Longitudinal Genomic Analyses In Chronic Lymphocytic Leukemia (CLL) Patients Reveal Clonal Relationship and Genomic Evolution In Disease Progression and After Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3605-3605
Author(s):  
Esteban Braggio ◽  
Neil E. Kay ◽  
Scott Van Wier ◽  
Stephanie Smoley ◽  
Jeanette Eckel-Passow ◽  
...  
Keyword(s):  
Disease Progression ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Clonal Evolution ◽  
The Other ◽  
Genomic Evolution ◽  
Advisory Committees ◽  
Time Points ◽  
Time Point

Abstract Abstract 3605 CLL is a malignant B-cell disorder characterized by the accumulation of small B lymphocytes with a mature appearance in blood, marrow and lymph nodes. Despite effective treatment options, all patients with CLL will eventually relapse after therapy. This could be due in part to the presence of subclones of the CLL cell population that harbor genetic abnormalities, which confer resistance to treatment. The aims of this study were to investigate the clonal evolution in longitudinal samples of CLL patients and to identify genetic alterations associated with disease progression and resistance to therapy. Sequential analyses were performed in 51 samples from 23 patients who were included in a previously reported clinical trial of pentostatin, cyclophosphamide and rituximab (PCR) given every 3 weeks for 6 cycles in previously untreated CLL (Blood 109:2007). In all cases the first sample analyzed was prior to therapy. In 5 of 23 patients, three time points were analyzed: >6 months prior to entry onto PCR trial (time point A), just before starting with the PCR regimen (time point B), and the time of relapse after PCR trial (time point C). Seven patients were analyzed at time points A and B; 9 at time points B and C and 2 at time points A and C. The median time between points A and B was 17.5 months (range 8–48 months) and between points B and C was 20.5 months (7–60 m). All samples were examined by array-based comparative genomic hybridization (aCGH) using the Agilent Sureprint G3 (1 million probe) array. aCGH findings were confirmed by interphase FISH using probes for D13S319 (MIR16–1/MIR15A), RB1, MDM2, CEP12, CEP6, MYB, TP53, NFKBIA, PERP and FGFR1 loci. Overall, we observed a small increase in the number of copy-number abnormalities (CNA) with disease progression. Twenty-two of the 23 patients with paired samples harbored at least one CNA that persisted in all samples, indicating clonal relationship between the sequential samples. In 15 of the 23 patients the tumor clone was stable and no CNA differences between time points were identified. Conversely, genomic evolution was found in 8 patients. In 3 cases the genetic differences were observed pre treatment (between time points A and B) and in the other 5 cases, the observed changes were found after therapy (between time points A and C or between B and C). One remarkable case with genome evolution exhibited two subclones sharing trisomies 12 and 19, but with several unique CNA confined to each subclone. The first subclone was characterized by deletions of 6q, RB1, MIR16-1/MIR15A and 3 other losses, while the second subclone showed homozygous deletion of MIR16–1/MIR15A and 5 other monoallelic deletions. The first subclone was predominant at time points A and B (60–70% of cells), but was present in only 10–20% of cells at time point C as confirmed by FISH. Conversely, the second subclone was observed in ~20% of cells at time points A and B and became predominant after therapy, found in ~80% of cells at time point C. Another case was characterized by deletion 11q32 (including ATM and others) as the sole abnormality at time point B. Significant genomic complexity was observed at time point C, including deletions of 11q32, 9p21 (CDKN2A), 9q12-q33, 14q13.2 (NFKBIA) and 17p (TP53), and gains of 2p16 (REL) and 9q34. Interestingly, the deletion 11q32 from both time points arose independently at each time point, as they exhibited different chromosomal breakpoints and copy number variants. Moreover, the other CNA found at relapse were not identified at diagnosis (confirmed by aCGH and FISH). For evolution of specific CNA, trisomy 12 was found in 5 cases at the first sample analyzed and was stable with no changes between time points. The frequency of deletions 13q14.3 (MIR16-1/MIR15A) and 17p increased at the later time points. Conversely, –6q decreased in frequency across time points (3 cases in time points A–B and 1 case in time point C). In summary, at least 35% of CLL patients exhibited clonal evolution and at least 9% showed evidence of multiple subclones. This subgroup of CLL patients provides an exceptional framework for comprehensive analysis of genome evolution during disease progression before and after therapy. Our observations also support the hypothesis of a common CLL progenitor cell can give rise to clonally related, but genetically evolving subpopulations of tumor cells. Finally, this study may bring novel information regarding the drug resistance pathways utilized by CLL B cell clones post therapy. Disclosures: Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding. Fonseca:Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

scholarly journals Clonal Evolution at First Sight: A Combined Visualization of Diverse Diagnostic Methods Improves Understanding of Leukemia Progression

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1293-1293
Author(s):  
Sarah Sandmann ◽  
Yvonne Lisa Behrens ◽  
Felicitas Thol ◽  
Michael Heuser ◽  
Doris Steinemann ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Genetic Architecture ◽  
Clonal Evolution ◽  
Copy Number Variations ◽  
Advisory Committees ◽  
Time Points ◽  
Myeloid Neoplasia ◽  
Bioinformatic Approach ◽  
Time Point

