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

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

Blood ◽

10.1182/blood-2021-145949 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3665-3665

Author(s):

Carmelo Gurnari ◽

Simona Pagliuca ◽

Yihong Guan ◽

Courtney E. Hershberger ◽

Ying Ni ◽

...

Keyword(s):

Board Of Directors ◽

High Frequency ◽

Research Funding ◽

Regression Analyses ◽

Review Committee ◽

Advisory Committees ◽

Mutational Burden ◽

Impact On Survival ◽

Independent Review

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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Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽

10.1182/blood-2020-136172 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Hanyin Wang ◽

Shulan Tian ◽

Qing Zhao ◽

Wendy Blumenschein ◽

Jennifer H. Yearley ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Activation ◽

Expression Profiles ◽

Lymphocytic Leukemia ◽

B Cell Activation ◽

Advisory Committees ◽

Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

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Multicenter Analysis of Treatment and Outcomes for Patient with TP53 Mutated AML in the Era of Novel Therapies; Significant Impact of Allogeneic Stem Cell Transplantation on Survival

Blood ◽

10.1182/blood-2021-147639 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 797-797

Author(s):

Talha Badar ◽

Mark R. Litzow ◽

Rory M. Shallis ◽

Jan Philipp Bewersdorf ◽

Antoine Saliba ◽

...

Keyword(s):

Stem Cell ◽

High Risk ◽

Stem Cell Transplantation ◽

Board Of Directors ◽

Research Funding ◽

Univariate Analysis ◽

Advisory Board ◽

Novel Therapies ◽

Advisory Committees ◽

Tp53 Mutations

Abstract Background: TP53 mutations occur in 10-20% of patients with AML, constitute high-risk disease as per ELN criteria, and confer poorer prognosis. Venetoclax combination therapies and CPX-351 were recently approved for AML treatment and lead to improved outcomes in subsets of high-risk AML, however the most effective approach for treatment of TP53-mutated (m) AML remains unclear. In this study we explored the clinical outcome of TP53m AML patients treated over the last 8 years as novel therapies have been introduced to our therapeutic armamentarium. Methods: We conducted a multicenter observational study in collaboration with 4 U.S. academic centers and analyzed clinical characteristics and outcome of 174 TP53m AML patients diagnosed between March 2013 and February 2021. Mutation analysis was performed on bone marrow specimens using 42, 49, 199, or 400 gene targeted next generation sequencing (NGS) panels. Patients with an initial diagnosis of AML were divided into 4 groups (GP) based on the progressive use of novel therapies in clinical trials and their approvals as AML induction therapy during different time periods: 2013-2017 (GP1, n= 37), 2018-2019 (GP2, n= 53), 2019-2020 (GP3, n= 48) and 2020-2021 (GP4, n= 36) to analyze difference in outcome. Results: Baseline characteristics were not significantly different across different GP, as shown in Table 1. Median age of patients was 68 (range [R], 18-83), 65 (R, 29-88), 69 (R, 37-90) and 70 (R, 51-97) years in GP1-4, respectively (p=0.40). The percentage of patients with de novo AML/secondary AML/therapy-related AML in GP1-4 was 40/40/20, 36/29/24, 37.5/37.5/25 and 28/52/20, respectively (p=0.82). The proportion of patients with complex cytogenetics (CG) was 92%, 89%, 96% and 94% in GP1-4, respectively (p=0.54). The median TP53m variant allele frequency (VAF) was 48% (range [R], 5-94), 42% (R, 5-91), 45% (R, 10-94) and 60% (R, 8-82) in GP1-4, respectively (p=0.38). Four (11%), 13 (24.5%), 10 (21%) and 9 (25%) patients had multiple