Bhatia, Ravi

Chronic myeloid leukemia (CML) is defined by the presence of Philadelphia chromosome resulting from a reciprocal translocation between chromosomes 9 and 22 [t9;22] that gives rise to a BCR::ABL1 fusion gene. CML occurs in 3 different phases (chronic, accelerated, and blast phase) and is usually diagnosed in the chronic phase in developed countries. Tyrosine kinase inhibitor (TKI) therapy is a highly effective treatment option for patients with chronic phase–CML. The primary goal of TKI therapy in patients with chronic phase–CML is to prevent disease progression to accelerated phase–CML or blast phase–CML. Discontinuation of TKI therapy with careful monitoring is feasible in selected patients. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with chronic phase–CML.

Chronic myeloid leukemia (CML) is defined by the presence of Philadelphia chromosome (Ph) which results from a reciprocal translocation between chromosomes 9 and 22 [t(9;22] that gives rise to a BCR-ABL1 fusion gene. CML occurs in 3 different phases (chronic, accelerated, and blast phase) and is usually diagnosed in the chronic phase. Tyrosine kinase inhibitor therapy is a highly effective first-line treatment option for all patients with newly diagnosed chronic phase CML. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with chronic phase CML.

Abstract Background Recent genome wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified several associations at both HLA and non-HLA loci. However, much of HL heritability remains unexplained. Methods To identify novel risk loci, we performed a meta-analysis of 3 HL GWAS including a total of 1,810 cases and 7,879 controls. Results were replicated in an independent set of 1,163 cases and 2,580 controls, for a total of 3,097 and 11,097 cases and controls combined, respectively. participants in discovery and replication stages were of European descent. quality control and imputation we conducted a meta-analysis addressing 1,004,829 variants (λ= 1.10, λ1000= 1.03). Associations between SNP genotypes and HL risk were evaluated under a log-additive model of inheritance adjusting for sex, study center and significant principal components to control for population stratification. We performed an analysis with all HL cases and then conducted stratified analyses by histological subtype (classical, nodular sclerosis and mixed cellularity), age at diagnosis (nodular sclerosis among those diagnosed at 15- 35 years in all studies, and those diagnosed at 35 and older years in the European Study only) and EBV tumor status (negative and positive). We then used a bioinformatic approach (FunciSNP) to identify potential functional variants associated with HD risk correlated with risk loci of interest. We extracted publically available ENCODE data on biofeatures to identify potential functional motifs associated with the index SNP or correlated SNPs. Finally, we measured expression levels of the two alternative mRNA transcripts in lymphoblastoid cell lines (LCLs) from 49 post-therapy HL patients and 25 unaffected controls. RT-PCR was carried out in triplicate. Relative expression levels were calculated relative to TBP as housekeeping gene. Linear models were used to assess correlation between genotype and TCF3expression levels. Results The meta-analysis identified a novel susceptibility variant at chromosome 19p13.3 (rs1860661) associated with HL risk (Odds Ratio [OR]= 0.78, P=2.0*10-8, I2=0%). variant is located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment. was also significantly associated with HL (OR= 0.85, P=0.002) in the replication series of 1,281 cases and 3,218 controls. the combined analysis consisting of the discovery and replication sets, rs1860661was strongly associated with HL (OR=0.81,=3.5*10-10), with no evidence of heterogeneity between contributing studies (Phom=0.41, I2=0%). The number of G alleles defined by rs1860661 was significantly associated with a reduced risk of each HL subtype except EBV positive HL. rs1860661 and two correlated SNPs, rs10413888 (r2=0.90) and rs8103453 (r2=0.89) identified by FunciSNP analysis map in or near marks of open chromatin and in DNAse hypersensitivity sites in TCF3 in CD20+ B cell lines., the protective minor alleles of these SNPs as defined by the G-G-G haplotype map to the binding sites for ZBTB7a (rs10413888 and rs1860661) and (rs8103453) transcription factors, likely improving the binding efficiency to the sites which may result in increased transcription rates of TCF3. TCF3 is encoded by two alternative transcripts (E12 and E47). Higher expression levels of TCF3-E47, whose transcription start site is located close to rs1860661, was associated with the rs1860661-G allele in controls (P=0.02), but not in HL patients (P=0.22). Conclusion/Discussion TCF3 is essential for the commitment of lymphoid progenitors to both B-cell and T-cell lineage development. A molecular and phenotypic hallmark of classical HL is the loss of the B-cell phenotype in HRS cells, including lack of demonstrable B-cell receptor and most B-cell specific markers such as CD19 or CD20. HRS cells have a low level of TCF3, particularly homodimers of the isoform E47, due to expression of the ABF-1 and ID2 inhibitors that bind to TCF3. Thus, higher TCF3 levels in HRS precursor cells may lead to enhanced retention of the B cell phenotype, thereby conferring a protective effect. These data suggest a link between the 19p13.3 locus including TCF3 and HL risk, indicating that TCF3 could be relevant to HL etiology and pathogenesis. Disclosures: Link: Genentech: Consultancy; Millenium: Consultancy; Pharmacyclics: Consultancy; Spectrum: Consultancy.