Abstract Introduction: Myeloid neoplasia, including acute myeloid leukemia and myelodysplastic syndrome are heterogeneous hematopoietic stem cell disorders which are marked by the acquisition of somatic alterations and clonal evolution 1. Patients with myeloid neoplasia are classified due to the WHO classification systems and besides clinical and hematological criteria, cytogenetic and molecular genetic alterations highly impact treatment stratification 2. In routine diagnostics, a combination of methods is used to decipher different types of genetic variants, i.e. single nucleotide variants (SNVs), insertions/deletions (indels), structural variants (SVs) and copy number variations (CNVs) which may not be detected using one single method. Methods: We used a bioinformatic approach to analyze clonal evolution and genetic architecture in patients with myeloid neoplasia using single nucleotide variants (SNVs), insertions/deletions (indels), structural variants (SVs) and copy number variations (CNVs). Six patients were comprehensively analyzed using karyotyping, fluorescence in situ hybridization (FISH), array-CGH and a custom NGS panel with 148 genes/ gene regions that are recurrently affected in patients with hematologic neoplasia. At the initial time point or during disease course all patients showed many genomic variants: Two patients (#1, #2) were analyzed at one time point (initial), two patients (#3, #4) were analyzed at two time points (initial and progression), one patient (#5) was analyzed at four time points (initial, progression, remission, relapse), and one patient (#6) was analyzed at five time points (initial, remission, relapse, progression, remission). Results and Conclusions: Clonal evolution was reconstructed manually, integrating all mutational information on SNVs, indels, SVs and CNVs 3. Cancer cell fractions (CCFs) for SNVs and indels were estimated based on VAFs, assuming heterozygous variants (2*VAF=CCF). CCFs for SVs and CNVs were estimated based on cell counts reported for karyotyping and FISH analyses. For SVs as well as CNVs, which were only detected by array-CGH, CCF was estimated based on logRatio. In case of a CNV overlapping the position of an SNV or indel, calculation of CCF is less straightforward. Altogether, we differentiate between three cases: 1) The CNV occurred prior to the SNV/indel, but in the same cells. 2) The SNV/indel occurred prior to the CNV, but in the same cells. 3) SNV/indel and CNV exist in parallel, independent of each other. The bioinformatic approach reconstructed clonal evolution (linear and/or branching) for all patients and the results were visualized by fishplots. We identified alterations, which play a role in the pathogenesis of the disease (driver) and alterations, which occur during disease development (passenger). On two samples, we showed that reconstruction of clonal evolution is possible even with data from one time point only. For other samples, providing data on more than one time point, the effect of therapy was estimated. This bioinformatic approach offers the possibility of analyzing clonal evolution and genetic architecture at one or more time points of analysis. The visualization of the results in fishplots contributes to a better understanding of genetic architecture and helps to identify possible targets for the disease (personalized therapy). Furthermore, this model can be used to identify markers in order to assess minimal residual disease (MRD). Figure 1 Reconstruction of clonal evolution (time point of analysis: black triangle) for patient #4 (diagnosis: secondary acute myeloid leukemia). References: 1. Doulatov S, Papapetrou EP. Studying clonal evolution of myeloid malignancies using induced pluripotent stem cells. Curr Opin Hematol. 2021;28(1):50-56. 2. Edited by Swerdlow SH CE, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer. 2017;Revised 4th edition. 3. Reutter K, Sandmann S, Rohde J, et al. Reconstructing clonal evolution in relapsed and non-relapsed Burkitt lymphoma. Leukemia. 2021;35(2):639-643. Figure 1 Figure 1. Disclosures Thol: Jazz: Honoraria; BMS/Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Astellas: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Heuser: Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding.

Analysis of Serial Patient Samples Reveals a Variety of Clonal Dynamics In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1923-1923
Author(s):  
Jonathan J Keats ◽  
Esteban Braggio ◽  
Scott Van Wier ◽  
Patrick Blackburn ◽  
Angela Baker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Leukemia ◽  
Advisory Committees ◽  
Tumor Mass ◽  
Time Points ◽  
Tumor Genome ◽  
Time Point

Abstract Abstract 1923 Our understanding of the genetic abnormalities associated with the development of multiple myeloma has increased significantly in the last decade. However, very little is known about how, or if, myeloma tumor genomes change with time and if therapeutic interventions influence these events. To address these issues we studied a cohort of 29 patients for whom at least two serial samples (1-65 months, median 19 months) were available for analysis. Each serial pair was analyzed by both array-based comparative genomic hybridization (aCGH) and microarray gene expression profiling (GEP) to identify DNA copy number abnormalities (CNA) at a 25kb resolution and gene expression differences present in the bulk of the tumor mass. Though this does not address the intra-clonal heterogeneity that may exist at a given time point, it does answer if the bulk of the tumor mass is changing with time. This study has unearthed several surprising and clinically relevant findings. First, myeloma tumor genomes are not as unstable as previous cytogenetic analyses suggest. In 40% of patients we observed no detectable CNA changes (1-37 months, median 12 months). In 24% of patients we observed the exclusive acquisition of new CNA (1-12, median 3.5) (3-22 months, median 18 months). In 36% of patients we observed both the loss (1-20, median 3) and gain (1-33, median 21) of CNA (5-43 months, median 20 months). Because time was not a significant influence on the detection of stable or unstable genomes we compared CNA changes with TC class and found patients with the high-risk 4p16 and maf IgH translocations were over-represented in the latter subset of patients. These observations raise the question of what happens between multiple rounds of therapy and if different regimens influence these phenotypes differently. For two patients with no CNA changes between the first two time points there was an additional sample that extended the follow-up by 52 and 12 months. Again no CNA changes were seen between diagnosis and these final samples taken 63 and 50 months later. For one patient with CNA changes (5 shared, 29 lost, and 32 gained) we have a detailed time course of 5 samples from diagnosis through to end-stage plasma cell leukemia. This patient received continuous lenalidomide-dexamethasone (Rd) for 20 months and progessed with a clone containing a BIRC2/3 deletion, which activates the NFKB pathway. The patient received single agent PR-171 and a bortezomib containing regimen and unexpectedly, the tumor genome observed in the third sample was almost identical (32 shared, 2 lost, and 4 gained CNA) to the first time point, including two copies of BIRC2/3. Subsequently, the patient received melphalan-prednisone-bortezomib (MPV) and the tumor genome observed in the fourth and fifth samples, which were identical, were similar to that seen in the second sample (24 shared, 13 lost, and 39 gained CNA). To understand these observations better we performed FISH to ascertain if the observed clones were detectable earlier, albeit at a low frequency. These experiments proved that the two dominant subclones observed at time points 1 and 3 versus 2, 4, 5 were mutually exclusive at the single cell level. Moreover, both of these clones were detectable at diagnosis with 12% of the tumor mass being the second subclone that eventually evolved into plasma cell leukemia. Interestingly, we assayed 5 of the 39 unique CNA observed in the final two samples and only one, the 17p13 deletion, was detectable earlier. This suggests the MPV regimen effectively eliminated a clone that was previously sensitive to Rd and selected for a dramatically evolved subclone that was previously sensitive to two different proteasome inhibitors. Although it is clear that the high-risk patients are enriched in the subset with the most changes, it is not clear if the specific drugs used (Melphalan vs IMID vs proteasome inhibitor) or intervention strategy (Cycled vs continuous/maintenance) and perhaps the response achieved (PR vs CR) influences these events. These observations do highlight two important clinical concepts that need to be considered in the future. First, the meaning of a partial response needs further investigation as this may reflect effective elimination of one subclone but not another. Second, because some patients are not changing or can revert back to a previous subclone we need to consider re-chanllenging patients with previously effective regimens when patients progress. Disclosures: Fonseca: Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties. Stewart:Millennium: Consultancy; Celgene: Honoraria. Bergsagel:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Millennium: Speakers Bureau; Novartis: Speakers Bureau.

