Kyle, Robert A.

Background Multiple myeloma (MM) is a hematological cancer characterized by the proliferation of plasma cells (PC). Progressive disease (PD) criteria are primarily characterized by an increase in (1) serum M-protein (SPEP), (2) urinary M-protein (UPEP), (3) serum free light chains (sFLC), or (4) PC percentage in the bone marrow. An essential aspect of the PD criteria is the requirement for confirmatory results from blood or urine analyses. However, in many cases, patients may be taken off treatment in favor of a new therapy after only one assessment. This can introduce clinical trial endpoint inaccuracies when confirmatory values are missing, resulting in increased censoring and underestimation of the progression rate (if the second assessment is not done). Moreover, in subjects with high tumor burden and rapidly progressing disease, it is often urgent to confirm PD before starting a subsequent line of treatment. This study assessed the necessity of repeating biomarkers to confirm PD if two biomarkers meet the progression criteria. Methods In this retrospective study, between 2002 and 2020, we assessed all consecutive patients with relapsed MM enrolled in any therapeutic clinical trial at Mayo Clinic. We included patients with measurable disease if SPEP and sFLC were available at the time of assessment. Patients required at least two consecutive assessments (by the same method) to be included in our study. PD was based on the IMWG criteria. Relapse characterized by the development of new bone lesions or extramedullary relapse without meeting biochemical progression criteria was an exclusion criterion. Patients starting a new line of treatment after non-confirmed PD were also excluded. Furthermore, patients who did not meet simultaneous progression criteria and had unconfirmed PD (did not meet IMWG criteria at the second assessment) were excluded from the analysis (n=15). The probability of meeting the progression threshold with 1 and 2 consecutive assessments was calculated. We then examined the proportions of patients meeting PD by 2 biomarkers simultaneously at the first assessment. Results We evaluated episodes of treatment among 434 patients enrolled in clinical trials, including 601 episodes: 593 episodes of biochemical confirmed PD and 8 episodes when patients did not progress but who met simultaneous progression criteria at the same time with two different biomarkers. Per measurable disease criteria, MM isotypes were 71.0% IgG, 16.8% IgA, 0.9% IgM, 1.2% IgD, 0.3% biclonal, and 9.8% light chain. According to the standard IMWG response criteria (sequential criteria), 78% and 9% of episodes reached the progression threshold with serum and urine M-components, respectively, with two consecutive assessments. For the FLC, 21% of episodes met the progression criteria. No patients had a second bone marrow examination for confirmation of PD. At the first available assessment, 82%, 19%, and 2% of episodes met progression threshold, respectively, with serum M-protein, urine M-protein, and both (serum + urine M-protein). Additionally, 58%, and 19%, episodes met the progression threshold at the first assessment with FLC and bone marrow examination, respectively. Moreover, 67% of patients with IgA myeloma meet the progression threshold with the total quantitative IgA. With the simultaneous criteria at time of first assessment, 403 episodes meet progression threshold: 45% with SPEP+sFLC (n=269/593), 4% with SPEP+UPEP (n=21/593), 9% with sFLC+UPEP (n=55/593), 11% with BME only (n=66/593), and 0.3% with qIgA +dFLC/UPEP (n=2/593). Thus, among these 413 episodes, which meet progression criteria by 2 simultaneous values at the time of the first assessment, 98% (n=413/(413+8)) of these patients (positive predictive value of the new criteria) subsequently had confirmed progression by sequential value. Conclusions In summary, we showed that the simultaneous criteria allow to identify patients with true progressive disease without overestimating the progression rate of myeloma. Therefore, confirmatory results of progression for relapsed MM patients having two biomarkers meeting criteria of progression at the same time is useless for determining disease progression.

