most patients had stage IV disease (49/70, 70%), and 43/70 (61%) had more than one extranodal site while 45/70 (64%) had IPI score ≥ 3. Forty-six out of 70 (66%) received central nervous system prophylaxis, most with intrathecal chemotherapy (44/70, 63%). Fifty-five out of 70 (79%) had iPET available while 6/70 (9%) had interim computerized tomography (CT) scans. Fifty-six out of 70 (80%) had EOT PET, and 4/70 (6%) had EOT CT scans. Sustained complete remission occurred in 46/70 (66%) after frontline DA-EPOCH ± R (CR1), and 12/70 (17%) were primary refractory while 5/70 (7%) had relapse after CR1. Four of 70 (6%) died before cycle 3, and 3/70 (4%) did not have long-term follow-up due to transition of care elsewhere. Median follow-up was 15.29 months (range 0.85 - 60.09 months). There was significantly better PFS observed if iPET showed DC 1-3 compared to DC 4-5 (Χ2=5.707, p=0.0169), and PFS was better if iPET was negative by IHP criteria (Χ2=4.254, p=0.0392) or ΔSUVmax method (Χ2=6.411, p=0.0113). Comparing iPET to EOT PET, there was significantly better PFS if iPET was negative with EOT PET negative (iPET-/EOT-) compared to iPET positive with EOT negative (iPET+/EOT-), and iPET+/EOT+ and iPET-/EOT+ had worse PFS after iPET-/EOT- and iPET+/EOT- respectively. This pattern in iPET/EOT PFS probability remained consistent when comparing DC (Χ2=30.041, p<0.0001), IHP (Χ2=49.078, p<0.0001), and ΔSUVmax method (Χ2=9.126, p=0.0104). These findings fit clinical expectations with positive EOT scans indicating primary refractory disease. There was no significant difference in PFS when comparing DLBCL versus non-DLBCL (Χ2=3.461, p=0.0628) or DHL/THL versus non-DHL/THL diagnoses (Χ2=2.850, p=0.0914). Conclusion: Our findings indicate a prognostic role of iPET during treatment with DA-EPOCH ± R for aggressive NHLs. Significant differences in PFS were seen when graded by DC, IHP, and ΔSUVmax methods used in prior studies and when comparing interim versus EOT response. Larger studies are needed to confirm these findings. Disclosures Bello: Celgene: Speakers Bureau. Shah:Novartis: Honoraria; AstraZeneca: Honoraria; Spectrum/Astrotech: Honoraria; Adaptive Biotechnologies: Honoraria; Pharmacyclics: Honoraria; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria. Sokol:EUSA: Consultancy. Chavez:Janssen Pharmaceuticals, Inc.: Speakers Bureau; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.136.136 ◽

2011 ◽

Vol 118 (21) ◽

pp. 136-136

Author(s):

Ze Tian ◽

Jian-Jun Zhao ◽

Jianhong Lin ◽

Dharminder Chauhan ◽

Kenneth C. Anderson

Keyword(s):

Multiple Myeloma ◽

Tumor Suppressor ◽

Board Of Directors ◽

Expression Profiling ◽

Research Funding ◽

Advisory Committees ◽

Myeloma Cells ◽

Investigational Agent ◽

Reporter Activity

Abstract Abstract 136 Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells Ze Tian, Jianjun Zhao, Jianhong Lin, Dharminder Chauhan, Kenneth C. Anderson Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115 MicroRNAs (miRNAs) are 19–25 nucleotide-long noncoding RNA molecules that regulate gene expression both at the level of messenger RNA degradation and translation. Emerging evidence shows that miRNAs play a critical role in tumor pathogenesis by functioning as either oncogene or tumor suppressor genes. The role of miRNA and their regulation in response to proteasome inhibitors treatment in Multiple Myeloma (MM) is unclear. Here, we utilized MLN9708, a selective orally bio-available proteasome inhibitor to examine its effects on miRNA alterations in MM.1S MM cells. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to its biologically active form MLN2238. Our previous study using both in vitro and in vivo models showed that MLN2238 inhibits tumor growth and triggers apoptosis via activation of caspases. Moreover, MLN2238 triggered apoptosis in bortezomib-resistant MM cells, and induced synergistic anti-MM activity when combined with HDAC inhibitor SAHA, dexamethasone, and lenalidomide. In the current study, we treated MM.1S cells with MLN2238 (12 nM) for 3 hours and harvested; total RNA was subjected to miRNA profiling using TaqMan® Array Human miRNA A-Card Set v3.0 and the data was analyzed using dChip analysis. Results showed that MLN2238 modulates miRNA expression with a total of 36 miRNA changing their expression profiling (δδCT>1.5 or δδCT <-1.5; 19 were upregulated and 17 showed a downregulation). Among all miRNA, miR-33b was highly (δδCT>7) upregulated in response to MLN2238 treatment. We therefore hypothesized that miR-33b may play a role in MM pathogenesis as well as during MLN2238-induced proteasome inhibition in MM cells. We first utilized quantitative polymerase chain reaction (q-PCR) to validate the changes in miRNA expression profiling. Results confirmed that MLN2238 treatment triggers significant increase in the miR-33b expression in MM.1S cells (2.1 and 2.2 folds at 3h and 6h, respectively; P<0.001). Examination of normal PBMCs and plasma cells showed higher expression of miR-33b than patient MM cells (P<0.001). We further investigated the functional role of miR-33b in MM cells at baseline and during MLN2238 treatment. Drug sensitivity, cell viability, apoptosis, colony formation, and migration assays were performed using cell TilTer-Glo, Annexin V-FITC/PI staining, MTT staining, and Transwell assays, respectively. Signaling pathways modulated post miR-33b overexpression were evaluated by q-PCR, immunoblot, and reporter assays. Our findings show that overexpression of miR-33b significantly decreased cell viability, cell migration, colony formation, as well as increased apoptosis and sensitivity of MM cells to MLN2238 treatment. Targetscan analysis predicted pim-1 as a putative downstream target of miR-33b. Overexpression of miR-33b downregulated pim-1 mRNA and protein expression. To further corroborate these data, we co-tranfected miR-33b and Pim-1-wt or Pim-1-mt in 293T and MM.1S cell lines. In concert with our earlier findings, miR-33b decreases pim-1-wt, but not pim-1-mt reporter activity in both cell lines. Reflecting the overexpression study results, MLN2238 treatment also decreases pim-1-wt, but not pim1-mt reporter activity. Moreover, a biochemical inhibitor of pim1/2 triggered apoptosis in MM cells. Finally, overexpression of miR-33b inhibits tumor growth (P<0.001) and prolongs survival (P<0.001) in both subcutaneous and disseminated human MM xenograft models. In summary, our study suggests that miR-33b is a tumor suppressor, which plays a role during MLN2238-induced apoptotic signaling in MM cells, and provide the basis for novel therapeutic strategies targeting miR-33b in MM. Disclosures: Anderson: Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership.

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