Familial MDS has been rarely reported, with few cases usually being in the context of early-onset disease and germline (GL) mutations. Patients with familial RUNX1 mutations show thrombocytopenia and ...progression towards MDS/AML. Similarly, germ line-(GL) CEBPA and GATA2 mutations are associated with MDS/AML. Within typical MDS, late presentation makes it difficult to distinguish hereditary factors from aging or environmental exposures. Only in rare cases, a strong family history may suggest a genetic predisposition.
Most of the NGS-based projects focused on discovery of somatic mutations, but it can also be used to search for disease prone GL-encoded sequence alterations. For the purpose of this study we hypothesized that in a proportion of seemingly sporadic MDS, known or new GL mutations may be present. They may predispose to disease evolution or constitute a first hit with a long latency, making assessment of penetrance difficult. Because of the tremendous amount of data generated through unbiased cross-sectional approaches and validation needed, we have devised various rational prioritization and analytic strategies: i) we stipulated that genes affected by somatic mutations may also be targets of GL alterations; ii) we screened for alterations in a panel of genes known to constitute predisposing factors to cancer and found in a limited WE NGS study (N=106) to be more frequent than expected in MDS. These genes were included in a somatic targeted panel of 81 genes.
In total we have analyzed 459 patients with typical MDS and focused on results of the above described analytic strategies. All identified alterations were independently validated to exclude somatic lesions and technical artifacts. Following sequencing, tier-1 lesions were defined as known disease-prone sequence alterations/mutations, new nonsense/frameshift mutations and highly recurrent missense mutations exceedingly rare in the general population. Tier-2 lesions were defined as missense mutations with general population frequency of <1% in the general population. Among those, the prioritization is according to the frequency and predicted impact on the protein function. For example, among tested genes, we found a total of 73 tier-1 lesions (>1) MDS. Altogether (tier-1/2) we identified 281 non-synonymous GL sequence alterations in 209 cases (45%). Surprisingly, high frequencies of mutations in DNA repair genes (including ATM, ATR or FA genes) were found in heterozygous and even (4 cases) in biallelic configuration. We also identified adult patients with otherwise typical Li-Fraumeni TP53/CHEK2 mutations. In total we found 40 tier-1 FA gene mutations, 7 ATM and 6 telomerase complex mutations and many others. One of FA genes was found to be affected by a novel, recurrent, frameshift GL variant present in 4 cases. Interestingly, the same locus is mutated in 2 non-hematological cell lines and there are proximal somatic mutations reported in that region. Several clinical and molecular associations were found when patients with various tier-1 mutations were studied. For instance, the presence of heterozygous GL mutations in FA/DNA repair genes were associated with somatic deletion of chr.7 or del5q (P<.001).
We also classified 208 tier-2 alterations. Among them, we focused our analysis on the most recurrent alterations. Not surprisingly some of the genes frequently affected by tier 1 mutations also showed common tier-2 mutations. Examples include heterozygous tier-2 genes such as, FA genes and ATM found in 9% and 14% patients, respectively. For some of the functionally related genes a combined tier-1/2 analysis was performed. For instance, analysis of telomerase machinery genes included NOP10, POT1, TERT, TERC and DKC1. Surprisingly, out of 5 different variants in TERT found in MDS patients, 2 were canonical, p.H412Y (N=3) and p.A279T (N=3) and reported to be pathogenic in hereditary aplastic anemia. Most frequent SNVs were found in ATM (N=66) and APC (N=20).
In conclusion, our results suggest that important GL alterations in cancer predisposition genes can be found in patients with otherwise seemingly sporadic MDS. Some of the genes thought to cause MDS in recessive fashion may contribute to MDS susceptibility in a delayed mode when present in heterozygous configuration. We conclude that already our very limited screen suggests inherited factors may play an important role in the pathogenesis of adult MDS.
Sekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen Corp: Membership on an entity’s Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity’s Board of Directors or advisory committees.
The rearrangements of core binding factors (CBF) are classical founder events in primary AML (pAML). In non-CBF AML, many new somatic mutations have been identified, some of which may also serve as ...initial ancestral events. In myelodysplastic syndromes (MDS) and secondary AML (sAML), the diversity of ancestral lesions may be even greater, with potential overlap with other myeloid neoplasm in initiating molecular events. E.g., endomorphic IDH1/IDH2 mutations have been discovered in pAML, but also can be present in sAML and MDS. Analysis of the clonal architecture of a limited number of AML cases suggested that IDH1/2 mutations are ancestral. Theoretically, any ancestral lesion could also occur at later stages as a subclone. It is not clear if IDH1/2 mutations play the same role in pAML vs. sAML and MDS. This is a critical distinction as drugs targeting founder events may be able to be curative, while targeting secondary events may reverse clonal evolution, but is unlikely to eliminate the founder clone. Moreover, initiating molecular lesions are more likely to be subcategory-defining than the secondary events.
