Influence of somatic mutations and pretransplant strategies in patients allografted for myelodysplastic syndrome or secondary acute myeloid leukemia
Authors: Christina Rautenberg, Ulrich Germing, Stefanie Stepanow, Michael Lauseker, Karl Köhrer, Paul S. Jäger, Stefanie Geyh, Min Fan, Rainer Haas, Guido Kobbe, Thomas Schroeder
Journal: American Journal of Hematology (2020)

Somatic mutations and pretransplant strategy both impact the outcome of patients with myelodysplastic syndromes (MDS), and acute myeloid leukemia derived from MDS (sAML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While the prognostic influence of several somatic mutations, especially TP53, as a disease-related variable is established,1 the optimal pretransplant strategy is less well defined due to the lack of prospective trials. In a recent analysis we showed that outcome after direct, so called upfront transplantation is at least not inferior compared to pretransplant cytoreduction, with AML-like induction chemotherapy (CTX) or hypomethylating agents (HMA).2 In the current analysis we aimed to comprehensively investigate the interplay of mutations and pretransplant strategy on outcome after allo-HSCT within one analysis. For this purpose, we examined pretransplant DNA samples from 128 of the 165 previously published patients with MDS (n = 97, 76%), sAML (n = 20, 15%) or chronic myelomonocytic leukemia (n = 11, 9%) for somatic mutations in 54 genes using the TruSight Myeloid panel (Illumina, San Diego, CA). Patients' characteristics, sequencing analysis and statistics are given in Tables S1-S4. Of these, 73 patients (57%) were transplanted without prior cytoreduction (upfront group), whereas 55 (43%, treatment group) had received either anthracycline-containing induction (n = 37, 29%, CTX group) or a median of four cycles (range: one to eight cycles) of Azacitidine (Aza, n = 18 14%, Aza group) prior transplant (Figure S1). Even though there was a higher frequency of sAML in the CTX group and a lower BM blast count in the upfront group at diagnosis, progression to advanced disease or even sAML between diagnosis and transplantation occurred in 14 (19%) and 7 (10%) patients within the upfront group (median 6.4 months; Tables S1 and S3). Consequently, at the time of cytoreductive treatment there was no statistically significant difference regarding the frequency of sAML between the upfront and treated group (15% vs 29%). With a median follow-up of 71 months estimated 5-year OS, RFS, CIR, and non-relapse mortality (NRM) probabilities of the entire cohort were 56%, 42%, 40% and 18%, respectively (Figure S2). First, we performed amplicon-based sequencing to adress the prognostic impact of somatic mutations. Hereby, we identified 285 mutations which affected 36 of the 54 investigated genes in 111 of 128 patients (87%, median two mutations per patient, range, zero to six) and reflected the clinical high-risk characteristics with RUNX1, TET2, ASXL1, TP53, SRSF2 and DNMT3A representing the most commonly mutated genes (Figures S3-S7; Table S5). With exception for RUNX1, TET2 and ASXL1, the mutation profile did not differ between treatment groups, even when focusing only on MDS patients (Figure S6, S8, S9). In those 17 genes mutated in ≥5% of patients we identified mutations in four individual genes (TP53, SF3B1, NRAS and DNMT3A), which negatively impacted OS and RFS (Figure 1A; Table S6, Figures S10-S12). Mutations in TP53 and SF3B1 were also associated with higher relapse incidence, while NRAS and SF3B1 mutations negatively influenced NRM (Table S7; Figure S13-S15). Consequently, mutations in these four genes, which were mutually exclusive to each other in three of four genes (TP53, NRAS, SF3B1), were summarized as poor-risk mutations for further analyses (Table S6-S8; Figures S16-S17). Acknowledging the negative prognostic impact of complex karyotype (CK, n = 25, Figure S18; Table S9) and the overlap between CK and poor-risk mutations (Figure S16), we analyzed their prognostic interaction. In accordance with recently reported findings3, 4 we show that the presence of poor risk mutations facilitates refinement of the prognostic information of CK with a dismal prognosis in patients carrying both CK and poor-risk mutations (Figure S19). Focusing in a next step on other disease-related, patient-associated and transplant-related factors (Tables S9,S10; Figures S20-S23) we found that pretransplant strategy was the most prominent factor significantly influencing OS. Patients, who were transplanted without prior cytoreduction, had longer survival compared to patients who received pretransplant cytoreduction (Figure 1A, Figure S15). In addition, patients undergoing upfront transplant had a trend towards a lower relapse rate (Figure 1A). Except for age (OS and RFS) and CK (RFS) no other factor impacted OS, RFS, relapse incidence or NRM (Tables S9-S10; Figures S20-S23). In multivariate analyses the presence of poor-risk mutations as well as pretransplant strategy confirmed their prognostic role with poor-risk mutation carriers and pretreated patients having inferior survival and higher risk of relapse. Additionally, BCOR and EZH2 mutations were associated with relapse and NRAS and SF3B1 mutations with NRM (Tables S11-S13). Based on this, we addressed the hypothesis that there might be an interaction between these two disease-related and procedure-related factors and re-analyzed our