Background
There have been concerns regarding increased peritransplantation complications, especially severe acute graft‐versus‐host disease (aGVHD), in patients with prior use of checkpoint ...inhibitors (CPI) before hematopoietic stem cell transplantation (HSCT).
Methods
The authors performed a retrospective study of 43 patients with acute myeloid leukemia and/or myelodysplastic syndromes who were treated with an antiprogrammed cell death protein 1 (PD‐1) (32 patients) or anticytotoxic T‐lymphocyte–associated protein 4 (CTLA‐4) (9 patients) blockade or both (2 patients) prior to HSCT with the primary outcome of aGVHD by day 100 after HSCT. Outcome analyses were stratified by GVHD prophylaxis as use of post‐HSCT cyclophosphamide (PTCy) (22 patients) or not (non‐PTCy) (21 patients).
Results
The PTCy group demonstrated a trend toward lower grade 3 to 4 aGVHD when compared with the non‐PTCy group (5% vs 22%), although the rates of grade 2 to 4 aGVHD were comparable (49% vs 56%). The interval between CPI and HSCT did not appear to impact the incidence of aGVHD. However, a higher incidence of grade 3 to 4 aGVHD was observed in patients who received >4 treatments of CPI prior to HSCT if they were not given PTCy as GVHD prophylaxis (43% vs 12%). Matched control analyses using patients with no prior use of CPI confirmed the increase in grade 3 to 4 aGVHD with those agents. However, that increased risk was limited to patients who did not receive PTCy and was not observed in patients who received PTCy as GVHD prophylaxis. Despite persistent improvement in GVHD with the use of PTCy, disease control was not compromised and progression‐free survival at 1 year was found to be superior for patients treated with PTCy compared with those not receiving PTCy among patients with prior use of CPI (55% vs 22%).
Conclusions
The results of the current study indicated that HSCT with prior use of CPI appears feasible in patients with acute myeloid leukemia and/or myelodysplastic syndromes and the use of PTCy as GVHD prophylaxis improves outcomes.
The use of checkpoint inhibitors (CPI) prior to hematopoietic stem cell transplantation (HSCT) in patients with acute myeloid leukemia and/or myelodysplastic syndromes affects transplantation outcomes, with an especially increased incidence of grade 3 to 4 acute graft‐versus‐host disease (aGVHD). The use of post‐HSCT cyclophosphamide as GVHD prophylaxis in patients treated with CPI prior to transplantation is found to lead to improved transplantation outcomes, including grade 3 to 4 aGVHD.
PGF is a devastating complication after allogeneic transplant. We retrospectively analyzed our haploidentical transplant registry to report the incidence and impact of DSA and anti-HLA on ...engraftment. 107 patients were identified. Median recipient-age of 22, median donor-age of 31. Sixty-two patients had AML (58%), 29 had ALL (27%), 16 (15%) had other malignancies. Sixty-one recipients (57%) had positive anti-HLA, 56 of them had the DSA results available, of these 17 patients had DSAs (15% of the total number of patients, or 28% of patients who have anti-HLA antibodies). The median cumulative MFI was 2062. Sixty-three percent of the DSA were against class-II HLA antigens. The OS, CIR, aGvHD, and cGvHD did not differ between patients with and without anti-HLA antibodies, nor between patients with and without DSA. The gender of the recipient and donor, as well as the gender mismatch between recipient and donor, were statistically associated with the incidence of anti-HLA antibodies. Three patients only developed GF (2.8%), one was primary (0.9%) and the other two secondary GF (1.9%). None of the GF cases was in patients with anti-HLA antibodies or DSA. The presence of anti-HLA or DSAs did not affect the outcomes including the incidence of PGF.
Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic neoplasm involving skin lesions and disseminated disease into bone marrow, peripheral blood, and lymph nodes, ...characterized by poor clinical outcomes and no standard therapeutic approaches. BPDCN is characterized by the malignant proliferation of precursor plasmacytoid dendritic cells (pDCs). It is now classified by WHO 2016 as a separate entity under myeloid malignancies owing to its unique clinico-pathologic nature, greater understanding of its distinct clinical course, but with some noted clinical, morphologic, and molecular similarities to AML and myelodysplastic syndrome (MDS). One of the most common molecular mutations observed by next-generation sequencing in the vast majority of patients with BPDCN has been the presence of TET2 mutations and variants. Notably, somatic missense and truncating mutations in TET2 have been reported in patients with both BPDCN and AML, yet their differential responses to similar therapeutic regimens in clinical trial testing indicates that there are likely key underlying etiologies that are yet to be determined.