We hypothesized that IDH1/2 mutations may occur at different stages in the clonal hierarchy in patients with various myeloid phenotypes and may affect both the nature of genetic landscape and clinical features should IDH1/2 mutations be ancestral. Conversely, should IDH1/2 be found to be subclonal it will be essential to identify the founder clone. To address these issues, we have investigated a cohort of 2033 patients with secondary AML (N=125), pAML (N=294), MDS (N=1404), MDS/MPN or MPN (N=201) for the presence of IDH1/2mutations and other clonal events using various forms of deep NGS.
IDH1 mutations were found in 3%, 1.3%, 7.8% and 8% of MDS, MDS/MPN, pAML and sAML, whereas those of IDH2 were found in 3.4%, 3.3%, 6.5% and 9.1%, respectively. While both IDH1/2 mutations occurred most frequently in pAML (P=.0001), IDH2 was commonly associated with higher-risk MDS (P=.013). IDH1 mutations coincided with younger age (P=.01) while IDH2 mutants were likely to have a normal karyotype (P=.01). The average copy number-adjusted clonal size was 58% for IDH2 and 56% for IDH1 (P=.78). DNMT3A and NPM1 mutations most frequently co-occurred with IDH1/2. RUNX1, ASXL1, SRSF2, and STAG2 were significantly associated with IDH2, whereas PHF6 mutations with IDH1.
To reconstruct the clonal architecture in IDH1/2 cases, variant allelic frequencies were compared, along with cross-sectional comparisons of mutational frequencies and serial samples. Cross-sectional analysis of the distribution of IDH1/2 mutations in patients with lower-risk and higher-risk MDS and sAML showed increasing proportions of both IDH1 and IDH2 mutant cases with more advanced stages of the disease, suggesting that in some cases IDH mutations evolved during progression. This finding was substantiated when we analyzed serial samples. In 5/6 cases, IDH1 mutations constituted ancestral events with PHF6, NPM1, JAK2, NRAS and SMC3 serving as the most common subsequent subclonal events. The sixth case had U2AF1 as the ancestral event. IDH2 was an ancestral event in 3/4 serial cases with STAG2, ASXL1, NRAS, TET2 and NPM1 as common subclonal events. When clonal hierarchy of concurrently mutated genes was analyzed using the diversity score calculation of VAFs, IDH1 mutation was identified as a dominant clone in 45% of cases; when it was subclonal, DNMT3A, SRSF2 and SF3B1 were the most commonly detected dominant mutations. In contrast, in pAML cases, IDH1/2 constituted dominant mutations is 80%, with NPM1, FLT3 mutations being the most common secondary events to IDH1, and RUNX1 being the most common secondary event to IDH2. When IDH1 mutations were analyzed, they constituted ancestral events in 59% of cases (P=.9, vs. IDH1). When IDH was found to be subclonal, the ancestral events were heterogeneous. OS was analyzed in 786 patients within this cohort. IDH1 mutational status had no effect on survival of pAML or MDS patients, whereas IDH2 mutant MDS patients had an inferior OS compared to WT patients (24 vs. 40 months, P=.05) in multivariate analysis.
In sum, our results suggest that in pAML, IDH1/2 mutations are primary events, whereas in sAML, MDS and MPN/MDS, IDH1/2 mutations may also be subclonal. As a primary event, IDH1 or IDH2 may influence clinical features and be used to categorize a proportion of patients in particular with regard to use of targeted therapies.
Haferlach:MLL: Equity Ownership.
The molecular pathogenesis of myeloid neoplasms characterized by 5q deletion (del(5q)) has not been completely elucidated. While some pathomorphologic features including e.g., megakaryocytic and ...erythroid dysplasia, have been associated with specific genes within minimal common deleted regions (CDR), genes responsible for clonal advantage and expansion have not been identified. It is not clear if haploinsufficiency of one or multiple genes within del(5q) is responsible for clonal evolution or whether mutations in those genes or other genes located in other genomic areas are present. Moreover, with the recognition of intra-tumor diversity and hierarchical clonal architecture, it may be possible to establish whether del(5q) or other lesions, including common somatic mutations, constitute the ancestral event in the pathophysiologic cascade.