cohort by combining the information about mutation status of these four genes and pretransplant strategy (Figure 1A). Indeed, the outcome of patients with poor-risk mutations, who had received pretransplant therapy, was poor with 5-year OS and RFS of 23% and 5.7% and significantly inferior compared to patients with poor-risk mutations in the upfront group (5-year OS: 45%, 5-year RFS: 27%). In contrast, among patients without poor-risk mutations the upfront group had a favorable 5-year OS of 83% compared to 62% in pretreated patients, while 5-year RFS was comparable (5-year RFS 61% vs 54%). These data were confirmed by a separate analysis of patients with MDS (Figure S24). Relapse, mainly driven by poor-risk mutations and pretransplant strategy (Table S12), was the major cause of treatment failure (Figure S2) with 62% of deaths being attributable to relapse. Therefore, we finally asked whether these two variables also influenced response to and survival following salvage therapy with HMA (Aza n = 40, decitabine n = 1) and donor lymphocytes infusions (DLI, Figure S25; Table S14). The negative prognostic impact of poor-risk mutations was indeed abrogated after salvage therapy, as indicated by a comparable CR rate (43% vs 53%) and survival (2-year OS 43% vs 53%) in mutation carriers and patients without poor-risk mutations. In contrast, the CR rate was significantly higher (73% vs 21%) and survival (2-year OS 69% vs 27%) significantly longer in the upfront group compared to pretreated patients (Figure 1B, Figure S26). A major criticism of our previous analysis, where we adressed the role of upfront transplantation in 165 patients with MDS and sAML,2 is a possible selection bias where poor-risk patients receive pretransplant cytoreduction whereas "better-risk "patients may be scheduled for upfront transplantation. To overcome this we now focused on the underlying disease biology in terms of somatic mutations in 128 of the previous cohort,2 which were selected based on DNA availability and exhibited comparable pretransplant CR rate and survival like the entire cohort (data not shown). We identified mutations in four genes (TP53, DNMT3A, NRAS and SF3B1) that were associated with poor post-transplant outcome primarily related to a higher relapse incidence. Our data regarding TP53, the NRAS/RAS pathway and in parts DNMT3A mutations are consistent with results from six other retrospective analyses in 62 to 1514 patients,1, 3-5 with discrepancies between the analyses probably related to imbalances regarding patient characteristics, treatment state, sampling and sequencing. The second major finding of our current, even though retrospective analysis, which also proved true when considering only the 97 MDS patients, is that upfront transplantation resulted in a significantly better survival. This is in line with previous retrospective analyses suggesting at least non-inferiority of upfront transplantation compared to pretransplant cytoreduction.2 By integrating molecular information and pretransplant strategy, we here show that patients with similar disease biology, namely poor-risk mutations have dismal outcome after pretransplant cytoreduction and seem to benefit from direct transplantation. It remains unclarified at this point, whether dose-intensification of conditioning regimen as another procedure-related, per-se modifiable variable may influence the prognosis associated with individual mutations such as RAS or TP53 mutations, as suggested by one,5 but not confirmed in our (Figure S27) and another analysis.3 Furthermore, whether pretransplant implementation of novel compounds like CPX-351, venetoclax or APR-246 may overcome chemoresistance implicated by the four poor-risk mutations will be subject of future investigations. Besides the trend towards a lower relapse rate the advantage of upfront transplantation in the entire, as well as in the molecularly defined poor-risk group, appears to result particularly from a higher efficacy of salvage therapy with HMA and DLI. A hypothesis explaining this phenomenon may be a Darwinian mechanism which selects resistant leukemic clones during pretransplant cytoreduction resulting in resistant relapse after transplant,6 but requires prospective confirmation by longitudinal sampling. In addition to disease burden and diagnosis of MDS previously reported as predictors for response and survival after post-transplant therapy with Aza, we here show for the first time that the pretransplant strategy, but not the four poor-risk mutations (Figure 1B, Figure S26 and S28) predict response and survival after HMA-based salvage therapy. We conclude that an individualized allografting concept for patients with MDS should incorporate molecular profiling at the time of decision for allo-SCT, and planning of pretransplant strategy. If feasible, upfront transplantation appears to be associated with favorable outcome particularly in patients with high-risk molecular characteristics and may be augmented by post-transplant HMA-based salvage therapy in case of relapse. These data build the hypothesis and rationale for confirmatory testing within a prospective study. C. R. received financial travel support from Celgene Deutschland GmbH. T. S. received financial travel support, lecture fees, research funding and participated in advisory boards for Celgene GmbH. T.S. received financial travel support, lecture fees, and participated in