Aims: We sought to investigate and identify critical differences between patients with BPDCN and AML at the molecular level, utilizing a series of advanced analyses including transcriptome microarray, serum multiplex immunoassays and cytokine analysis.
Methods: In order to discern these differences, we profiled bone marrow, peripheral blood and serum samples from primary patients samples with BPDCN (N = 16) and TET2-mutated AML (AMLTET2m) (N = 9) using 3 different assays. We first ascertained somatic point mutations and copy number alterations of 300 genes in our BPDCN specimens using an in-house hematologic malignancy panel (“T300” panel). Next, we confirmed the prevalence of compound truncating TET2 mutations in patients with BPDCN and few copy number alterations in the genes profiled. We then used the transcriptome microarray (ThermoFisher Scientific ClariomTM D Pico Assay, and serum multiplex immunoassays (Cytokine/Chemokine/Growth Factor 45-Plex Human ProcartaPlex™ Panel 1 (ThermoFisher Scientific, formerly Affymetrix) with the addition of IL-3 Human ProcartaPlex™ Simplex Kit, formerly Affymetrix) to compare BPDCN specimens against those from TET2-mutated AML patients.
Results: With the microarray analysis, we found 920 genes to be up-regulated and 791 genes down-regulated in BPDCN specimens as compared to AMLTET2m. We corroborated known differentially expressed marker genes: higher levels of IL3Ra and TCL1A and lower levels of MPO in BPDCN as compared to AMLTET2m specimens. Genes specific to dendritic cells (PTPRS, LTK, LAMP5) were highly expressed in BPDCN than in AMLTET2m specimens. Of interest, two of these genes, PTPRS and LTK, provide possible links to the skin lesions as PTPRS is implicated in the progression of melanoma and LTK is involved in pigmentation of melanocytes. The serum cytokine profile analysis showed significantly elevated levels of eotaxin and RANTES in the BPDCN cohort as compared to the AMLTET2m cohort (Figure 1a,b). Both of these are implicated in allergic and autoimmune reactions by behaving as eosinophil chemo-attractants. Along with the higher levels of PTPRS and dendritic nature of the tumor cells, these findings suggest a possible autoimmune background which exists in the context of disease.
Conclusions: In this novel analysis, we observed elevated levels of eotaxin and RANTES in patients with BPDCN as compared to AMLTET2m. These findings may represent an important aspect of pDC functioning even outside of BPDCN, as pDCs may contribute to the pathogenesis of systemic lupus erythematosus (SLE), an autoimmune disorder with hallmark cutaneous lesions. Moreover, autoimmune pathologies have been hypothesized to damage the bone marrow and induce destruction of myeloid precursor cells. This may incorporate some of the dendritic cell nature since in its natural context, as pDCs serve to recognize foreign particles such as viruses and synthetic oligonucleotides through Toll-like Receptors TLR7/9. These findings suggest that further study into these markers are warranted in patients with BPDCN.
Figure 1. Differential serum cytokine levels between BPDCN and AMLTET2m (a) Eotaxin (pg/mL), Wilcox rank test P < 0.01 (b) RANTES (pg/mL), Wilcox rank test P < 0.05.
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Konopleva:Stemline Therapeutics: Research Funding. Pemmaraju:stemline: Consultancy, Honoraria, Research Funding; plexxikon: Research Funding; SagerStrong Foundation: Research Funding; daiichi sankyo: Research Funding; celgene: Consultancy, Honoraria; Affymetrix: Research Funding; samus: Research Funding; cellectis: Research Funding; abbvie: Research Funding; novartis: Research Funding.
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, male-predominant hematologic malignancy with poor outcomes and with just one recently approved agent (tagraxofusp). It is characterized ...by the abnormal proliferation of precursor plasmacytoid dendritic cells (pDCs) with morphologic and molecular similarities to acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) in its presentation within the bone marrow and peripheral blood. To identify disease-specific molecular features of BPDCN, we profiled the bone marrow, peripheral blood, and serum samples from primary patient samples using an in-house hematologic malignancy panel ("T300" panel), transcriptome microarray, and serum multiplex immunoassays. TET2 mutations (5/8, 63%) were the most prevalent in our cohort. Using the transcriptome microarray, genes specific to pDCs (LAMP5, CCDC50) were more highly expressed in BPDCN than in AML specimens. Finally, the serum cytokine profile analysis showed significantly elevated levels of eosinophil chemoattractants eotaxin and RANTES in BPDCN as compared with AML. Along with the high levels of PTPRS and dendritic nature of the tumor cells, these findings suggest a possible pre-inflammatory context of this disease, in which BPDCN features nonactivated pDCs.