scholarly journals Outcomes, Causes of Discontinuation and Mutation Profile of Patients with Mantle Cell Lymphoma Who Progressed on Acalabrutinib

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4151-4151 ◽  
Author(s):  
Preetesh Jain ◽  
Rashmi Kanagal-Shamanna ◽  
Shaojun Zhang ◽  
Chi Young Ok ◽  
Makhdum Ahmed ◽  
...  
Keyword(s):  
Disease Progression ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Median Number ◽  
Copy Number Alterations ◽  
Advisory Committees ◽  
Mantle Cell

Abstract Introduction: Acalabrutinib is a Bruton tyrosine kinase (BTK) inhibitor approved for treatment of relapsed patients (pts) with mantle cell lymphoma. We have reported previously that ibrutinib refractory MCL pts have poor survival. However, outcomes, causes of discontinuation, management and the genomic landscape of MCL in pts who discontinued acalabrutinib are rarely reported. Method: We reviewed charts from all MCL pts treated with single agent acalabrutinib (n=28) in the relapsed setting and identified 15 pts who discontinued acalabrutinib and who are described in this analysis. Outcome after discontinuing acalabrutinib is reported. Whole-exome sequencing (WES) with SureSelect Human All Exon V6 was performed on 10 tumor specimens and 5 matched germline samples collected from 9 pts whose MCL progressed on acalabrutinib; among these pts 4 tumors were collected at baseline and 6 were collected after disease progression. One patient had sufficient DNAs available for both time points (baseline and progression). Results: The median duration on treatment with acalabrutinib was 6.5 months (1 to 29 months) and the median number of cycles of acalabrutinib treatment was 6 (range, 1-30). Seven pts had complete remission (CR) as their best response on acalabrutinib, 5 were primary refractory and 3 achieved partial remission. In 12 pts (80%) acalabrutinib was discontinued due to disease progression (2 pts transformed from classic to blastoid and pleomorphic type at progression) and 3 pts were discontinued due to intolerance (one for fatigue and idiopathic encephalopathy, one due to unrelated severe aortic stenosis and another for cytopenias secondary to therapy related myelodysplasia; all three pts were in CR). Nine pts had classic and 3 pts each had blastoid or pleomorphic features before starting acalabrutinib. Overall, median Ki-67 expression was 50% (range, 5-100) and all pts had high a MIPI score. The median number of prior treatments was 1 (range, 1-3); all chemo-immunotherapy (10 pts were previously treated with rituximab-hyper-CVAD) and none with ibrutinib. Two pts who transformed on acalabrutinib received acalabrutinib for a median duration of 12 months (range, 8-16.5). Median follow up after discontinuation was 27 months and the median survival was 25 months (26 months for progression and 1.5 months for intolerance; p <0.001, Figure-1A). Patients who discontinued due to intolerance did not get subsequent treatment for MCL. Among the 12 pts who progressed on acalabrutinib, 11 pts received systemic therapy for MCL [seven received ibrutinib based therapies (2 non responders, 3 achieved CR and 2 were PR and all pts progressed subsequently), 3 got chemo-immunotherapy and progressed and one pt did not receive any treatment and was lost to follow up and died. Six patients received a clinical trial with CAR-T cells (results will be reported separately). Overall, at the time of last follow up, 8 pts were alive and 7 were in CR. Recurrently mutated genes in these tumors included ATM (6/10; 60%), TP53 (4/10; 40%), KMT2C (3/10), MYCN (2/10), NOTCH1 (2/10), NOTCH3 (2/10), and MEF2B (2/10) (Fig. 1B). We did not detect any mutation or copy number alterations in BTK, PLCG2, TRAF2/3 and MYD88 that have been reported previously to be associated with ibrutinib resistance. Compared to tumors at baseline, ATM was mutated at a higher frequency in samples at progression (67% vs. 50%; p=NS). To investigate the mutation evolution on acalabrutinib treatment, mutation profiles, particularly the mutation variant allelic fractions (VAFs), were compared between the baseline and progression samples from pt-1 (Fig. 1C). Mutation of MYCN, MEF2B, ATM, and NOTCH1 were identified in both tumors at similar VAFs, whereas mutation of CARD11 (two mutations), NLRC5 and B2M were detected only at progression. In pt-1, both the NLRC5 and β2M mutations acquired at disease progression were truncating, suggesting loss-of-function alterations. Copy number analysis reveals frequent whole-genome doubling and intensive copy number alterations in all tumors, including recurrent losses of chromosome 9p, 17p, and chromosome 13, indicating chromosomal instability as a driver of disease progression. Conclusion: Patients who progress on acalabrutinib have a poor outcome, and newer therapies are required for their treatment. In this small cohort, we observed non-BTK mutations associated with acalabrutinib resistance and disease progression. Disclosures Nastoupil: Genentech: Honoraria, Research Funding; TG Therappeutics: Research Funding; Spectrum: Honoraria; Gilead: Honoraria; Merck: Honoraria, Research Funding; Janssen: Research Funding; Celgene: Honoraria, Research Funding; Karus: Research Funding; Novartis: Honoraria; Juno: Honoraria. Neelapu:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Fowler:Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Wang:Acerta Pharma: Honoraria, Research Funding; MoreHealth: Consultancy; AstraZeneca: Consultancy, Research Funding; Kite Pharma: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Whole Genome Sequencing Defines the Clonal Architecture and Genomic Evolution in Myeloma: Tumor Heterogeneity with Continued Acquisition of New Mutational Change