We performed a comprehensive mutational screen in 124 patients with del(5q), including 59 patients studied by whole exome sequencing (WES) and 65 by targeted deep NGS of genes within the deleted area and the other most commonly mutated genes as previously determined in WES cohorts. To identify pathogenic genes, those most consistently found to be haploinsufficient in del(5q) were matched for the presence of mutations in diploid cases. For the purpose of this study haploinsufficiency was quantitated based on the number of cases with del(5q) showing <60% expression of the corresponding genes. E.g.,HDAC3 in 81%, PPP2CA in 62% and RPS14 in 14% of cases with del(5q). For all somatic mutations, we also describe the clonal composition based on deep sequencing in serial samples and analyses of variant allelic frequency. Finally, we compare the clonal size for individual mutations with that of del(5q). The latter was accomplished by calculation of clonal size based on allelic imbalance for informative SNPs present within deleted regions in heterozygous configurations in germ line samples. The average deviation from the ideal 50/50 distribution in tumor samples allowed for precise calculation of the proportion of cells in the sample affected by the deletion. Using this approach, there was a good correlation to the size of del(5q) clone by FISH (r=.94)
Our results demonstrate that 10/14 genes were haploinsufficient within the CDR, but only 2 hemizygous somatic mutations were identified. However, 12 mutations in 7 genes (MATR3, SH3TC2, CSNK1A1, PDGFRB, CD74, FAT2 and G3BP1) were present with the area corresponding to the CDR in diploid cases. TP53 mutations were more commonly associated with del(5q) (73%, vs. 27% in diploid 5, p<.001) and were particularly frequent in patients affected with 2 commonly retained regions (CRR1;5q11.1-5q14.2 and CRR2; 5q34-qter), where they were found in 81% of cases (30/37) vs. 19% (7/30) among CDR deletions (p<.001). In lower-risk MDS, mutations were detected in 11% of deletion cases, whereas they were only found in 5% of diploid chr5 (p<.0001). In higher-risk MDS, TP53 mutation were found in 42% of del(5q) vs. 4% of diploid chr5 (p<.0001). Similarly, 45% patients with concomitant -7/del(7q) and del(5q) had TP53 mutations. The most common mutation associated with del(5q) was TP53, while mutations of FLT3, NRAS or TET2 were significantly mutually exclusive (p=0.03, 0.04 and 0.03; respectively). Next we determined the earliest somatic event by comparing of clonal size of the associated lesions. Del(5q) was present in 17-98% of tumor cells. We identified three theoretical possibilities as to the clonal architecture of del(5q) myeloid neoplasms: i) Tumors in which driver somatic mutations precede del(5q) (35%), ii) those in which del(5q) appears to precede any other somatic mutation (6%) and iii) the succession cannot be determined because of very expanded clones of similar size (“clonal saturation”) i.e., these cases were not informative. For cases in which del(5q) was a secondary lesion, TP53 was the ancestral event 64% of the time, and DNMT3A 27% of the time. The TP53 mutation was detected as a secondary event in 1 of 2 samples in which del(5q) was found to be ancestral.
In sum, our results suggest that del(5q) is not universally an ancestral event. The TP53 mutation is the most common mutation in del(5q) and may also serve as ancestral event. While UPD17p and hemizygocity for TP53 can be found in 33% of TP53 mutant cases, most of the detected TP53 mutations were likely to heterozygous, and therefore the clonal size was not overestimated.
Sekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen Corp: Membership on an entity’s Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity’s Board of Directors or advisory committees.
Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. The extent of individual defects vary, which may account for observed clinical ...diversity. Del(5q) pathogenesis has been related to haploinsufficiency of genes contained in the common deleted regions (CDR), including RPS14, miR-145/146a and SPARC. Driver mutations or pathogenic microdeletions were not identified for these genes, suggesting that multiple genes must function in combination to promote clonal evolution and phenotypic heterogeneity.
