Background: Multiple myeloma (MM) is a remitting-relapsing malignancy with variable clinical outcome. Certain cytogenetic abnormalities, either present at diagnosis or emerged at later stages, ...predict for poor outcome and highlight the clinical importance of MM genetic heterogeneity. Whole genome and exome sequencing studies reveal a complex intraclonal genetic landscape, organised in linear and branching Darwinian patterns, which evolves in space and time. Clones with a more complex genetic architecture may be more fit to escape treatment and those patients are likely to have a worse clinical outcome. Clinical multicolour flow cytometry (MFC) is routinely used in MM diagnosis and detection of minimal residual disease. Previous studies have shown that MM cell subpopulations with discrete phenotypic features correspond to genetic subclones, therefore it is plausible that MFC data captures clonal heterogeneity. On that basis, we propose that clustering analysis of MM phenotypic subpopulations could be clinically relevant.
Methods: We retrospectively analysed clinical MCF data at diagnosis from 44 patients eligible for autologous stem cell transplantation (AutoSCT) and 14 ineligible patients and data from 52 relapsed patients after first AutoSCT. All patients were treated between 2012 - 2018. The 8-colour MCF marker panel included CD138, CD38, CD56, CD45, CD20, CD19, cytoplasmic kappa and lambda light chains (cytLC). Data was analysed in FlowJo software and MM plasma cells were identified as CD38high, CD19-, cytLC+, within their FSC-A/SSC-A physical gate. The gated events were exported in a new fcs file. Clustering analysis was performed in Cytofkit, a R-based Bioconductor package, using the Rphenograph, Cluster-X and FlowSOM algorithms. All fcs files were subjected in the same clustering analysis, but CD56 positive and CD56 negative cases were analysed separately to offset bias from differential CD56 expression. Parameters inserted in the algorithms were FSC-A, CD138, CD38, CD45, CD20 and CD56. The number of clusters was produced by FlowSOM (k=4) and only clusters with size >1% of the total events were accepted.
Results: At diagnosis, FlowSOM identified 1 (n=32, 56.1%) or 2 clusters (n=19, 33.3%) in most cases. Three clusters were found only in 5 patients (8.8%) and 4 clusters in 1 patient (1.8%). The number of clusters at diagnosis did not correlate with cytogenetic risk group or ISS. Also, the number of clusters did not predict for depth of response or relapse free survival post AutoSCT. On the contrary, phenotypic patterns at relapse post AutoSCT were more complex, with 1 cluster identified in 2 patients only (3.8%), 2 clusters in 23 (44.2%), 3 clusters in 24 (46.2%) and 4 clusters in 3 patients (5.8%). Patients with >2 clusters (n=27) had a shorter survival post relapse (median 17 months - 95% CI, 7-26.6) compared to those (n=25) with 1-2 phenotypic clusters (median not reached, Log rank p=0.06). A phenotypic cluster characterised by CD138low/- at relapse, was also associated with adverse outcome and higher risk cytogenetics. In 14 patients with available serial samples at diagnosis and relapse we observed 3 patterns of phenotypic evolution: a. sustained pattern, b. change of dominant cluster and c. emergence of completely new subpopulations. These evolving phenotypes resemble changes in clonal composition over time observed in genetic studies.
Conclusion: Clinical MCF, in addition to routine diagnostics, can be informative of MM biological and clinical heterogeneity. Particularly in the relapsed setting, complex phenotypic patterns identified by clustering analysis may be of prognostic value. Validation of this preliminary study results in larger patient cohorts or clinical trials, could provide a useful and readily available tool for patient stratification and prognosis.
Apperley:Novartis: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Karadimitris:Celgene: Research Funding; GSK: Research Funding; Gilead: Honoraria.
We analyzed a cohort of 26 patients with chronic myeloid leukemia who had failed imatinib and a second tyrosine kinase inhibitor but were still in first chronic phase and identified prognostic ...factors for response and outcomes. The achievement of a prior cytogenetic response on imatinib or on second-line therapy were the only independent predictors for the achievement of complete cytogenetic responses on third-line therapy. Younger age and the achievement of a cytogenetic response on second line were the only independent predictors for overall survival (OS). At 3 months, the 9 patients who had achieved a cytogenetic response had better 30-month probabilities of complete cytogenetic responses and OS than the patients who had failed to do so. Factors measurable before starting treatment with third line therapy and cytogenetic responses at 3 months can accurately predict subsequent outcome and thus guide clinical decisions.