TP53 mutations in GP1-4, respectively (p=0.33). The proportion of patients who received 3+7 (30%, 16%, 6% & 8%; p=0.01), HMA only (11%, 18%, 2% & 8%; p=0.06), venetoclax-based (2.5%, 12%, 48%, & 61%; p <0.01) and CPX-351 induction (16%, 40%, 28% & 5%; p<0.001) were varied in GP1-4, respectively. The rate of CR/CRi was 22%, 26%, 28% and 18% in GP1-4, respectively (p=0.63). Treatment related mortality during induction was observed in 3%, 7%, 10% and 17% of patients in GP1-4, respectively (p=0.18). Overall, 28 (16%) patients received allogeneic hematopoietic stem cell transplantation (alloHCT) after induction/consolidation: 22%, 15%, 17% and 11% in GP1-4, respectively (p=0.67). In subset analysis, there was no difference in the rate of CR/CRi with venetoclax-based regimens vs. others (39% vs 61%, p=0.18) or with CPX-351 vs. others (25% vs 75%, p=0.84). The median progression-free survival was 7.7, 7.0, 5.1 and 6.6 months in GP1-4, respectively (p=0.60, Fig 1A). The median overall survival (OS) was 9.4, 6.1, 4.0 and 8.0 months in GP1-4, respectively (p=0.29, Fig 1B). In univariate analysis for OS, achievement of CR/CRi (p<0.001) and alloHCT in CR1 (p<0.001) associated with favorable outcome, whereas complex CG (p=0.01) and primary refractory disease (p<0.001) associated with poor outcome. Multiple TP53 mutations (p=0.73), concurrent ASXL1m (p=0.86), extra-medullary disease (p=0.92), ≥ 3 non-TP53m mutations (p=0.72), TP53m VAF ≥ 40% vs. < 40% (p=0.25), induction with CPX-351 vs. others (p=0.59) or venetoclax-based regimen vs. others (p=0.14) did not show significance for favorable or poor OS in univariate analysis. In multivariable analysis, alloHCT in CR1 (hazard ratio [HR]=0.28, 95% CI: 0.15-0.53; p=0.001) retained an association with favorable OS and complex CG (HR 4.23, 95%CI: 1.79-10.0; p=0.001) retained an association with dismal OS. Conclusion: We present the largest experience with TP53m AML patients analyzed by NGS. Although outcomes were almost universally dismal, alloHCT appears to improve the long-term survival in a subset of these patients. Effective therapies are warranted to successfully bridge patients to alloHCT and to prolong survival for transplant ineligible patients. Figure 1 Figure 1. Disclosures Badar: Pfizer Hematology-Oncology: Membership on an entity's Board of Directors or advisory committees. Litzow: Omeros: Other: Advisory Board; Pluristem: Research Funding; Actinium: Research Funding; Amgen: Research Funding; Jazz: Other: Advisory Board; AbbVie: Research Funding; Astellas: Research Funding; Biosight: Other: Data monitoring committee. Shallis: Curis: Divested equity in a private or publicly-traded company in the past 24 months. Goldberg: Celularity: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aprea: Research Funding; Arog: Research Funding; DAVA Oncology: Honoraria; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Prelude Therapeutics: Research Funding; Aptose: Consultancy, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Atallah: BMS: Honoraria, Speakers Bureau; Takeda: Consultancy, Research Funding; Amgen: Consultancy; Abbvie: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Research Funding. Foran: revolution medicine: Honoraria; gamida: Honoraria; bms: Honoraria; pfizer: Honoraria; novartis: Honoraria; takeda: Research Funding; kura: Research Funding; h3bioscience: Research Funding; OncLive: Honoraria; servier: Honoraria; aptose: Research Funding; actinium: Research Funding; abbvie: Research Funding; trillium: Research Funding; sanofi aventis: Honoraria; certara: Honoraria; syros: Honoraria; taiho: Honoraria; boehringer ingelheim: Research Funding; aprea: Research Funding; sellas: Research Funding; stemline: Research Funding.