Abstract Introduction B progenitor acute lymphoblastic leukemia (B-ALL) is a leading cause of childhood cancer death. Many chimeric genes have been identified and led to a refined classification of B-ALL and tailored therapies. Still, up to 30% of B-ALL cannot be classified into established subtypes, and the outcome for many is poor. Methods To identify novel subtypes of B-ALL, we performed integrative genomic analysis including transcriptome sequencing (RNA-seq) of 1,988 cases from St. Jude, Children's Oncology Group and adult cooperative group studies and analyzed chromosomal rearrangements, gene-expression profiles (GEP), somatic mutations and chromosome-level copy-number alterations. Cases lacking known or putative subtype-defining alterations underwent whole genome sequencing. Effects on proliferation and transformation of novel lesions were assessed by retroviral expression in cell lines and point-mutation knock-in mice using CRISPR/Cas9 genome editing. Results Using integrated genetic alterations and gene expression profiling, we classified 23 B-ALL subtypes (Table and Figure). Three groups included cases with similar GEP as canonical subtypes (ETV6-RUNX1, KMT2A-rearranged, and ZNF384-rearranged), but lacking the expected drivers (e.g., ETV6-RUNX1-like, n=42). Eighteen cases (0.9%) had rearrangements of BCL2, MYC and/or BCL6 showing a distinct GEP; they were mostly adults (n=16) with very poor outcome. These alterations, rarely seen in ALL, are identical to those observed in "double/triple hit" lymphoma, and are of pre-B immunophenotype. Eight cases with tightly clustered GEP comprised a novel subtype defined by IKZF1 N159Y missense mutation. N159Y is in the DNA-binding domain of IKZF1, and is known to perturb IKZF1 function, with distinct nuclear mislocalization and induction of aberrant intercellular adhesion. We identified two subtypes with distinct GEP characterized by PAX5 alterations. One, herein termed PAX5 altered (PAX5alt), comprised 148 (7.4%) cases, was characterized by diverse PAX5 alterations including rearrangements (n=57), sequence mutations (n=46) and/or focal intragenic amplifications (n=8). These PAX5 alterations were found in 73.6% of PAX5alt cases and different alteration types were mutually exclusive. Other PAX5 alterations, including deletions and large-scale amplifications were also assessed using SNP array, but were not enriched in the PAX5alt group. Clinically, PAX5alt pediatric and adult patients had favorable (96.8±3.2%) and intermediate (42.1±10.2%) 5-year overall survival (OS), respectively. The other GEP distinct subtype comprised 44 cases, all with PAX5 P80R missense mutations. In 30 of these cases, PAX5 P80R was homozygous due to deletion of the wild-type (WT) PAX5 allele or copy-neutral loss of heterozygosity. Of the other 14 cases with heterozygous PAX5 P80R mutations, 13 had a second frameshift (n=7), nonsense (n=2) or deleterious missense (n=4) PAX5 mutation. Four of the remaining 1,944 cases also had the PAX5 P80R mutation, but all were heterozygous with preservation of a WT PAX5 allele, consistent with the notion that homozygous or compound heterozygous PAX5 P80R mutation is the hallmark of this subtype. Adult PAX5 P80R cases (n=14) showed better 5-year OS (61.9±13.4%) than those in PAX5alt subtype (42.1±10.2%). To examine the effects of PAX5 P80R on B-cell maturation, WT PAX5, PAX5 P80R, V26G and P34Q were expressed in Pax5-/- lineage-depleted bone marrow cells. Expression of WT PAX5, PAX5 V26G and P34Q resulted in near complete rescue of B cell differentiation; however, expression of PAX5 P80R blocked the differentiation at the pre-pro-B stage of B-cell maturation. Further, Pax5 P80R heterozygous or homozygous mice developed pre-B-ALL with