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 297-297
Author(s):  
Nikhil C. Munshi ◽  
Hervé Avet-Loiseau ◽  
Philip J. Stephens ◽  
Graham R Bignell ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Copy Number ◽  
Genomic Evolution ◽  
Copy Number Alterations ◽  
Advisory Committees ◽  
Genomic Changes ◽  
Time Points ◽  
Progression Of Disease ◽  
Serial Samples ◽  
Generation Sequencing

Abstract Abstract 297 Genetic instability, a central feature of malignant cells, plays an important role in oncogenesis by perturbing critical cell signaling pathways, including activation of oncogene and/or deletion of tumor suppressor genes; moreover, ongoing genomic changes are associated with tumor progression, invasiveness, and drug resistance. We hypothesized that the inherent genomic instability in tumors would lead to a heterogeneous tumor cell population at diagnosis, thereby providing a substantial substrate for ongoing selection during progression of the disease. We have here investigated serial samples from patients with multiple myeloma (MM) using a variety of methodologies to study the genomic evolution. Purified MM cells, as well as matching normal samples from the same patients, were collected at 2 time points at least 4 months apart and subjected to genomic analyses. To compare the changes between matching normal and MM cells collected at two time points (range 5–18 months apart), we utilized SNP 6.0 array to identify copy number alterations (CAN); identified genome-wide rearrangements utilizing a low-coverage whole genome shotgun approach generated via next-generation sequencing; and, importantly, for the first time in 13 patients performed whole exome sequencing based on a solution phase capture and next generation sequencing. Variants identified in both the rearrangement and exome screens were validated on orthogonal platform. Our analysis demonstrates: 1) a significant intratumoural heterogeneity at the initial time of evaluation, suggesting that even at diagnosis multiple sub-clones may be co-existing; 2) discernable shifts in the clonal structure of disease at the time of progression (2nd sample) that indicates appearance of previously undetected sub-clones. We have observed frequent mutational changes (3 or more samples) involving CCND1, DTX1, KRAS genes. The changes are irrespective of intervention and disease status. We have also observed appearance of new copy number alterations and heterozygosity between 2 serial samples, ranging from 0.021 – 2.674 % (i.e. per 100 informative loci investigated), as well as insertion/deletion changes. These data therefore confirm evolution of genomic changes in MM patients over time and identify molecular alterations associated with progression of disease and development of drug resistance. This study begins to define the clonal architecture of MM and will provide insights into the impact of this structure and heterogeneity on pathogenesis and progression of disease. Disclosures: Munshi: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Ibrutinib-Based Therapy Improves Anti-Tumor T Cell Killing Function Allowing Effective Pairing with Anti-PD-L1 Immunotherapy Compared to Traditional FCR Chemoimmunotherapy; Implications for Therapy and Correlative Immune Functional Data from the Phase III E1912 Trial

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 236-236 ◽  
Author(s):  
Despoina Papazoglou ◽  
Connie E. Lesnick ◽  
Victoria Wang ◽  
Neil E. Kay ◽  
Tait D. Shanafelt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Phase Iii ◽  
Advisory Committees ◽  
Time Points ◽  
Killing Function ◽  
Time Point