Hence, we performed a comprehensive analysis of somatic mutations in genes located on chromosome 5 (chr5), both in patients with diploid 5q and in those with del(5q), to clarify the role of germline and somatic mutations in disease pathogenesis. In parallel, expression analysis was performed to correlate haploinsufficiency with the frequency of mutational events, in particular for diploid 5q cases. Applying SNP-array karyotyping to samples from 146 patients with del(5q), the lesion was identified in 5q31.1q33.1. Two retained regions (CRRs) were also observed in q11.1q14.2 (CRR1) and q34qter (CRR2). Lower-risk MDS is frequently affected by CDR, while in higher-risk MDS and secondary AML CRR1/2 are commonly co-involved. Using whole exome sequencing, we identified 11 hemizygous mutations located within the deleted area in del(5q) (N=59), while in cases diploid for 5q (N=330), 243 heterozygous mutations were found. One of the mutations discovered on chr5q afflicted a gene G3BP1 (5q33.1), located within the CDR and present in 2 patients. Both were missense mutations (one heterozygous and the other homo/hemizygous). A mutant case showed good responses to lenalidomide even though diploid 5. In addition, other somatic mutations of driver genes including TET2, CUX1 and EZH2 were concomitantly observed. Whole transcriptome sequencing demonstrated hemizygous loss of G3BP1 resulting in haploinsufficiency. G3BP1 was haploinsufficient in 48% of RAEB as well as low-risk MDS cases with del(5q). Overall, defective G3BP1 is associated with shorter overall survival (P<.001) in AML, consistent with the reports that del(5q) is a worse prognostic factor in myeloid neoplasms with aggressive phenotype.
G3BP1 is a nuclear RNA-binding protein and is ubiquitously expressed in bone marrow, CD34+ progenitors and leukemic cell lines. Furthermore, G3BP1 binds to TP53 and its expression leads to the redistribution of TP53 from the nucleus to the cytoplasm. Similar to RPS14, haploinsufficient of G3BP1 resulted in TP53 up-modulation. Moreover, low expression of G3BP1 in diploid 5q cases was indeed associated with higher TP53 expression. Next, we generated haploinsufficient G3BP1 cell lines using shRNA transduction. Decreased expression of G3BP1 led to growth inhibition and impaired colony formation by transduced cells lines and hematopoietic progenitor cells, respectively. Knockdown of G3BP1 in K562 cell line increased TP53 in the nucleus, and when treated with CPT11, DNA-damaged induced G1-arrest was more prominent in knockdown cells. Furthermore, after knockdown of G3BP1 in TP53-null HL60 cells, we observed increased aneuploidy, suggesting that the loss of function of G3BP1 and TP53 may result in chromosomal instability. Most significantly, G3bp1-/+ mice showed lower blood counts and defective, dysplastic hematopoiesis, similar to lower-risk MDS. As previously described, TP53 defects are associated with advanced disease but recently it became apparent that TP53 may be one of the most common somatic lesions found in the context of del(5q). We stipulate that loss of TP53 function might overcome TP53 tumor suppressor effects and induce leukemic evolution in the defective G3BP1 status. In our cohort, TP53 mutations were more frequently present in high-risk phenotype with G3BP1 haploinsufficient expression.
In conclusion, novel somatic mutations of G3BP1 suggest that it could be a candidate gene associated with the clonal evolution of del(5q). Loss of function or low expression of G3BP1 has been shown to up-modulate TP53 and result in dysplasia and growth inhibition, hallmarks of early stages of MDS. Additional events constitute loss of function of TP53, resulting in chromosomal instability, which is associated with leukemogenesis.
Sekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen Corp: Membership on an entity’s Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity’s Board of Directors or advisory committees.
Background: The majority of MDS patients (pts) have anemia and are treated initially with ESAs. Particularly for lower-risk MDS pts (International Prognostic Scoring System (IPSS) Low and Int-1), ...once ESAs are no longer effective, treatment options are limited to drugs commonly used for higher-risk MDS, such as hypomethylating agents, or off-label use of immunomodulatory drugs. As a result, most pts receive only transfusion support post-ESA, representing a pt group with an unmet medical need frequently targeted for drug development, for whom long-term outcome is unknown.
Methods: We studied pts diagnosed with lower-risk MDS from 1997-2014 at MDS CRC institutions and treated with ESAs (epoetin alpha (epo) or darbepoetin (darb)). The best response to treatment was categorized per International Working Group 2006 response criteria (hematological improvement (HI), complete response (CR), or partial response (PR)). The primary endpoint was overall survival (OS) at the time of ESA failure, defined as cessation of treatment due to relapse or refractoriness; a secondary endpoint was time to AML transformation or death, from time of response (for responders) or failure (for nonresponders) determination. Descriptive statistics were used for baseline characteristics. The Kaplan Meier method was used to estimate OS and a log rank analysis was used to compare response categories. Cox regression analysis was performed for multivariable analysis.