a median latency of 166 and 87 days, respectively, with heterozygous mice acquiring alterations on the second allele. In contrast, Pax5+/- mice, and those harboring G183S mutation observed in familial leukemia, do not spontaneously develop B-ALL. Conclusions These results show the utility of transcriptome sequencing in defining subtypes and founding genetic alterations in B-ALL, provide a revised taxonomy of the disease across the age spectrum, and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis. Disclosures McKay: ImmunoGen Inc.: Employment. Tallman:Orsenix: Other: Advisory board; AROG: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Konopleva:Stemline Therapeutics: Research Funding. Relling:Shire Pharmaceuticals: Research Funding. Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Abstract Background: Nilotinib, a 2nd-generation BCR-ABL inhibitor, is 20–30 times more potent than imatinib in vitro. A phase II, open-label study of safety/efficacy of nilotinib in pts with imatinib-resistant/intolerant Ph+ CML-CP or -AP was reported. Planned starting dose was 400mg BID, with escalation to 600mg BID if response was inadequate. The objective of the post-hoc analysis was to evaluate safety/efficacy of nilotinib 600mg BID in CML-CP pts who had dose escalation. Methods: Starting dose was 400mg BID. If response was inadequate (failure to achieve complete hematologic response [CHR] by 3 mos, minimal cytogenetic response [CyR] by 6 mos, or major cytogenetic response [MCyR] by 12 mos; loss of HR, CyR, or progression), nilotinib to 600mg BID dosing was permitted. Imatinib resistance was defined as treatment with imatinib ≥600 mg/d with disease progression (≥50% increase in WBC, blasts, basophils, or platelets) or no HR in peripheral blood and/or bone marrow after 4 wks. Results: Of 320 enrolled pts in CP, 269 remained on nilotinib 400mg BID; 51 pts were dose-escalated to 600mg BID for inadequate responses (mean age 57.7 yrs; 68.6% male). All pts met the study criteria for imatinib resistance. Prior imatinib dose was ≥600mg QD in 98% pts and ≥800 mg QD in 53%. 43/51 (84%) pts had no baseline CHR; 38% had no prior history of complete cytogenetic response (CCyR). 35% had prior MCyR. Median time to dose escalation was 169 (114–211) d. 14 (27.5%) pts remain on therapy; median time to discontinuation from study was 307 (235–414) d. 5 pts with disease progression and no deaths were observed among pts who received 600mg BID. CHR was achieved in 58% of evaluable pts without CHR at baseline (n=43). MCyR occurred in 29% of evaluable pts (15/51) on 600mg BID; 10% (5/51) CCyR. Disease progression was low, occurring in 10% (5/51) of pts on 600mg BID. Median time to progression for CP pts on 600mg BID was 12 mos, substantially less than for pts who stayed on 400mg BID (median not observed yet, at least 20 mo). Nonhematologic AEs in 600mg BID pts were similar to those reported for the entire CP cohort from the phase II study. Grade 3/4 hematologic lab abnormalities were: anemia 13.8%, neutropenia 7.9%, thrombocytopenia 13.7%. Hyperglycemia and elevated bilirubin occurred in 6% and 10% of pts, respectively, on 600mg BID. Conclusions: Some HR and CyR after dose escalation to 600mg BID were observed. Safety/tolerability for 600mg BID were similar to the entire CP cohort. Based on these preliminary data, nilotinib dose escalation to 600mg BID in imatinib-resistant Ph+ CML-CP pts may be effective in pts with inadequate responses to 400mg BID. As might be expected in a more resistant population, response duration was shorter than reported for the phase II CP cohort. Additional follow-up is necessary. Use of 600mg BID is not currently recommended for imatinib-resistant CML-CP pts outside clinical studies.