Abstract The targeted therapy ibrutinib inhibits B cell receptor signaling (BTK inhibitor) and has yielded high response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL). However, it is widely believed that the addition of immune therapies to targeted drugs will be required to activate anti-tumor immunity and work towards curative therapy. Identifying effective combinations of targeted drugs and/or standard chemotherapy with immunotherapy is a priority research area and particularly relevant for CLL, as patients' T cells have been shown to exhibit profound tolerance/exhaustion and notably, no activity was reported in a recent trial of anti-PD-1 immunotherapy for relapsed disease. Ibrutinib has shown beneficial immunomodulatory activity in CLL by inhibiting IL-2-inducible T cell kinase (ITK) as well BTK that is associated with increased effector CD4+and CD8+ T cell numbers and decreased expression of inhibitory checkpoint receptors such as PD-1 on patient T cells. Here we have performed comparative immune bioassays from a randomized phase III trial comparing ibrutinib-based therapy to traditional FCR chemoimmunotherapy to assess the effects of treatments on anti-tumor T cell function. Viable peripheral blood mononuclear cell samples were collected serially (baseline, 6 months and 12 months) from CLL patients on the randomized phase III E1912 trial of ibrutinib and rituximab versus FCR for previously untreated disease to allow longitudinal batched immune analysis. Cytotoxicity assays revealed that highly purified CD3+ T cells from the FCR treated patients at 6 and 12-month time-points did not change their activated killing function against autologous baseline CD19+ CLL tumor B cells (acting as target antigen-presenting cells pulsed with superantigen, sAg) compared to pre-treatment/baseline exhausted T cells (n=22). In contrast, patients treated with ibrutinib-based therapy had a significant increase in activated anti-tumor T cell killing function (P<.01, n=22) at both 6-month (66% increase) and 12-month (89% increase) time-points. Flow cytometric analysis of circulating immune subsets revealed that the percentage of PD-1 and PD-L1 positive cells among CD8+ and CD4+ T cells (particularly effector compartments) were reduced with ibrutinib-based therapy, whereas only a partial reduction was detected following FCR treatment. However, patients' T cells from both treatment arms responded normally to T cell receptor engagement by upregulating these checkpoint molecules. This led us to explore ex vivo treatment of highly purified CD3+ T cells and CD19+ CLL B cells from both treatment arms with anti-PD-L1 or anti-PD-1 immunotherapy prior to cytotoxicity assays. Our functional data revealed that the T cells from both FCR time-points (6 and 12-months) were not sensitive to either anti-PD-L1 (n=14) or anti-PD-1 (n=14) treatment. In contrast, ibrutinib-based treatment sensitized anti-tumor T function (23% increase in killing) following anti-PD-L1 treatment (n=14) at the 6-month time-point only (P<.01) but not with anti-PD-1. To investigate the mechanism underlying these effector function differences, we compared the ability of highly purified CD3+ T cells from each treatment arm (n=45) time-point to form F-actin immunological synapses with baseline autologous CLL tumor B cells. Quantitative confocal image analysis revealed that ibrutinib-based therapy significantly (P<.01) enhanced polarization of F-actin, tyrosine-phosphorylated proteins and granzyme B at immune synapses with tumor cells at both 6 and 12-month time-points, whereas FCR treated patient T cells failed to mobilize these lytic synapse molecules. Importantly, our assays have revealed that T cells from both FCR treatment time-points formed "non-polarized" immune synapses with tumor cells, in keeping with cytotoxic dysfunction and insensitivity to additional checkpoint immunotherapy. In contrast, our functional correlative bioassays have revealed that ibrutinib-based therapy can reactivate exhausted cytolytic T cell function and suggest to us, a potential therapeutic window for anti-PD-L1 immunotherapy at the earlier 6-month time-point. We believe this data supports the concept of incorporating functional bioassays to immune-monitoring assays associated to clinical trials that should aid knowledge-led design of future combination immunotherapy. Disclosures Kay: Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Shanafelt:Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding; MedImmune: Research Funding.

scholarly journals High Clonal Complexity of Resistance Mechanisms Occurring at Progression after Single-Agent Targeted Therapy Strategies in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-16
Author(s):  
Ella R Thompson ◽  
Tamia Nguyen ◽  
Yamuna Kankanige ◽  
Mary Ann Anderson ◽  
Sasanka M. Handunnetti ◽  
...  
Keyword(s):  
Single Cell ◽  
Disease Progression ◽  
Board Of Directors ◽  
Research Funding ◽  
Single Cell Analysis ◽  
Clonal Evolution ◽  
Single Agent ◽  
Resistance Mechanisms ◽  
Lymphocytic Leukemia ◽  
Advisory Committees