Results: Of 206 patients included in analyses, median age was 71.6 years (range: 25.3-88.1), 36% were female, 5% were African-American, and 11% had t-MDS. WHO categories included RA (14%), RARS (16%), RCMD (42%), MDS-u (6%), del (5q) (4%), RAEB-1 (9%), RAEB-2 (2%), RARS-T (2%), MDS/MPN-u (3%), and CMML-1 (2%), with pts classified as IPSS Low (39%), Int-1 (61%), or IPSS-R Very Low (16%), Low (55%), Intermediate (26%), and High (4%). IPSS cytogenetic risk groups were Good (72%), Intermediate (22%), and Poor (6%). Baseline median hemoglobin was 9.4 g/dl (range: 5.5-14.2), serum epo level was 97.2 (range: 14.2-3899.0), and 11% were transfusion-dependent. Treatment included darb (59%) and epo (41%) at median doses of 300 mcg (range: 100-500) and 40,000 units (range: 5,000-80,000), respectively. Pts remained on therapy for a median of 30.4 weeks (range: 0.0-447.7) and had a median follow-up of 28.4 months (95% confidence interval (CI): 24.5, 45.4). First treatments following ESA failure included azacitidine (41.7%), decitabine (10.2%), lenalidomide (16.6%), experimental drugs (3.1%), other growth factors (13.6%), ATG and/or other immunosuppressants (8%), chemotherapy (0.1%) , transplant (0.1%) and others (6.6%). The overall response rate (ORR) to ESAs was 18.8%, with 0% achieving CR; 0.1% PR; and 18.7% HI. Responses for epo were 17.3% and for darb were 19.8% (p=.67 for difference). For both ESAs, 81.2% of patients had disease refractory to treatment: 69.4% with stable disease and 12% with progressive disease with no significant differences between epo and darb by responder status. Median response duration for epo and darb were 21.9 weeks (range: 3.0 - 447.7) and 39.1 weeks (range: 0.0 - 350.7) respectively (p=0.045). Median survival from the date of diagnosis was 28.4 months (95% CI: 24.5, 45.4), and from ESA failure was 23.9 months (95% CI: 19.9, 33.0): 21.6 months (95% CI: 15.6, 39.2) for epo and 28.8 months (95% CI: 21.2, 39.7) for darb (p=0.99) (Figure). Median time to AML transformation or death was 17.4 Months (95% CI: 14.1, 22.9): 25.4 months for responders and 16.8 months for non-responders (p=.069). For patients who received ESAs for a minimum of 4 months (39% of pts for epo and 61% for darb), ORR was 16.5%, and median survival from ESA failure was 23.0 months (95% CI: 14.7, 33.0): 22.3 months (95% CI: 13.1, NA) for epo and 24.7 months (95% CI: 14.3, 39.7) for darb (p=0.87).
Conclusion: In this large, but uncontrolled cohort, response rates were similar for lower-risk MDS patients treated with epo and darb, though duration was longer for darb. There was a trend for improved outcomes in patients who responded to ESAs. Lower-risk MDS patients treated with ESAs have an OS of less than 2 years from the time of failure, and can thus be considered a high-risk MDS group for whom subsequent therapies are not standardized, representing an unmet medical need.
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Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Steensma:Incyte: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onconova: Consultancy. Komrokji:Incyte: Consultancy, Honoraria, Research Funding; Novartis: Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics: Speakers Bureau.
Historically, the evolution rate of aplastic anemia (AA) to MDS approaches 15% in 10 yrs; thus, AA can be considered a major predisposition factor for secondary MDS (sMDS). The likely etiology ...includes expansion of a preexisting clone or truly late clonogenic events. In both instances, progression can be a result of a clonal escape, but confirmation of the presence of mutant cells at presentation would indicate that initial autoimmune processes may represent a tumor surveillance reaction.