Lineage-specific growth factors mobilize peripheral blood progenitor cells (PBPC) and accelerate hematopoietic recovery after high-dose chemotherapy. Recombinant human thrombopoietin (rhTPO) may further increase the progenitor-cell content and regenerating potential of PBPC products. We evaluated the safety and activity of rhTPO as a PBPC mobilizer in combination with granulocyte colony-stimulating factor (G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 μg/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 μg/kg on days −3, −1, and 1, or 0.6 μg/kg on days −1 and 1. G-CSF, 5 μg/kg IV or subcutaneously (SC) twice daily, was started on day 3 and continued through aphereses. Twenty comparable, concurrently and identically treated patients (who were eligible and would have been treated on protocol but for the lack of study opening) mobilized with G-CSF alone served as comparisons. CD34+ cell yields were substantially higher with the first apheresis following rhTPO and G-CSF versus G-CSF alone: 4.1 × 106/kg (range, 1.3 to 17.6) versus 0.8 × 106/ kg (range, 0.3 to 4.2), P = .0003. The targeted minimum yield of 3 × 106CD34+ cells/kg was procured following a single apheresis procedure in 61% of the rhTPO and G-CSF–mobilized group versus 10% of G-CSF–mobilized patients (P = .001). In rhTPO and G-CSF mobilized patients, granulocyte (day 8 v 9, P= .0001) and platelet recovery (day 9 v 10, P= .07) were accelerated, and fewer erythrocyte (3 v 4,P = .02) and platelet (4 v 5, P = .02) transfusions were needed compared with G-CSF–mobilized patients. Peripheral blood platelet counts, following rhTPO and G-CSF, were increased by greater than 100% and the platelet content of PBPC products by 60% to 110% on the first and second days of aphereses (P < .0001) with the greatest effect seen with repeated dosing of rhTPO at 0.6 μg/kg. rhTPO is safe and well tolerated as a mobilizing agent before PBPC collection. Mobilization with rhTPO and G-CSF, in comparison to a comparable, nonrandomized G-CSF–mobilized group of patients, decreases the number of apheresis procedures required, may accelerate hematopoietic recovery, and may reduce the number of transfusions required following high-dose chemotherapy for breast cancer.

Abstract DNA methylation at cytosine position 5 (5mC) is a genetic modification that regulates gene expression and is critical for cancer initiation and development. The methylcytosine dioxygenase TET2 participates in active DNA demethylation by converting 5mC to 5-hydroxymethylcytosine (5hmC) and its subsequent intermediates thereby regulating gene expression. TET2 gene is frequently mutated in hematological disorders, including 15-25% of myelodysplastic syndrome (MDS) cases. Loss of TET2 function leads to DNA hypermethylation and subsequent dysregulated gene expression in hematopoietic stem cells, and has been considered as an initial step of myeloid malignant transformation including MDS and acute myeloid leukemia (AML). While enzymatic activity of TET2 is well studied, little is known about post-translational modifications (PTMs) that regulate its activity in hematopoietic cells. Lysine acetylation, one of the most crucial PTMs, occurs in a variety of proteins and modulates protein-protein or protein-nucleic acids interactions. Here, we first observed that ectopic expression of histone acetyltransferase EP300 or CREB binding protein (CBP) increased the endogenous TET2 acetylation in 293T cells. Mass spectrometry analysis of TET2 identified several conserved acetylated lysine residues located on the exterior surface of its catalytic domain (CD), which is predicted to be essential for the interaction of TET2 with DNA. Modeling of TET2/DNA interaction using x-ray crystal structure (PDB code: 4NM6) indicates acetylated TET2 interferes with DNA binding thereby impairing the catalytic activity of the enzyme. Like other acetylated proteins, TET2 modification is reversible, since ectopic expression of the protein deacetylase Sirtuin1 (SIRT1) significantly decreases acetylation of TET2. A domain mapping analysis revealed that SIRT1 preferentially interacts with TET2 CD, further supporting the regulatory role of SIRT1 on TET2. To explore the role of SIRT1 mediated deacetylaion of TET2 in more physiological condition, SIRT1 knockdown (KD) MDS-L cells as well as SIRT1 knockout (KO) murine hematopoietic c-kit+ cells were used. In MDS-L cells derived from a MDS patient, SIRT1 KD promoted hyperacetylation