Background
The combination of the αPD-1 (nivolumab) and hypomethylating agent azacytidine demonstrated encouraging response rate and overall survival in relapsed/refractory acute myeloid leukemia ...(AML) patients, compared to azacytidine alone1 (NCT02397720). However, the percentage of the patient who achieved an IWG 2016 response to such therapy was still limited (overall response rate = 33%), so it is desirable to have early predictive biomarkers to facilitate patient stratification and selection for future trials. A better understanding of T cells (primary targets of αPD-1 therapy) from bone marrow (BM) and peripheral blood (PB) in AML pre-therapy and on-therapy should yield valuable insights on the treatment-induced anti-tumor response.
Methods
We performed whole transcriptomic profiling (RNA-sequencing, RNA-seq) on T cells from a cohort of AML patients who were enrolled in the clinical trial mentioned above, and treated with azacytidine and nivolumab (Table 1). Sixty-four FACS-sorted patient-derived T cell samples from cryopreserved peripheral blood (PB) or bone marrow (BM) aspirates, which were collected pre-therapy (T0) and after the first round of treatment (end of cycle one, EOC1), were evaluated. By leveraging subset definitions based on scRNA-seq results from T cells of cancer patients, we implemented deconvolution of our bulk T-cell RNA-seq data to obtain the relative abundance of different T-cell subsets (in-silico dissection without physical isolation).
Results
We performed ex vivo cDNA library preparation on 2,000-100,000 sorted T cells and yielded a minimal of 17 million sequencing reads per BM T-cell library and 2.6 million sequencing reads per PBMC T-cell library. We compared the gene expression profile of peripheral blood T cells from AML patients (CD4: n = 16; CD8: n = 15) and healthy donors (HD, n = 8)2,3 to validate our methodology. The deconvolution results were consistent with previously published flow-cytometry data profiling cancer patients4,5 (Figure 1). In comparison with HDs, circulating CD4 T cells from AML patients consisted of a higher frequency of Treg (AML versus HD: 5.1% versus 0.6%, P<0.0001)4. Peripheral blood CD8 T cells from AML patients were with a significantly lower frequency of naïve (AML versus HD: 22.1% versus 55.9%, P<0.0001), a higher frequency of effector phenotype (AML versus HD: 35.6% versus 6.7%, P =0.0011), and an increasing frequency of exhausted phenotype (AML versus HD: 19.1% versus 0.0%, P<0.0001)5.
Independent of the clinical responses attained on the azacytidine and nivolumab, comparison of the pre-therapy CD8 T cells from BM and PB from all AML patients using both gene set enrichment analysis and deconvolution indicated that the BM CD8 T cells were more activated/differentiated compared with PBMC CD8 T cells, likely reflective of an ongoing immune response against the AML. We also found treatment-induced gene expression profile changes in the AML circulating CD8 T cells, characterized by increased cell metabolism and cell proliferation. Deconvolution identified that pre-therapy relative abundance of exhausted (CD3+CD8+PD-1+CD45RO+) and effector (CD3+CD8+CD45RA+Tbet+PD-1lo) CD8 T cells (plasticity) could serve as subpopulations relevant for patient stratification (Figure 2). We further validated these results using CyTOF wherein these same subpopulations were differentially abundant between responders and non-responders at the pre-therapy time-point.
Conclusions
Collectively, the analyses of RNA-seq of CD8 T cells from AML patients on the azacytidine with nivolumab trial revealed that (1) the PD-1 blockade-based treatment-induced gene expression profiling changes (increased cell proliferation and metabolism) of CD8 T cells are detectable as early as EOC1 in the peripheral blood; (2) specific subpopulations of plastic CD8 T cells identified may have the potential to serve as an actionable biomarker to select AML patients most likely to benefit from such immune checkpoint therapies. These findings need to be confirmed in larger studies, planned or currently being conducted, with αPD-1 based therapies in AML and MDS.
References
1. Daver, N. et al. Cancer Discov.9, 370-383 (2019).
2. Corces, M. R. et al. Nat. Genet.48, 1193-1203 (2016).
3. Linsley, P. S. et al. PLoS One9, (2014).
4. Szczepanski, M. J. et al. Clin. Cancer Res.15, 3325-3332 (2009).
5. Knaus, H. A. et al. JCI Insight3, (2018).
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Varadarajan:CellChorus: Employment, Equity Ownership.