We studied 326 patients with AA and 47 patients with paroxysmal nocturnal hemoglobinuria (PNH) and identified 36 cases (progression rate: 11% in median follow up of 6 years) that evolved to MDS or AML (median time to progression: 3.2 yrs.; transplanted patients were not censored). Cytogenetic analysis upon progression showed abnormal karyotype in 83% of cases; 7% had complex karyotype and -7/del(7q) was present in 62% of cases. The presence of a PNH clone was detected in 17% of cases that transformed to sMDS vs. 35% in non-progressors (P =.1). For comparison, we have also analyzed primary de novo cases of MDS (pMDS) with (N=94) and without (N=557) -7/del(7q). In contrast to sMDS, -7/del(7q) was present in 14.4% of cases in pMDS.
Because sMDS following AA or PNH included a high proportion of patients with -7/del(7q), we compared sMDS with -7/del(7q) to pMDS with -7/del(7q) for coexisting mutational events. Mutations in RUNX1, CBL, SETBP1 and ASXL1 appeared to be more frequent in sMDS vs. pMDS (28.6% vs. 2.1%, 21.4% vs. 2.1%, 21.4% vs. 5.3%, 21.4% vs. 10.6%, P =.003, P =.02, P =.07, P =.37, respectively). In contrast, TP53 and DMT3A were more common in pMDS (7.1% for sMDS vs. 17%, 0% for sMDS vs. 8.5%, P =.69, P =.59). Similarly, there were several other distinctive differences between all sMDS and pMDS irrespective of the cytogenetics: mutations in SF3B1, SRSF2, NPM1, DNMT3A were common in primary AML but entirely absent from cases after AA; mutations in RUNX1 and SETBP1 appeared to be more frequent in sMDS vs. pMDS (26.3% vs. 8.3%, 21.1% vs. 3.2%, 15.8% vs. 3.9%, P =.03, P =.005, respectively).
Whole exome NGS was performed after progression, with confirmed somatic mutations subsequently tracked back by targeted deep NGS applied to serial samples starting at initial presentation. Confirmed mutational events and chromosomal aberrations were found in 19/36 patients with sMDS; 17/19 cases of sMDS had at least 1 confirmed somatic mutation. Remarkably, in retrospective analysis in 6/7 cases studied serially, at least one of the identified mutations was detectable at presentation when deep targeted sequencing (depth 5,000~20,000 reads) was performed. In 5 of these cases the alterations appeared to be ancestral events for sMDS evolution. When anadditional 77 AA or PNH cases were studied by deep sequencing, somatic mutations were present in 48% of AA patients at presentation. Detection of clonal events at presentation was associated with an increased risk of subsequent MDS evolution (14/37 mutant cases vs. 3/40 nonclonal cases evolved, P =.002). Mutations found at both initial presentation and upon evolution were suggestive of a slow expansion of previously cryptic clones (ASXL1, CUX1, TET2, CBL, RUNX1, and SETBP1). Patients with these genes (n=18) had worseoverall survival compared to patients without these mutations (P =.03). To assess the potential impact of immunosuppressive therapies (IST), we also investigated a subset (out of 77) of 53 patients (39 responders and 14 refractory cases) following IST. Clonal somatic events were identified in 27 of them, but there was no association between the response to IST and somatic mutations at presentation.
Our results demonstrate that while subclonal mutations indicative of oligoclonal hematopoiesis are frequent in AA, the presence of permissive ancestral somatic events at the outset of AA predisposes patients to sMDS, a feature that had diagnostic and prognostic implications.
Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.
Background
Achieving a complete remission (CR) in patients with newly diagnosed acute myeloid leukemia (AML) after induction chemotherapy with cytarabine and an anthracycline (7+3) remains an ...important treatment goal associated with better overall survival (OS). Approximately 25-30% of younger, and up to 50% of older patients (pts) fail to achieve CR. AML pts with residual leukemia at day 14 receive a second cycle of the same regimen; whether these pts have worse survival than pts not requiring re-induction is unclear. Information on pts with primary refractory AML and the best treatment strategy in this setting are limited.
Methods
Pts with newly diagnosed AML treated at our institution between 1/2000 and 1/2015 were included. Pts received standard induction chemotherapy with cytarabine for 7 days and an anthracycline for 3 days (7+3). Bone marrow biopsies were obtained at day 14 and a second cycle of the same regimen (7+3 for younger adults, 5+2 for older adults) was given to pts with residual leukemia (blasts > 5%). All responses were assessed at day 30 +/- 5 days post induction. Response was defined as CR and CR with incomplete hematologic recovery (CRi) or platelet recovery (CRp) per International Working Group (IWG) 2003 response criteria. Cytogenetic risk stratifications were based on CALGB/Alliance criteria. OS was calculated from the time of diagnosis to time of death or last follow up. A panel of 62 gene mutations that have been described as recurrent mutations in myeloid malignancies was used to evaluate whether genomic data can be used to predict response.