Background: A deeper understanding of the cytogenetic features has enabled robust risk stratification for patients with newly diagnosed multiple myeloma (NDMM). However, heterogeneity persists in the currently available prognostic tools and there is further room for personalization. We assess the utility of PC-PRO, a test to measure the proportion of monotypic plasma cells in the synthetic phase (S-phase) of cell cycle, as a prognostic marker for NDMM. Methods: Patients with active MM diagnosed between January 2013 and January 2023 with a flow cytometry-based S-phase assessment at diagnosis were included. Flow cytometric immunophenotyping was performed using the following antibodies: CD19, CD38, CD45, CD138, cytoplasmic kappa and lambda immunoglobulin, and DAPI (4',6-diamidino-2-phenylindole). The percentage of plasma cells in S-phase was determined by measuring the proportion of cells with DNA content of the monotypic plasma cells between the G0/G1 and G2/M peaks. Plasma cell clonality was detected through demonstrating CD38 and CD138 positivity along with immunoglobulin light chain restriction, abnormality of CD19/ CD45 expression and/or ploidy difference by DAPI staining. High-risk cytogenetic abnormalities (HRCA) by FISH were defined as follows: presence of deletion 17p (del17p), t(4;14), t(14;16), t(14;20), 1q gain/amplification, 1p deletion (when available). Additionally, the second iteration of the revised ISS (R2-ISS), incorporating both laboratory and cytogenetic features as well an adaptation of the recently proposed International Myeloma Working Group (IMWG) Classification for high-risk disease, was utilized for risk stratification. All time-to-event analyses were performed using the Kaplan Meier method and compared using the log-rank test. Univariable and multivariable Cox proportional hazards analyses were performed to assess the independent prognostic impact of S-phase. Results: A total of 823 patients were included in the study. The median follow-up was 6 years [95% Confidence interval (CI): 5.5-6.3 years] and estimated median overall survival (OS) was 8.3 years. The median S-phase value for the cohort was 0.8 % (interquartile range: 0.5-1.4%), with 135 (16%) patients harboring an S-phase of ≥2% at diagnosis. Applying the R2-ISS prognostic model to our cohort, 11% (79/706) patients were stratified to the R2-ISS high-risk cohort; 93 patients (out of 788, 12%) had 2 or more HRCA at diagnosis. Patients with a S-phase ≥2% at diagnosis had a median OS of 3.9 years (95% CI: 2.9-5.7) vs. 9.2 years [95% CI: 7.9-not reached (NR)] for patients with a S-phase <2% [Hazard ratio (HR): 2.2 (95% CI: 1.7-2.8), p<0.0001]. The median OS was inferior for cohorts with an increasing S-phase value: S-phase of 0.1-1%: HR: 1 (standard), S-phase 1.1-3%: HR 1.7 (95% CI: 1.3-2.1), S-phase >3%: HR 3.0 (95% CI: 2.2-4.1)]. On a multivariable analysis including S-phase ≥2%, age at diagnosis of MM (>65 years) and R2-ISS high-risk status, S-phase ≥2% was an independent predictor of inferior OS [hazard ratio (HR) 1.9 (95% CI: 1.4-2.6), p<0.0001]. Age >65 years [HR 1.9 (95% CI: 1.5-2.5), p<0.0001] and R2-ISS high-risk status [HR 1.8 (1.3-2.6), p=0.001] were also independent predictors of OS. Within the sub cohort of patients with 2 or more of the listed high-risk cytogenetic features (n=93, 12%), a S-phase ≥2% (n=24) was associated with a worse median OS of 1.6 years (95% CI: 0.7-NR) vs. 6.2 years (95 % CI:3.3-NR) for S-phase <2% (p=0.002). Conclusion: Flow cytometry-based determination of the proportion of monotypic plasma cells in S-phase is an easy-to-study and powerful prognostic tool for NDMM. A S-phase of ≥2% at diagnosis is a predictor of OS independent of age and R2-ISS high-risk stage. An S-phase of ≥2% was able to identify a distinct group with markedly inferior outcomes even within the sub cohort of patients with 2 or more high-risk cytogenetic features.