Progression of chronic lymphocytic leukemia (CLL) on venetoclax (VEN) and BTK inhibitors (BTKi) is associated with acquired genomic variants in BCL2/MCL1/BCL2L1 and BTK/PLCG2, respectively, in some patients. We aimed to assess the clonal structure and evolution of resistance in patients (pts) with progressive disease treated with single agent VEN or BTKi (or both as sequential monotherapies) using next generation sequencing (NGS) and single cell sequencing. Seven pts with CLL and 1 with mantle cell lymphoma (MCL) with disease progression on VEN, ibrutinib (IBR) or zanubrutinib (ZANU) were identified from patients treated at our institutions. Pts were selected on the basis of multiple known resistance mechanisms from previous analysis of mutations (muts) and copy number changes detected using clinical bulk NGS targeting genes of interest including BCL2, MCL1, BCL2L1, BAX, BAK1, BTK, PLCG2, CXCR4, as well as TP53 and SF3B1. Of the 8 pts selected for single cell analysis, all had disease that was relapsed/refractory to chemotherapy prior to receiving either VEN (3 pts), BTKi (2 pts) or sequential VEN-BTKi (3 pts). 6,520-16,378 individual cells from 9 samples (8 pts) were analyzed (total 103,388 cells) using a custom panel targeting pt-specific muts on the Tapestri platform (Mission Bio). A summary of genomic abnormalities detected across the cohort is presented in Figure 1. We first evaluated the relationship between genomic resistance mechanisms within the context of single agent (VEN or BTKi) as well as sequential VEN-BTKi treatment. In CLL pts treated with a single agent, all BCL2 muts in VEN pts and BTK muts in IBR or ZANU pts were identified in different subclones consistent with an oligoclonal pattern of disease progression with independent clonal acquisition of resistance mechanisms. Both pts who received ZANU (either as a single agent or sequentially) harbored the BTK L528W mut (previously described as enriched in ZANU progressors; Handunnetti ASH 2019) in independent clones from BTK C481 muts. In pts who received sequential VEN-BTKi treatment, clones were observed that harbored established or novel dual genomic resistance mechanisms within the same cell (BTK mut/MCL1 amp in CLL, BTK/BAX muts in MCL). However, this was not observed in all clones or for all pts, suggesting the presence of further undetected resistance mechanisms (genetic or other). Given the unique ability of single cell sequencing to resolve mut context within a clonal hierarchy, we next assessed this phenomenon within our cohort utilizing other muts known to be present in these tumors. Analysis of TP53 muts exemplified the diversity of clonal patterns observed, with resistance muts being detected subclonally to parental TP53 muts in some pts and independently of TP53 muts in others. In addition, further evolution of resistant clones was observed through the development of TP53 muts within clones harboring acquired resistance muts, consistent with continued clonal evolution within the resistant disease compartment. In one pt, post-resistance clonal evolution was identified through the clonal acquisition of a CXCR4 mut within a BTK mutated population. Finally, to understand the contribution of BTK zygosity and gender to BTKi resistance (given its location on the X-chromosome), we performed single cell analysis on a disease specimen from a female pt with progressive MCL harboring multiple BTK mutations following treatment with sequential VEN-BTKi. Analysis revealed four clonally independent heterozygous BTK muts inferring the sufficiency of a single mutant allele to drive resistance in this context. Interestingly, this pt also harbored a BCL2 mut and a BAX mut, the latter co-occurring with a BTK mut (BCL2 not assessable). This pt therefore represents the first description of BCL2 or BAX muts occurring in a pt with progressive MCL on VEN and the first of a BTK L528W mut in MCL progressing on ZANU. In summary, these data highlight the significant clonal complexity of CLL progression on VEN and BTKi. Our data show that disease progression in this context is consistently oligoclonal with separate clones harboring distinct identifiable resistance mechanisms. These data have pt-specific implications for the potential utility of cycling back to previously efficacious targeted therapies as well as providing a strong rationale for the early use of disease-appropriate combination targeted therapies. Disclosures Anderson: Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Handunnetti:AbbVie: Other: Travel expenses; Roche: Honoraria; Gilead: Honoraria. Yeh:Novartis: Honoraria; Gilead: Research Funding. Tam:BeiGene: Honoraria; Janssen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Seymour:Morphosys: Consultancy, Honoraria; Mei Pharma: Consultancy, Honoraria; Gilead: Consultancy; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nurix: Honoraria. Roberts:Janssen: Research Funding; Servier: Research Funding; AbbVie: Research Funding; Genentech: Patents & Royalties: for venetoclax to one of my employers (Walter & Eliza Hall Institute); I receive a share of these royalties. Blombery:Amgen: Consultancy; Novartis: Consultancy; Invivoscribe: Honoraria; Janssen: Honoraria.

scholarly journals Mutational Type and Configuration of an Individual Gene May Differentially Impact the Clinical and Phenotypic Features

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2992-2992
Author(s):  
Yasunobu Nagata ◽  
Hideki Makishima ◽  
Cassandra M Kerr ◽  
Bhumika J. Patel ◽  
Hassan Awada ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Somatic Mutations ◽  
The Other ◽  
Missense Mutations ◽  
Advisory Committees ◽  
Different Types ◽  
Equity Ownership ◽  
The Impact

Myelodysplastic syndromes (MDS) arise in older adults through the stepwise acquisition of multiple somatic mutations. The genetic heterogeneity that results includes mutations of diverse genes and their combinations, clonal hierarchy, genetic configuration (e.g., bi-allelic or compound heterozygous, hemizygous lesions), specific positions within a gene including canonical hotspots vs. other positions, and types of mutation (truncations vs. missense), all of which could differentially affect pathogenesis. Given the binary status (e.g. mutated vs. wild-type) used in many clinical analyses, the true impact of specific types of mutations may be obscured and their specific roles underestimated. Deep targeted NGS was carried out for a panel of the 36 most frequently mutated genes in 1,809 MDS patients (low-risk MDS (n=839) vs. high-risk MDS (n=607), MDS/MPN (n=212), and sAML n=151). Copy number alterations (CNA) were also evaluated by combining karyotyping, microarray, and digital copy number analysis. With a mean coverage of 862x, after removing SNPs and errors, 3,971 somatic mutations were identified, the most common (>10% of cases) being TET2, SF3B1, ASXL1, del(5q), SRSF2, complex karyotype, and del(7q). For the purpose of this proof of concept analysis we focused on illustrative genes (TP53, RUNX1, TET2, and EZH2) affected by 2 recurrent hits. Bi-allelic TET2 or TP53 mutations were found in 15% (271/1,809) and 4% (72/1,809) of patients, respectively. TET2 and RUNX1 were most likely biallelic, whereas TP53 and EZH2 were most often affected by mutations and somatic deletion. Comparing the distribution of canonical vs. other types of mutations in genes, DNMT3A mutations affected the canonical site (R882) in 17% (35/203) of patients, were truncating in 39% (79/203) and missense in 44% (89/203) have also been found; deletions affecting the DNMT3A locus are rare. Within U2AF1, U2AF1Q157 are more frequent than U2AF1S34 (54% vs. 35%). Next, we checked correlation between these different types of mutations of one gene. 78 significant combinations were found. For instance, U2AF1Q157 mutations more commonly accompanied ASXL1 mutations and del(7q) and less frequently DNMT3A and BCOR mutations, trisomy8 and del(20) when compared to U2AF1S34 mutations [ASXL1 mutations 53% (42/80) in U2AF1Q157 vs. 16% (8/49) in U2AF1S34, P < .0001]. TET2 Bi-allelic mutations were more commonly associated with ZRSR2 and SRSF2 mutations, and less frequently del(5q) when compared to TET2 mono-allelic mutations [SRSF2 mutations 29% (80/276) in TET2-bi vs. 15% (34/227) in TET2-mono, P = .003]. In addition, patients with SRSF2 missense mutations were more likely to have RUNX1 bi-allelic mutations than those with SRSF2 in-frame mutations. We evaluated the impact of different types of mutations and combinations of them on disease phenotypes and survival. We then evaluated the impact of different types of mutations and their combinations on clinical phenotypes including dichotomous morphological (MDS vs. MDS/MPN) features, progressive (low- vs. high risk) subtypes. EZH2 bi-allelic alterations were more commonly associated with myleoproliferative features` compared to EZH2 mono-allelic alteration (q=.016). TET2 bi-allelic alterations and truncating mutations were found more frequently in higher-risk subtypes than TET2 mono-allelic and missense mutations (q<.001). In survival analyses, patients with DNMT3AR882 mutations had a poorer prognosis than those with truncating and the other missense mutations [P = .033, HR 1.86 (1.05-3.3)]. Next, using the PyClone bioanalytic pipeline, we recapitulated for each patient the clonal hierarchy and defined "dominant" vs. "secondary" mutations. DNMT3AR882 mutations were likely to be dominant/founder lesions compared to truncating or the other missense mutations: 77% (27/35) for R882 vs. 51% (40/79) for truncating vs. 45% (47/98) for the other missense, p=.0046. Specific dominant and secondary mutational pairs also differentially affected survival compared to the reverse configuration (q<.1) including EZH2 and RUNX1 or BCOR and U2AF1 or RUNX1 and BCOR. In conclusion, we report a comprehensive analysis of various types and configurations of lesions of individual commonly affected genes. Our results indicate that establishment of clinical or phenotypic correlations requires consideration of the type, rank and configuration of somatic mutations. Disclosures Mukherjee: McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy. Nazha:Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