Results:
Among 227 pts with AML, 123 received 7+3 and had clinical and mutational data available. Median age was 60 years (range, 23-82). Median baseline WBC was 8.2 X 109/L (range, 0.3-227), hemoglobin 8.9 g/L (range, 4.7-13.8), platelets 47 X 109/L (range, 9-326), and BM blasts 46% (range, 20-95). Cytogenetic risk groups were: favorable in 12 (10%), intermediate in 68 (56%) normal karyotype in 44 (36%), and unfavorable in 42 (34%). A total of 93 pts (76%) responded, 69 (74%) received 1 cycle of induction and 24 (26%) required re-induction at day 14 due to residual leukemia.
A total of 39 pts (32%) received allogeneic stem cell transplant (ASCT): 18 (46%) from a matched sibling donor, 16 (41%) from a matched unrelated donor and 5 (13%) had an umbilical cord transplant. With a median follow up of 13.5 months, the median OS for the entire group was 13 months (m, range, 0.1-120). The median OS for pts who failed 1-2 cycles of 7+3 was significantly worse than pts who responded (median 2.6 vs 16.9 m, p = 0.002). When pts undergoing ASCT were censored, the median OS was 2.3 vs 9.9 m, p= 0.003, respectively. Overall, 33 pts (27%) had residual leukemia at day 14 and received re-induction, 24 (72%) achieved a response at day 30+/- 5 days. The median OS for pts who received re-induction was inferior compared to pts who did not (10.1 vs. 16.1 months, p= 0.02). When pts who received ASCT were censored, the OS was similar (8.5 vs. 7.4 months, p = 0.49, respectively). Among the 30 pts with persistent disease following induction therapy at day 30, 11 (37%) died from induction complications, 6 (20%) received salvage therapy with mitoxantrone/etoposide/cytarabine, 3 (10%) received high dose cytarabine, 2 (7%) received azacitidine, and 8 (27%) received best supportive care. Among pts who received salvage chemotherapy 56% achieved CR and proceeded with ASCT. Two pts had ASCT with residual leukemia and relapsed within 3 m of ASCT. Pts who received ASCT after induction failure had a significantly better OS compared to non-transplant pts (median OS 22.0 vs. 1.4 months, p < 0.001, respectively); however, this benefit was only seen in pts who had ASCT in CR.
We then investigated if genomic mutations can predict response or resistance to chemotherapy. Out of the 62 genes tested, only a TP53 mutation was associated with resistance, p = 0.02. Further, pts with TP53 mutations had significantly inferior OS compared to TP53 wild type regardless of ASCT status (1.4 vs 14.8 m, p< 0.001)
Conclusion:
Pts with newly diagnosed AML who fail induction chemotherapy with a 7+3 regimen have a poor outcome. Re-induction with the same regimen at day 14 for residual leukemia converted most non-responders to responders, but was associated with worse OS. ASCT improves outcome only in pts who achieve CR with salvage therapy. TP53 mutations predicted resistance to chemotherapy with 7+3.
Carew:Boehringer Ingelheim: Research Funding. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.
CMML is heterogeneous clinically (a varying degree of dysplastic or proliferative clinical features) and in terms of its molecular pathogenesis. Analysis of the spectrum of genomic lesions in CMML ...may contribute to understanding of the pathogenesis and help identify certain mutations as diagnostic biomarkers. Apart of the mere presence of a somatic lesion, phenotypic features may be shaped by initial hits. Conversely sub-clonal events may determine the phenotype or progression. Finally, initial hits may predetermine e.g., mutator phenotype, or differentiation block and therefore selecting for specific sub-clonal hits.
We have selected a large CMML cohort to establish generalizable pathogenetic and clinical associations to account for molecular heterogeneity and serial samples have to be analyzed to assess clonal dynamics and hierarchy. The study group consisted of 242 patients, including 150 CMML cases (96 CMML-1, 27 CMML-2, 27 post CMML sAML) and JMML (N =92); 15 patients were studied serially. We also used comparison cohorts of M4/M5 AML (N =64) and advanced (N= 231) and low risk MDS (N= 199) serving as risk adjusted match for CMML subtypes. The CMML entity was further sub-classified based on clinical parameters and pathomorphologic features, 57% dysplastic (MD-CMML) and 43% proliferative form (MP-CMML). Analysis was performed using WES (paired germ line/tumor samples) and multiamplicon NGS targeting top 60 most commonly affected genes. For clonal architecture analysis, cross-sectional variant allelic frequency (VAF) concept based analysis was performed including assessment of affected genes by ranking of the corresponding clonal burden rather than the absolute cellular frequency. The results of this analysis were confirmed in serial samples to identify expanding, declining and stable subclones.