Introduction MYD88 L265P mutations (MUT) are found in 90% of patients with Waldenstrom macroglobulinemia (WM) and lead to the activation of pro-survival signaling. Limited literature has demonstrated that patients with MYD88 wild-type (WT) WM have poorer responses to conventional therapies and a higher likelihood of histological transformation. Increasingly the role of non-coding genomics in lymphomagenesis has demonstrated importance and currently, the epigenetic landscape of MYD88 WT WM remains underexplored. This study utilized a multi-omics approach to assess DNA methylation in MYD88 WT WM compared to MYD88 MUT patients and controls. Methods Bone marrow samples were prospectively collected from 20 subjects [3 MYD88 WT/CXCR4 WT WM, 12 MYD88 MUT WM and 5 normal controls] and positive selection for CD19+ and/or CD138+ B cells was performed. The analysis focused on the comparison between MYD88 WT WM to normal controls and MYD88-WT to MUT WM. Genome-wide DNA methylation analysis was performed, and CpG methylation ratios were segmented into 200-bp regions. Differentially methylated regions (DMRs) were determined using a Q-value <0.05 and absolute methylation difference ≥10%. Next, RNA-seq data was interrogated and differentially expressed genes (log2fold change>0.5 or <-0.5, false discovery rate<0.05) with corresponding promoter hypo- or hypermethylation were assessed. Ingenuity Pathway Analysis was used to determine potentially epigenetically regulated pathways using a corrected p<0.05. Results Comparing MYD88 WT WM samples to controls, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (29129 vs 6300), 3'untranslated region (UTR) (2068 vs 1551), 5'UTR (10169 vs 735), intron (30344 vs 24511), exon (2224 vs 6855), CpG island (30372 vs 1240) and CpG shore (14477 vs 5872) (all p<0.001). Next, mRNA analysis demonstrated that 214 genes were differentially up-/downregulated with concordant promoter hypo-/hypermethylation. On the assessment of promoter-methylation-based pathways, multiple pathways involved in cell signaling, proliferation, gene regulation, cytokine and immune signaling and metabolism were found to be deregulated. Most pathways were found to be negatively enriched in MYD88 WT patients compared to controls including several members of interleukin (IL) signaling (IL-2, IL-3, IL,7) and regulators of cell proliferation and signaling including mTOR, PI3K/AKT, and p53. Positive enrichment of the PTEN, FGFR3 and ERK/MAPK pathways were observed. Underlying multiple pathways was promoter hypermethylation with corresponding mRNA downregulation of AKT3 and PIK3CA. Furthermore, promoter hypomethylation with transcript upregulation of PTEN was observed. Next, in comparison of MYD88 WT to MUT samples, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (24829 vs 2544), 3'UTR (3534 vs 378), 5'UTR (6111 vs 605), intron (52292 vs 5360), exon (21066 vs 2187), CpG island (18334 vs 1551) and CpG shore (16351 vs 1957) (all p<0.001). Assessing mRNA expression, 354 genes were up-/downregulated with concordant promoter hypo-/hypermethylation. Pathway analysis revealed positive enrichment of the mitotic G1 phase to G1/S transition pathway with upregulation of the CDK4, LYN, and PSMA6 transcripts in MYD88 WT compared to MUT samples. Further, negative enrichment of cell signaling regulators including the RHO GTPase family, IL pathways (IL-4, IL-8, IL-13, IL-17) and integrin signaling were found in MYD88 WT compared to MUT samples. Underlying multiple pathways were downregulation with promoter hypermethylation of BCL2, an important regulator of apoptosis. Conclusion Genome-wide methylation analysis of MYD88 WT WM revealed a unique DNA methylation profile, with potential epigenetic alteration of key oncogenic transcripts and pathways. The negative enrichment of cell signaling and cytokine/inflammatory pathways in MYD88 WT samples compared to controls and MYD88 MUT WM suggests an epigenetic mechanism for immune evasion and potentially underlies the reduced response to conventional WM-directed therapies. Overall, this study reveals that MYD88 WT WM is epigenetically distinct from MYD88 MUT WM and regulators such as methylation may underlie the unique clinical and phenotypic properties observed.

Abstract As patients with relapsed/refractory multiple myeloma (RRMM) continue to live longer, they might get exposed to most available drugs and drug classes during the disease course. For such late line RRMM or among patients without access to novel therapies, retreatment with a drug that the disease had previously been refractory to might be one option. In this retrospective study, we describe 315 patients with RRMM at our institution who were retreated with a drug that the disease had been previously refractory to. We found an overall response rate of 56.2% and a median progression-free survival (PFS) of 11 months with retreatment. Patients with a longer time on initial therapy with the index drug (>28.4 months) had a superior PFS with retreatment (median PFS, 16.9 vs 8.1 months; P < .001). Similarly, patients with a longer time gap between the initial line of therapy with index drug and retreatment with index drug (>46.1 months) had better PFS with retreatment (28.2 vs 8.9 months; P = .016). In conclusion, retreatment with a previously refractory drug is a viable therapeutic option for RRMM, with the most significant benefit derived in disease demonstrating sensitivity to initial drug exposure and among those with a longer gap between initial drug exposure and retreatment.