scholarly journals Whole Genome Sequencing of Extramedullary Myeloma Autopsy Tumors Reveals a Genomic Portrait at Culmination of Clonal Convergence

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3169-3169
Author(s):  
Venkata D Yellapantula ◽  
Even H Rustad ◽  
Dominik Glozdik ◽  
Gunes Gundem ◽  
Jun Fan ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Clonal Evolution ◽  
Tumor Development ◽  
Advisory Committees ◽  
Site Specific ◽  
Metastatic Dissemination ◽  
Metastatic Lesions

Abstract Introduction Multiple Myeloma (MM) pathogenesis is characterised by extensive genetic and clonal evolution with frequent on-treatment progression. To date, most studies have focused on single diagnostic or paired diagnostic relapse biopsies, and the molecular mechanisms eventually resulting in treatment failure are poorly understood. To determine the molecular underpinnings of disease in its most advanced stage, we performed comprehensive genome profiling of 4 patients with extra-medullary metastatic disease. Methods A total of 8 patients with extramedullary myeloma with 188 (median = 22) distinct metastatic lesions were enrolled as part of the medical donation program at MSK. Here, we present results from 4 patients. Patients 1 and 2 had an indolent disease with a total survival of ~10 years whereas patients 3 and 4 had very aggressive disease and 2-3 years survival. All patients had received a sequence of multi-modal myeloma treatments. Targeted gene sequencing using a myeloma specific targeted panel myTYPE was performed in 28 samples from all 4 patients to a median coverage of 667x. Additionally, 6 tumors from patients 1 and 2 were subject to WGS to a median coverage 92x. Results Driver events: Aberrations across all 28 samples sequenced using myTYPE were examined. We found t(4;14) in Patients 3 and 4 across all the metastatic lesions consistent with previous knowledge that these are early initiating events. Besides IGH translocations, we found copy number changes involving 1p-, 1q+ and 13q-. Patient 1 and 2 had 17p- and 8p- shared across all the metastatic lesions. All patients had RAS/RAF pathway mutations and additional mutations were found in FAM46C, TP53 and BIRC3. In WGS, we observed a median SNV, Indel & SV burden of 12150, 1196 & 70.5 respectively. This mutational load is greater than two-fold higher to previously published estimates derived from primary diagnostic samples. Clonal structure using WGS: Clonal phylogeny was constructed using nDirichlet process clustering. Evaluation of mutation and clonal spectra showed evidence of clonal diversification amongst sites but within each sample all mutations had fully clonal cancer cell fractions, i.e. there were no subclones. For Patient 1, the phylogenetic tree was dominated by 9,099 truncal mutations, and 150-462 site specific yet clonal mutations. For Patient 2, the tree was dominated by 8,540 truncal mutations and site specific clonal mutations (n= 356; 1,186). However, evaluation of copy number alterations showed evidence of subclonal emergence of copy number aberrations implicating chromosomes 5, 8,16,18, 20, 21. This suggests that in these patients late stage tumor development and metastatic dissemination is further shaped by accrual of CNAs. Mutational processes using WGS: Signature analysis was performed by deconvolution of observed WGS mutations on the set of mutational signatures reported by Alexandrov et al. Consistent with previous reports, Signature 9 was identified as the dominant mutation signature, contributing to a median of 27% of all mutations in our cohort. Signature 9 is related to AID and has been previously implicated in early myeloma pathogenesis. Whilst canonical IGH translocations were not identified in Patient 1 or 2, Patient 1 showed evidence for chromoplexy with closed chain translocations having breakpoints spanning chromosomes 1, 4, 11, 16, 17, 19 across all 4 sites. Patient 2 presented with localised hypermutation on chromosomes 1, 5 and 22 which are shared between the 2 sites. These results suggest that subsequent clonal sweeps have acted upon the genome since disease initiation. Conclusion Preliminary data from multi-region WGS of the evolutionary end-stage in MM shows a single dominant clone with known driver events in each patient. This is in contrast to the subclonal heterogeneity characteristic of early disease, and presents opportunities for targeted therapies. Our observations are consistent with convergent evolution, where selective pressure from many years of therapy results in a relatively homogenous genomic landscape. A larger cohort of samples will ascertain patterns of biological processes we present here. These early investigations provides new insights on MM pathogenesis and metastatic dissemination. Disclosures Landgren: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Karyopharm: Consultancy; Pfizer: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lesokhin:Genentech: Research Funding; Serametrix, inc.: Patents & Royalties: Royalties; Janssen: Research Funding; Takeda: Consultancy, Honoraria; Squibb: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding.