Comparison of mutational spectra between the disease entity show profound differences in morphologically similar entities as particularly evident in comparison of CMML to JMML (TET2, ASXL1) or to lesser degree low risk MDS and CMML1 while progression in advanced cases was often associated with similar spectrum of additional subclonal events. However, the differences were more striking when clonal hierarchy was assessed to identify dominant/codominant and subclonal mutational events. We found that top 4 dominant/codominant clonal events, included TET2 (56%), SRSF2 (42%), ASXL1 (46%), DNMT3A (45% of patients), while in MDS corresponding frequency of these dominant events was TET2 (15%) SRSF2 (8%), ASXL1 (11%) and DNMT3A (8%), with most common ancestral events ranked SF3B1, TET2, ASXL1 etc. The clinical importance of these dominant events in CMML is highlighted by their impact on survival in KM analysis (p=.018).
Our analysis also demonstrated that for certain founding events not pathognomonic for CMML either codominant or subsequent subclonal events determine the phenotypic features (1st generation) or progression (2nd generation). For instance initial TET2 in CMML was followed often by SRSF2 or in conjunction, RAS pathway mutations while MDS was driven by TET2, SF3B1, TP53, and many other events. Progression in our cohort was driven in both CMML and MDS by ASXL1, RUNX1, NPM1. When other common mutations were categorized by their role in individual patients 27% EZH2, 20% of CBL and 22% of SETBP1 were dominant. Serial analysis further qualified the cross-sectional analysis and allowed for categorization of subclonal events. For instance, CMML-1 cases initially presented with dominant TET2 followed by subtype specific subclonal SRSF2 and progression event IDH2 progressed to sAML with new NPM1 acquisition and expansion of IDH2 c lone. In our serial sample analysis we observed that increasing ASXL1 and RUNX1 clones correspond to clinical progression, ancestral events may remain stable (TET2, SRSF2, SETBP1), while non-permissive subclones can smolder or even decline.
In sum, deep NGS allows for identification of specific ancestral events, which may determine the subsequent secondary mutational events in CMML. Classification of CMML based on ancestral events and subclonal events rather than on the global mutational spectrum correlates with clinical features and prognosis and may contribute to further clinical resolution of CMML based on the presence of specific founder mutations ultimately help establish therapeutic interventions.
Sekeres:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.
Public health surveillance is the monitoring of data to detect and quantify unusual health events. Monitoring pre-diagnostic data, such as emergency department (ED) patient chief complaints, enables ...rapid detection of disease outbreaks. There are many sources of variation in such data; statistical methods need to accurately model them as a basis for timely and accurate disease outbreak methods.
Our new methods for modeling daily chief complaint counts are based on a seasonal-trend decomposition procedure based on loess (STL) and were developed using data from the 76 EDs of the Indiana surveillance program from 2004 to 2008. Square root counts are decomposed into inter-annual, yearly-seasonal, day-of-the-week, and random-error components. Using this decomposition method, we develop a new synoptic-scale (days to weeks) outbreak detection method and carry out a simulation study to compare detection performance to four well-known methods for nine outbreak scenarios.
The components of the STL decomposition reveal insights into the variability of the Indiana ED data. Day-of-the-week components tend to peak Sunday or Monday, fall steadily to a minimum Thursday or Friday, and then rise to the peak. Yearly-seasonal components show seasonal influenza, some with bimodal peaks.Some inter-annual components increase slightly due to increasing patient populations. A new outbreak detection method based on the decomposition modeling performs well with 90 days or more of data. Control limits were set empirically so that all methods had a specificity of 97%. STL had the largest sensitivity in all nine outbreak scenarios. The STL method also exhibited a well-behaved false positive rate when run on the data with no outbreaks injected.
The STL decomposition method for chief complaint counts leads to a rapid and accurate detection method for disease outbreaks, and requires only 90 days of historical data to be put into operation. The visualization tools that accompany the decomposition and outbreak methods provide much insight into patterns in the data, which is useful for surveillance operations.
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