Natural killer (NK) cells exert antimyeloma cytotoxicity. The balance between inhibition and activation of NK-cells played by the inherited repertoire of killer immunoglobulin-like receptor (KIR) ...genes therefore may influence prognosis. One hundred eighty-two patients with multiple myeloma (MM) were analyzed for KIR repertoire. Multivariate analysis showed that progression-free survival (PFS) after autologous stem cell transplantation (ASCT) was significantly shorter for patients who are KIR3DS1+ (P = .01). This was most evident for patients in complete or partial remission (good risk; GR) at ASCT. The relative risk (RR) of progression or death for patients with KIR3DS1+ compared with KIR3DS1− was 1.9 (95% CI, 1.3-3.1; P = .002). The most significant difference in PFS was observed in patients with GR KIR3DS1+ in whom HLA-Bw4, the ligand for the corresponding inhibitory receptor KIR3DL1, was missing. Patients with KIR3DS1+KIR3DL1+HLA-Bw4− had a significantly shorter PFS than patients who were KIR3DS1−, translating to a difference in median PFS of 12 months (12.2 vs 24 months; P = .002). Our data show that KIR–human leukocyte antigen immunogenetics represent a novel prognostic tool for patients with myeloma, shown here in the context of ASCT, and that KIR3DS1 positivity may identify patients at greater risk of progression.
Abstract 68▪▪This icon denotes a clinically relevant abstract
Several groups have shown that that the BCR-ABL1 transcript level measured at 3 or 6 months after starting TKI therapy strongly predicts ...for the achievement of cytogenetic and molecular responses and for PFS and OS. In particular, we have shown that CML patients treated with imatinib who at 3 months have a transcript level lower than 9.8% on the international scale or lower than 1.67% at 6 months fare significantly better. We have also shown that the molecular assessment made at 3 months on imatinib therapy is a better predictor of the prognosis of patients than the analysis of BCR-ABL1 transcripts at 6 months. Here we investigate whether it is possible to improve the prognostic accuracy of early measurement of the transcript level by combining the 3 and 6 month results.
Between June 2000 and December 2010 282 consecutive adult patients with CML in CP seen at our institution received imatinib 400 mg daily as first line therapy. The median follow-up was 69 months (range 17–131). During follow-up 118 patients discontinued imatinib and received nilotinib (n=37), dasatinib (n=72) or an allogeneic stem cell transplant (n=9). BCR-ABL1 transcripts were measured in the blood at 6 to 12 week intervals using RQ-PCR and results were expressed as percent ratios relative to an ABL1 internal control with original laboratory values converted to the international scale.
Two hundred and seventy-four patients were still alive in chronic phase and receiving imatinib at 6 months. We classified these patients according to their transcript levels at 3 months (lower or higher than 9.8%) and 6 months (lower or higher than 1.67%). 181 (66%) patients had low transcripts both at 3 and 6 months; these patients had an excellent outcome with a OS of 93.5% and a 100% cumulative incidence (CI) of CCyR. Fifty-seven (21%) of the patients had high transcript levels on both occasions; these patients had a significantly worse outcome than the previous cohort, namely an OS of 55.6% (p<0.001) and a CI of CCyR of 14.9% (p<0.001). Thirty (11%) patients had low transcript levels at 3 months but high transcript levels at 6 months; these patients had a prognosis similar to those of the patients with low transcripts at both the 3 and 6 month time points with an OS of 92.4 (p=0.78) and a 8-year CI of CCyR of 99.5% (p=0.001), although the kinetics of the response in this cohort was slower. Only 6 patients (2%) had high transcript levels at 3 months but low levels at 6 months; these patients had an outcome similar to the patients with low transcript levels at the two time points (OS= 100%, PFS=83.3% and CI of CCyR = 85%).