scholarly journals Favorable Long-Term Outcomes after Daratumumab Discontinuation in AL Amyloidosis Patients Achieving Deep Responses

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1884-1884 ◽  
Author(s):  
Alfred Chung ◽  
Gregory P. Kaufman ◽  
Surbhi Sidana ◽  
David Iberri ◽  
Erik Eckhert ◽  
...  
Keyword(s):  
Disease Progression ◽  
Board Of Directors ◽  
Median Duration ◽  
Research Funding ◽  
Control Group ◽  
Al Amyloidosis ◽  
Cell Transplant ◽  
Advisory Committees ◽  
Free Interval ◽  
Significant Difference

Daratumumab (DARA) is a CD38-targeted antibody FDA-approved for the treatment of multiple myeloma (MM) and its efficacy has recently been demonstrated in the treatment of AL amyloidosis. DARA is conventionally given indefinitely until evidence of disease progression or intolerance for the treatment of MM. In AL amyloidosis, the optimal duration of therapy is not known, and patients may be treated indefinitely on maintenance, extrapolating from MM data. However, the plasma cell burden observed in AL amyloidosis is often lower than in MM, and thus certain patients achieving deep responses may have durable responses with time-limited treatment. Outcomes for patients who are observed after DARA discontinuation are not known. We report the outcomes of patients at our institution who received time-limited DARA. A retrospective analysis of AL amyloidosis patients treated at Stanford University from 2016 to 2019 with DARA monotherapy and dexamethasone for at least 2 months was performed, and patients who subsequently had DARA discontinued for reasons other than disease progression or lack of response were selected for the study. Hematologic responses were assessed by consensus guidelines. Duration on and off therapy were explored, along with time-to-next treatment or death (TTNT), defined as the time from DARA initiation to restarting/switching therapy or death. An exploratory analysis comparing TTNT between the study population and a control cohort who achieved hematologic CR and were maintained on DARA was conducted with the Kaplan-Meier method and log-rank testing. 67 patients received at least 2 months of DARA monotherapy and dexamethasone; among these, 15 patients discontinued therapy for reasons other than disease progression and were included. Median age was 66 years old and median lines of prior therapies was 4 (range: 1 - 6). Baseline difference between involved and uninvolved free light chains (dFLC) prior to DARA initiation was 2.6 mg/dL (range: 0 - 16.8 mg/dL). 10 of 15 patients had cardiac involvement with median NT-proBNP of 1982 pg/mL and 9 of 15 patients had renal involvement with median 24-hour proteinuria of 6.2 g and eGFR of 32 mL/min/1.73m2 at DARA initiation. Median duration from starting to stopping DARA was 7.8 months (range: 2 - 21 months). Median duration from achieving best hematologic response to stopping DARA was 3 months (range: 0 - 17 months). Reasons for discontinuation included: patient preference (5), fatigue/body aches (4), infection (2), other active medical comorbidities (3), and lack of perceived further benefit (1). At DARA discontinuation, median dFLC was 0.1 mg/dL (range: 0 - 2.2 mg/dL) and there were 12 hematologic CR, 1 VGPR, 1 PR, and 1 not assessable for response. Outcomes for all 15 patients are shown in Figure 1. The median treatment-free interval was 17.5 months (range: 5 - 34 months); estimated 2-year TTNT-free survival was 83% (95% CI: 61 - 100%). All 14 evaluable patients eventually achieved CR. 3 patients restarted DARA for rising dFLC, and all 3 patients demonstrated response to retreatment (2 achieving CR and 1 near PR with ongoing follow-up). There were 2 deaths. One patient with severe baseline cardiac amyloidosis developed sudden rise in dFLC after treatment-free interval of 21 months; although he rapidly achieved hematologic CR on retreatment, he died of heart failure within 2 months of restarting DARA. The other patient developed therapy-related AML while off therapy and underwent allogenic stem cell transplant but died of leukemia (censored for AL amyloidosis outcomes at transplant). There was no significant difference in the TTNT between the study group and a control group of 16 patients who achieved CR and were on continuous maintenance (Figure 2; p=0.807). AL amyloidosis patients achieving deep responses with DARA can have favorable outcomes after treatment discontinuation, including a long treatment-free interval. Although our sample size is small, the outcomes of these patients appeared comparable to those achieving CR on continuous DARA maintenance, and patients were able to regain responses when retreatment was necessary. These results suggest that DARA may be safely discontinued in patents achieving deep hematologic responses, which has significant implications for quality of life and financial burden of treatment. Future studies evaluating time-limited versus continuous DARA maintenance after achievement of deep responses are warranted. Disclosures Kaufman: Janssen: Other: travel/lodging, Research Funding. Liedtke:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; IQVIA/Jazz: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celator: Research Funding; Caelum: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Agios: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Daratumumab for treatment of AL amyloidosis

scholarly journals Patterns of Clonal Evolution Assessed By Whole Exome Sequencing during Progression from MDS to AML Are Related to Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4309-4309
Author(s):  
María Abáigar ◽  
Jesús M Hernández-Sánchez ◽  
David Tamborero ◽  
Marta Martín-Izquierdo ◽  
María Díez-Campelo ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Clonal Evolution ◽  
Clonal Expansion ◽  
Driver Mutations ◽  
Advisory Committees ◽  
Disease Evolution ◽  
Mutational Burden ◽  
Whole Exome ◽  
Leukemic Phase

Abstract Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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