Subsets of acute myelogenous leukemia (AML) are characterized by molecular alterations with prognostic significance, however, little is known about how modifiable behaviors, such as cigarette ...smoking, intersect with genetic factors. Mutations rendering the Fms-like tyrosine kinase 3 (FLT3) constitutively active, such as internal tandem duplication (ITD), are associated with refractory disease and therapy resistance. Inhibition of the FLT3 kinase shows some benefit in this population, as highlighted by the FDA approvals of midostaurin and gilteritinib, but overall outcomes remain poor. Cigarette smoke exposure (CSE) also marks a population of patients with poor prognosis. Current and former smokers who develop AML are known to have worse survival as compared to never smokers (Alfayez M., et al. ASCO 2019), but the impact of FLT3 mutation and subsequent associated treatment response has not been studied. Also, the underlying mechanism of how history of cigarette smoking influences leukemia biology and response to therapy is poorly understood.
In order to model a history of smoking in AML patients, NOD-SCID mice (n=25) were exposed to CSE using a smoking robot for 2 hours, 5 days/week, for 2 weeks. Mice were then inoculated via tail-vein injection with luciferase tagged human FLT3-ITD cells and leukemia burden was monitored through noninvasive imaging. CSE continued through the duration of the experiment, post engraftment. Control “non-smoking” mice (n=15) were only injected with leukemia cells. Within one week post leukemic introduction, a significant increase in leukemic burden as measured by bioluminescence was apparent in mice exposed to CSE versus control mice (P-value<0.0001). To model the impact of smoking cessation upon diagnosis of AML in patients, experiments were modified to halt CSE once leukemic engraftment was detectable by non-invasive imaging. Smoking cessation versus continuous smoke exposure yielded reduced relative leukemic burden. Mice with continuous smoke exposure had higher rates of leukemia compared to mice who ceased smoking (n=10) one week prior (P-value =0.0064). These rapid changes in leukemic burden suggest that CSE may prime the microenvironment to promote leukemia progression or directly affect leukemia cells. To address the latter possibility, human AML cells were exposed to cigarette smoke condensate (CSC), which contains the chemicals present in cigarettes, for two weeks before introducing the cells into mice. A significant increase in leukemic burden was observed in mice injected cells exposed to CSC compared to mice injected with unexposed leukemia cells (P-value <0.001), highlighting a direct role for the chemicals in cigarettes on in vivo leukemia proliferative factors.
Smokers are known to carry altered global DNA methylation signatures that persist decades after quitting. To measure DNA methylation changes in the in vivo models described above, we examined spleens of non-smoking and smoke exposed mice by reduced representative bisulfite sequencing (RRBS). Sequences were mapped to either the human or mouse genome, (enabling identification of leukemia specific versus microenvironment specific alterations) and were compared in the smoking and non-smoking mice. Over 200 genes exhibited significant DNA methylation alterations in their promoter regions. Genes involved in RNA polymerase activity and chromatin remodeling were highly represented amongst those with altered DNA methylation.
The clinical significance of our observations was confirmed in a cohort of 58 treatment naïve FLT3-ITD AML patients at MD Anderson receiving intensive induction therapy: 41 never smokers and 17 ever smokers. Smokers had significantly reduced survival as compared to the never smokers (median overall survival of 18 vs 23 months, P-value 0.0092).
Collectively our findings indicate that short-term CSE is sufficient to alter DNA methylation patterns and accelerate the early progression of FLT3-ITD AML in vivo. Smoking cessation upon diagnosis may slow leukemic growth relative to smoking throughout AML therapy prompting the consideration of behavioral interventions for smokers with AML. Improved understanding of the mechanism of leukemic progression and drug resistance from CSE is expected to lead to improved treatment paradigms designed for patients with a history of cigarette smoking.
Konopleva:Genentech: Honoraria, Research Funding; Kisoji: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. DiNardo:agios: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; jazz: Honoraria; medimmune: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; daiichi sankyo: Honoraria; syros: Honoraria; celgene: Consultancy, Honoraria.
(
) mutations occur in approximately a third of acute myeloid leukemia (AML) patients and confer an adverse prognosis. Numerous studies have evaluated
targeting as single agent and in combination ...approaches in frontline and relapsed AML. At this time, midostaurin, a multikinase inhibitor, is the only FLT3-inhibitor that is US FDA approved to be used in combination with induction therapy in the frontline
mutated AML setting based on improved overall survival noted in the RATIFY Phase III trial. The utility of midostaurin in maintenance post stem cell transplantation has shown promising results and further studies are still ongoing. In this review, we discuss the studies that led to the inception of midostaurin as a targeted kinase inhibitor, its evaluation in AML, the early clinical trials and the large Phase III clinical trial that led to its eventual US FDA-approval in
mutated AML. Our review also discusses data on midostaurin adverse effects, mechanisms of resistance and limitations of its utility. We further discuss emerging second-generation FLT3 inhibitors, with a focus on quizartinib and gilteritinib and future directions to enhance FLT3-inhibitor efficacy and overcome mechanisms of resistance.
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