The measurement of the transcript level at 6 months adds very little prognostic discrimination to the measurement already taken at 3 months. The 11% of patients who met the three month milestone but failed the 6 month milestone had an OS and PFS identical to the patients who achieved both milestones. The CI of CCyR was also similar (although slower, median time to CCyR 12 months vs 6 months for the patients who met both milestones, p=0.001). The 2% of patients who failed the first milestone at 3 months but who met the second one at 6 months also seemed to fare well, although this group is too small to clearly establish whether patients with high 3 month transcript levels are ‘rescued' by meeting the 6 month milestone. In summary, the prognosis of patients can be accurately established by assessing the transcript level purely at 3 months, although the analysis at 6 months may improve the prognostic classification of 2% of the patients.
Goldman:Novartis, Bristol Myers-Squibb, and Amgen: Honoraria. Marin:BMS: Research Funding; Novartis: Research Funding.
Abstract 3760
The introduction of tyrosine kinase inhibitors (TKIs) has proved to be a major advance in the management of patients with chronic myeloid leukemia in chronic phase (CML-CP) although the ...clinical benefit seems to be limited to those patients who achieve complete cytogenetic remission (CCyR). Patients who achieve CCyR are believed to have an excellent prognosis, but a very small proportion of those patients may lose the response and progress to blastic phase. In this work we studied a cohort of 210 patients who achieved CCyR (142 of them were in MMR) on imatinib as first line therapy in order to identify the incidence of blastic transformation in this population.
Between June 2000 and December 2010, 282 consecutive adult patients with CML-CP were seen at our institution, of whom, 210 achieved CCyR on imatinib 400 mg daily as first-line therapy. The median follow-up was 69 months. Of the 210 patients in CCyR, 5 (2.3%) progressed to blastic transformation (BT) (3 myeloid and 2 lymphoid). The Sokal risk was low in 3 patients and intermediate in two. The median time to achievement of CCyR in these 5 patients was 18 months (range 9–20 months). Two of these patients also achieved a major molecular response (MR3), one achieved a 4 log reduction on the international scale and two patients had a CCyR with no MR3. The median time to achievement of MR3 (or better) in the three patients was 24 months (range 18–28 months). After achieving CCyR all 5 patients had progressive increases in their BCR-ABL1 transcript levels over a period of a few months leading to BT without an intervening accelerated phase. None of the patients had a ABL1 kinase domain mutation prior to transformation to advanced phase, but two patients subsequently developed a new mutation at the time of BT (M244V and T315I, respectively). The 8-year cumulative incidence (CI) of BT after reaching CCyR was 2.3% and was 2.8% when calculated from diagnosis (Figure 1). The median time from achieving CCyR to BT, was 18 months (range 12–40 months). The median time from the onset of an increase in BCR-ABL1 transcripts to BT was 6 months (range 4–7 months). All 5 patients had good compliance with TKIs.
All 5 patients were treated by allogeneic stem cell transplantation after BT in their second chronic phase. One patient proceeded to a transplant after combination chemotherapy and TKI, but 4 others only received a TKI prior to transplant (3 dasatinib and 1 nilotinib). All 5 patients subsequently died due to transplant related causes. The 8-year CI of BT after attaining CCyR was 2.3%. MR3 does not appear to completely protect against BT as 3 of the 5 patients with MR3 lost the response and progressed to BT. In our series we could not identify a time point beyond which patients may be safe from blastic transformation, which continues to be a rare but catastrophic event in responding patients.
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Goldman:Novartis, Bristol Myers-Squibb, and Amgen: Honoraria. Marin:Novartis: Research Funding; BMS: Research Funding.
Summary
BCR‐ABL1 transcript numbers were monitored in 161 patients who started treatment with imatinib early after diagnosis of chronic myeloid leukaemia in chronic phase and achieved complete ...cytogenetic responses (CCyR). A confirmed doubling in BCR‐ABL1/ABL1 transcript levels was found to be a significant factor for predicting loss of CCyR relative risk (RR) 8·3, P < 0·0001 and progression to advanced phase (RR 0·07, P = 0·03) provided that the eventual BCR‐ABL1/ABL1 transcript level exceeded 0·05%; increases that never exceeded 0·05% had no predictive value. The finding of a kinase domain mutation in a patient in CCyR, though rare, also predicted for loss of CCyR.
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