advisory boards for Janssen-Cilag GmbH. U.G. received lecture fees from Celgene Deutschland GmbH, Novartis, Jazz Pharmaceuticals and Janssen-Cilag GmbH. U.G. received research funding from Celgene Deutschland GmbH and Novartis. Christina Rautenberg, Guido Kobbe and Thomas Schroeder designed the research study. Christina Rautenberg, Stefanie Stepanow, Karl Köhrer, and Thomas Schroeder performed the research. Christina Rautenberg, Ulrich Germing, Paul S. Jäger, Stefanie Geyh, Guido Kobbe and Thomas Schroeder contributed essential data. Christina Rautenberg, Ulrich Germing, Michael Lauseker, Min Fan and Thomas Schroeder analyzed the data. Christina Rautenberg, Ulrich Germing, Guido Kobbe and Thomas Schroeder wrote the paper. All authors critically revised the manuscript. This work was generously supported by the Leukämie Lymphom Liga Düsseldorf e. V. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1Consort diagram representing the study population. Figure S2Post-transplant outcome of the whole study population regarding survival and relapse. Figure S3Landscape of moleculargenetic alterations in 128 patients with MDS/sAML. Figure S4Comparison of the absolute number of genetic alterations and variant allele frequencies depending on the sample source. Figure S5Frequency distribution of the number of mutations detected per patient (A) and frequency distribution of distinct functional pathways being affected by respective gene mutations (B). Figure S6Frequency of molecular alterations among three groups of pre-transplant strategies. Figure S7Variant allele frequency of molecular alterations in the study population. Figure S8Comparison of frequency and distribution of mutated genes among two groups of pretransplant strategies. Figure S9Mutational landscape in 97 patients with MDS. Figure S10 Post-transplant outcome depending on the number of genetic alterations pretransplant. Figure S11 Impact of moleculargenetic alterations on post-transplant overall survival. Figure S12 Impact of moleculargenetic alterations on post-transplant relapse free survival. Figure S13 Impact of moleculargenetic alterations on post-transplant relapse incidence. Figure S14Impact of moleculargenetic alterations on post-transplant non-relapse mortality. Figure S15Effects of "poor risk mutations" and pre-transplant strategy on survival and relapse in 97 patients with MDS. Figure S16Genetic profile of patients harboring any poor risk mutations. Figure S17Post-transplant outcome depending on the number of poor risk mutations pretransplant. Figure S18Post-transplant outcome depending on complex karyotype cytogenetics. Figure S19Interaction between "poor risk mutations" and complex karyotype regarding survival and relapse. Figure S20Impact of clinical, patient and transplant related characteristics on post-transplant overall survival. Figure S21Impact of clinical, patient and transplant related characteristics on post-transplant relapse free survival. Figure S22Impact of clinical, patient and transplant related characteristics on post-transplant cumulative incidence of relapse. Figure S23Impact of distinct clinical, patient and transplant related characteristics on non-relapse mortality. Figure S24 Interaction between "poor risk mutations" and complex karyotype as well as interplay between "poor risk mutations" and pre-transplant strategy regarding survival and relapse in 97 patients with MDS. Figure S25Consort diagram representing the study population and patients treated with HMA +/− DLI for relapse after allo-HSCT. Figure S26Effects of "poor risk mutations" and pretransplant strategy on outcome after HMA-based salvage therapy for relapse after allo-HSCT in 29 patients with MDS. Figure S27Impact of conditioning regimen on post-transplant survival and relapse in 50 patients harboring any poor risk mutation. Figure S28Outcome of patients receiving HMA-based salvage therapy for relapse after allo-HSCT depending on TP53 mutation status. Table S1 Patient Demographics, Disease and Transplant Characteristics. Table S2 Gene regions assessed by TruSight Myeloid Sequencing Panel. Table S3 Patient demographics, disease and transplant characteristics in the distinct groups of pretransplant strategies. Table S4 Conditioning regimen. Table S5 Comprehensive list of genetic alterations and sequencing depth informations. Table S6 Impact of gene mutations on post-transplant overall and relapse-free survival - univariate analysis. Table S7 Impact of gene mutations on post-transplant cumulative incidence of relapse and non-relapse mortality - univariate analysis. Table S8 Characteristics of patients with poor risk mutations depending on pretransplant strategy. Table S9 Impact of clinical parameters on post-transplant overall and relapse free survival - univariate analysis. Table S10 Impact of clinical parameters on post-transplant cumulative incidence of relapse and non-relapse mortality - univariate analysis. Table S11 Multivariate analysis for post-transplant overall and relapse-free survival. Table S12 Multivariate analysis for post-transplant cumulative incidence of relapse and non-relapse mortality. Table S13 Moleculargenetic profile and causes of death of patients harboring any poor risk mutation - TP53mut patients. Table S14 Relapse Characteristics of 41 patients treated with HMA +/− DLI for MDS/sAML relapse after allo-HSCT. Appendix S1: Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.