of endogenous TET2 which is associated with 50% decrease in 5hmC levels compared to control (p=0.03, n=3). Similarly, SIRT1 KO murine c-Kit+ cells showed TET2 hyperacetylation and 62% decrease in 5hmC compared to wildtype (WT) counterpart (p=0.009, n=5). Phenotypically, the transforming ability of SIRT1 shRNA transduced MDS-L cells was evaluated in vitro through serial replating assay. In contrast to first plating which showed no difference between two groups, the number of colonies derived from SIRT1 KD cells was 2.39 fold higher than that from the control shRNA transduced group after two rounds of replating (p=0.0002, n=3). Importantly, we tested the in-vivo effect of SIRT1 KD on MDS-L cells engrafted in immunodeficient NSGS mice. SIRT1 KD significantly enhanced MDS-L cell engraftment compared to control shRNA (50±5% of human CD45+ cells in SIRT1 KD group vs. 19±4% human CD45+ cells in control group, p=0.003, n=6). Interestingly, increased transformation capacity of SIRT1 KD MDS-L cells was associated with decreased expression of TET2 regulated tumor suppressor genes. For example, expression levels of Mtss1 and Dusp6 were significantly decreased in SIRT1 KD MDS-L cells compared to control shRNA counterpart (Mtss1, 49±5% decrease compared to control, p=0.005; Dusp6, 25±2% decrease compared to control, p=0.01). Similar results were observed in SIRT1 KO cells. The validation of physical binding of TET2 to its targets is ongoing. Moreover, the effect of an SIRT1 allosteric activator-SRT1720 in MDS-L cells is similar to that of Vitamin C, a known TET2 activator. Specifically, SRT1720 significantly increased Mtss1 expression in MDSL cells (p=0.0005, n=3), indicating that SIRT1 agonist may lead to activation of TET2 downstream tumor suppressor genes. In summary, our data demonstrate that TET2 activity can be functionally modified by acetylation and may be enhanced in MDS patients that do not harbor loss-of-function mutations through the SIRT1 mediated deacetylation. These results support further exploration of molecular mechanisms inducing TET2 acetylation, and evaluation of SIRT1 activation as a potential therapeutic approach in MDS. Disclosures No relevant conflicts of interest to declare.

Abstract Lineage-specific growth factors mobilize peripheral blood progenitor cells (PBPC) and accelerate hematopoietic recovery after high-dose chemotherapy. Recombinant human thrombopoietin (rhTPO) may further increase the progenitor-cell content and regenerating potential of PBPC products. We evaluated the safety and activity of rhTPO as a PBPC mobilizer in combination with granulocyte colony-stimulating factor (G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 μg/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 μg/kg on days −3, −1, and 1, or 0.6 μg/kg on days −1 and 1. G-CSF, 5 μg/kg IV or subcutaneously (SC) twice daily, was started on day 3 and continued through aphereses. Twenty comparable, concurrently and identically treated patients (who were eligible and would have been treated on protocol but for the lack of study opening) mobilized with G-CSF alone served as comparisons. CD34+ cell yields were substantially higher with the first apheresis following rhTPO and G-CSF versus G-CSF alone: 4.1 × 106/kg (range, 1.3 to 17.6) versus 0.8 × 106/ kg (range, 0.3 to 4.2), P = .0003. The targeted minimum yield of 3 × 106CD34+ cells/kg was procured following a single apheresis procedure in 61% of the rhTPO and G-CSF–mobilized group versus 10% of G-CSF–mobilized patients (P = .001). In rhTPO and G-CSF mobilized patients, granulocyte (day 8 v 9, P= .0001) and platelet recovery (day 9 v 10, P= .07) were accelerated, and fewer erythrocyte (3 v 4,P = .02) and platelet (4 v 5, P = .02) transfusions were needed compared with G-CSF–mobilized patients. Peripheral blood platelet counts, following rhTPO and G-CSF, were increased by greater than 100% and the platelet content of PBPC products by 60% to 110% on the first and second days of aphereses (P &lt; .0001) with the greatest effect seen with repeated dosing of rhTPO at 0.6 μg/kg. rhTPO is safe and well tolerated as a mobilizing agent before PBPC collection. Mobilization with rhTPO and G-CSF, in comparison to a comparable, nonrandomized G-CSF–mobilized group of patients, decreases the number of apheresis procedures required, may accelerate hematopoietic recovery, and may reduce the number of transfusions required following high-dose chemotherapy for breast cancer.