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic premalignant disorder. The current standard of care is not to screen for MGUS, so it is often incidentally diagnosed in the clinic. It is unknown whether the outcomes of screened vs clinically detected MGUS differ. We compared the progression risk between screened vs clinical MGUS cohorts and assessed whether the MGUS detection method affected risk prediction of established clinical factors (score). We included 379 screened MGUS cases from the Olmsted County population-based study and 1384 patients with MGUS diagnosed during routine clinical evaluation at Mayo Clinic. Median follow-up time for the screened vs clinical cohort was 26.6 and 40.1 years, respectively. Accounting for death as a competing risk, the cumulative incidence of progression at 25 years was similar in the screened (11.1% [95% confidence interval [CI], 8.3-14.8]) vs clinical (10.1% [95% CI, 8.6-11.8]) MGUS cohorts, even when stratified by sex, age, or the baseline MGUS risk score. Overall, 0.9 (95% CI, 0.6-1.2) of patients with screened MGUS vs 1.0 (95% CI, 0.9-1.2) of those with clinically detected MGUS experienced disease progression for every 100 person-years of follow-up. MGUS detection method did not modify the association between MGUS risk score and progression risk (pinteraction = 0.217) and did not add to known risk factors for progression (likelihood ratio test; P = .839). Here, we show that progression risk among patients with screened vs clinically detected heavy-chain MGUS was similar. Future studies are needed to assess whether tailored follow-up of patients with screened MGUS affects clinical outcomes.

Abstract Currently, the role of DNA methylation in the immunoglobulin M (IgM) monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multiomics prospective analysis was conducted integrating DNA methylation, RNA sequencing (RNA-seq), and whole-exome sequencing data in 34 subjects (23 with Waldenström macroglobulinemia [WM], 6 with IgM monoclonal gammopathy of undetermined significance [MGUS], and 5 normal controls). Analysis was focused on defining differences between IgM gammopathies (WM/IgM-MGUS) compared with controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions that were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of the CXCR4 signaling pathway along with several interleukin (interleukin 6 [IL-6], IL-8, and IL-15) signaling pathways in WM compared with IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions.

PURPOSE Patients with Waldenström macroglobulinemia (WM) have disparate outcomes. Newer therapies have emerged since the development of International Prognostic Scoring System, and MYD88 L265P mutation is now frequently assessed at diagnosis, warranting reexamination of the prognostic parameters. PATIENTS AND METHODS We reviewed records of 889 treatment-naïve patients with active WM, consecutively seen between January 01, 1996, and December 31, 2017, to identify clinical predictors of overall survival (OS) in univariate analyses. Patients with complete data for the parameters significant on the univariate analyses (n = 341) were included in a multivariable analysis to derive a prognostic model, subsequently validated in a multi-institutional cohort. RESULTS In the derivation cohort (n = 341), age (hazard ratio [HR], 1.9 [95% CI, 1.2 to 2.1]; P = .0009), serum lactate dehydrogenase (LDH) above upper limit of normal (HR, 2.3 [95% CI, 1.3 to 4.5]; P = .007), and serum albumin <3.5 g/dL (HR, 1.5 [95% CI, 0.99 to 2.3]; P = .056) were independently prognostic. By assigning a score of 1 point each to albumin <3.5 g/dL (HR, 1.5) and age 66-75 years (HR 1.4) and 2 points for age >75 years (HR, 2.6) or elevated LDH (HR, 2.3), four groups with distinct outcomes were observed on the basis of the composite scores. Five-year OS was 93% for the low-risk (score 0), 82% for low-intermediate risk (score 1), 69% for intermediate-risk (score 2), and 55% for the high-risk (score ≥3; P < .0001) groups. In the validation cohort (N = 335), the model maintained its prognostic value, with a 5-year OS of 93%, 90%, 75%, and 57% for the four groups, respectively ( P < .0001). CONCLUSION Modified Staging System for WM (MSS-WM), utilizing age, albumin, and LDH is a simple, clinically useful, and externally validated prognostic model that reliably risk-stratifies patients with symptomatic WM into four groups with distinct prognosis.