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Dose-Adjusted EPOCH and Rituximab (DA-EPOCH-R) Treatment in Dual Expressor Diffuse Large B-Cell and Double/Triple Hit Lymphomas: TP53 Mutations Influence on Clinical Outcome

Blood ◽

10.1182/blood-2019-124350 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4116-4116

Author(s):

Anna Dodero ◽

Anna Guidetti ◽

Fabrizio Marino ◽

Cristiana Carniti ◽

Stefania Banfi ◽

...

Keyword(s):

High Risk ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Poor Prognosis ◽

Tp53 Mutation ◽

Advisory Committees ◽

Tp53 Mutations ◽

Travel Costs ◽

Large B Cell

Introduction: Diffuse Large B-Cell Lymphoma (DLBCL) is an heterogeneous disease: 30-40% of cases have high expression of MYC and BCL2 proteins (Dual Expressor, DE) and 5-10% have chromosomal rearrangements involving MYC, BCL2 and/or BCL6 (Double-/ Triple-Hit, DH/TH). Although the optimal treatment for those high-risk lymphomas remains undefined, DA-EPOCH-R produces durable remission with acceptable toxicity (Dunleauvy K, Lancet 2018). TP53 mutation is an independent marker of poor prognosis in patients (pts) with DLBCL treated with R-CHOP therapy. However, its prognostic value in poor prognosis lymphomas, receiving intensive therapy, has not been investigated yet. Methods: A series of consecutive pts (n=87) with biopsy proven diagnosis of DE DLBCL (MYC expression ≥40% and BCL2 expression ≥ 50% of tumor cells) or DE-Single Hit (DE-SH, i.e., DE-DLBCL with a single rearrangement of either MYC, BCL2 or BCL6 oncogenes) or DE-DH/TH (MYC, BCL2 and/or BCL6 rearrangements obtained by FISH) were treated with 6 cycles of DA-EPOCH-R and central nervous system (CNS) prophylaxis consisting of two courses of high-dose intravenous Methotrexate. Additional eligibility criteria included age ≥18 years and adequate organ functions. Cell of origin (COO) was defined according to Hans algorithm [germinal center B cell like (GCB) and non GCB)]. TP3 mutations were evaluated by next generation sequencing (NGS) based on AmpliseqTM technology or Sanger sequencing and considered positive when a variant allelic frequency ≥10% was detected. Results: Eighty-seven pts were included [n=36 DE only, n=32 DE-SH (n=8 MYC, n=10 BCL2, n=14 BCL6), n=19 DE-DH/TH] with 40 patients (46%) showing a non GCB COO. Pts had a median age of 59 years (range, 24-79 years). Seventy-three pts (84%) had advanced disease and 44 (50%) an high-intermediate/high-risk score as defined by International Prognostic Index (IPI). Only 8 of 87 pts (9%) were consolidated in first clinical remission with autologous stem cell transplantation following DA-EPOCH-R. After a median follow-up of 24 months, 73 are alive (84%) and 14 died [n=12 disease (n=2 CNS disease); n=1 pneumonia; n=1 suicide]. The 2-year PFS and OS were 71% (95%CI, 60-80%) and 76% (95%CI, 61%-85%) for the entire population. For those with IPI 3-5 the PFS and OS were not significant different for DE and DE-SH pts versus DE-DH/TH pts [64% vs 57% p=0.77); 78% vs 57% p=0.12)]. The COO did not influence the outcome for DE only and DE-SH [PFS: 78% vs 71% (p=0.71); 92% vs 86% (p=0.16) for GCB vs non -GCB, respectively]. Fourty-six pts (53%;n=18 DE only, n=18 DE-SH, n=10 DE-DH/TH ) were evaluated for TP53 mutations with 11 pts (24%) carrying a clonal mutation (n=6 in DE, n=3 in DE-SH, n=2 in DE-DH/TH). The 2-year PFS and OS did not significantly change for pts DE and DE-SH TP53 wild type as compared to DE and DE-SH mutated [PFS: 84 % vs 77%, (p=0.45); OS: 87% vs 88%, (p=0.92)]. The two pts DE-DH/TH with TP53 mutation are alive and in complete remission.Conclusions: High risk DLBCL pts treated with DA-EPOCH-R have a favourable outcome independently from high IPI score, DE-SH and DE-DH/TH. Also the presence of TP53 mutations does not negatively affect the outcome of pts treated with this intensive regimen. The efficacy of DA-EPOCH-R in overcoming poor prognostic genetic features in DLBCL should be confirmed in a larger prospective clinical trial. Disclosures Rossi: Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria. Carlo-Stella:Takeda: Other: Travel, accommodations; F. Hoffmann-La Roche Ltd: Honoraria, Other: Travel, accommodations, Research Funding; Rhizen Pharmaceuticals: Research Funding; Celgene: Research Funding; Amgen: Honoraria; AstraZeneca: Honoraria; Janssen Oncology: Honoraria; MSD: Honoraria; BMS: Honoraria; Genenta Science srl: Consultancy; Janssen: Other: Travel, accommodations; Servier: Consultancy, Honoraria, Other: Travel, accommodations; Sanofi: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Other: Travel, accommodations, Research Funding; Novartis: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy. Corradini:AbbVie: Consultancy, Honoraria, Other: Travel Costs; KiowaKirin: Honoraria; Gilead: Honoraria, Other: Travel Costs; Amgen: Honoraria; Celgene: Honoraria, Other: Travel Costs; Daiichi Sankyo: Honoraria; Janssen: Honoraria, Other: Travel Costs; Jazz Pharmaceutics: Honoraria; Kite: Honoraria; Novartis: Honoraria, Other: Travel Costs; Roche: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Other: Travel Costs; Servier: Honoraria; BMS: Other: Travel Costs.

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DYRK1A Is Regulated By Oncogenic KMT2A and Required for Survival of KMT2A-Rearranged Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-123475 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2742-2742

Author(s):

Christian Hurtz ◽

Gerald Wertheim ◽

Rahul S. Bhansali ◽

Anne Lehman ◽

Grace Jeschke ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Negative Regulator ◽

Advisory Committees ◽

Normal Tissues ◽

Pharmacologic Inhibition

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

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