AbstractSmoldering multiple myeloma (SMM) is a precursor stage that precedes multiple myeloma (MM). SMM is heterogenous with nearly 40% of patients progressing to MM in the first 5 years. The high rate of progression of SMM patients highlights the need for early intervention, which underscores the importance of identifying SMM patients with the highest risk of progression. Several risk stratification models showed utility in identifying high‐risk SMM patients; however, these systems showed limited sensitivity. To date, identifying high‐risk SMM patients remains an important clinical need. In this study, we present the 3‐dimensional telomere profiling as a structural biomarker capable of stratifying SMM patients as a function of genomic instability. Quantifying telomere dysfunction using the TeloView technology showed utility in risk stratification of cancer patients, particularly hematological malignancies. In this study, we analyzed 168 SMM patients. We report an AUC in ROC analysis of 0.8 using a subset of the patients as a training dataset. We then conducted a blind validation on a different cohort and demonstrated a positive predictive value of 85% and negative predictive value of 73%, with sensitivity and specificity of 83% and 76%, respectively. We examined the correlation between the TeloView prediction and the 20‐2‐20 scoring system, and cytogenetic abnormalities. We report a correlation of 53% with the 20‐2‐20 scores and over 60% correlation with cytogenetic abnormalities. The result of this study presents the telomere profiling as an effective biomarker able to stratify SMM patients to their respective risk groups with high sensitivity and specificity.

AbstractThe presence of a serum immunoglobulin D (IgD) monoclonal protein (M‐protein) is seen in < 1% of patients with monoclonal gammopathies and is usually indicative of a malignant plasma cell disorder. Only a few cases of well‐documented benign monoclonal gammopathy of undetermined significance (MGUS) of IgD subtype have been reported, and only 2 of those had over 5 years of follow‐up at the time they were reported. Herein we describe longer‐term follow‐up of one of those 2 patients who has subsequently passed away from unrelated causes but never developed multiple myeloma or amyloidosis after 26 years of follow‐up. Although IgD MGUS is extremely rare, this case confirms that presence of an IgD M‐Protein is not always synonymous with a malignant plasma cell process.

Background:Waldenström Macroglobulinemia (WM) is a rare, indolent B‐cell lymphoplasmacytic malignancy. Patient outcomes are highly heterogeneous, with a proportion of patients surviving less than 5 years (short survivors) and another proportion that survive longer than 10 years. The laboratory parameters in the currently used International Prognostic Scoring System for WM (IPSSWM) include hemoglobin, serum IgM, platelet count, and serum β2‐microglobulin.Aims:We aim to identify unique patient characteristics and prognostic features associated with short survival in WM.Methods:The medical records of patients with active WM that were diagnosed at Mayo Clinic Rochester, Arizona, and Florida between January 1st, 1996 and December 31st, 2013 were reviewed. The patients with smoldering WM were excluded. Cohorts of patients who either survived ≤ 5 years (Short Survival Group) or those with an overall survival (OS) ≥ 10 years (Long Survival Group) from the diagnosis of active disease were compared. Survivors with a follow‐up of ≤ 5 years were not included in the analyses. Two‐sided Wilcoxon rank sum test and Chi square/Fisher's exact test were used to compare the continuous and categorical variables, respectively. The variables that were statistically significant on univariate analysis (P < 0.05) were included in a multivariate logisitic regression analysis.Results:Of 893 consecutively seen WM patients (median follow‐up of 9 years [95% CI 8.4–9.7]), we identified 387 patients who could be categorized into either the Short Survival Group (n = 171 patients, 44%) or the Long Survival Group (n = 216, 56%). The Short Survival Group had a median OS of 2.4 years (2.1–2.8) and median age at diagnosis of 73 years (42–93), while the Long Survival Group (n = 216, 56%) had a median OS of 18.9 years (16‐NR) and median age at diagnosis of 62 years (31–85). The baseline characteristics and significant findings between the two groups are listed in the Table. Parameters significant on univariate analysis included age >65 years, albumin < 3.5 g/dL, β2‐microglobulin >3 μg/ml, and LDH > upper limit of normal (ULN). On multivariate analysis, albumin <3.5 g/dL and LDH > ULN were independently associated with short survival. Among the Long Survivors, 60 (28%) patients survived 15 years or more.Summary/Conclusion:Lactate dehydrogenase above the ULN and serum albumin <3.5 g/dL are independent biomarkers of short survival in WM. The MYD88 L265P status and the size of serum IgM are not prognostic in WM. Our data suggest the need for simplification of the current staging system with the use of widely available markers: serum albumin and lactate dehydrogenase.image