Introduction

autologous stem cell transplant (ASCT) remains an important element of the treatment paradigm for patients with multiple myeloma (MM), offering deeper responses and improved progression free survival (PFS) [1,2,3,4]. However, some patients experience suboptimal outcomes following ASCT. Studies have shown that early relapses, occurring within one to two years post-ASCT, are associated with inferior outcomes [5]. Some pre and post- transplant prognostic factors that have been associated with earlier relapses following ASCT include the baseline presence of high-risk fluorescence in situ hybridization (HR-FISH) cytogenetics, higher international staging system (ISS) stage, elevated LDH (Lactate dehydrogenase) levels, higher number of prior lines of therapy and achieving less than a complete response (CR) after ASCT [6, 7]. However, there are limited studies addressing factors present at the time of ASCT that may influence relapse risk and survival outcomes in patients with MM. Our group has previously demonstrated that a higher percentage of plasma cell labeling index (PCLI) at the time of ASCT is associated with earlier relapses following ASCT [8]. The PCLI measures the percentage of plasma cells actively synthesizing DNA, reflecting their proliferative activity and providing insight into the biology of plasma cells. However, the use of PCLI has been limited due to its labor-intensive nature. As a result, the plasma cell proliferation (PCPRO) test, a multiparametric flow cytometry-based assay, has replaced PCLI in clinical practice. The PCPRO test offers a more convenient method to quantify the proportion or percentage of monotypic plasma cells in the S-phase, effectively capturing their proliferation rate [9]. Recently, our group evaluated the impact of S-phase% at the time of MM diagnosis on outcomes, considering more recent prognostic stratification and treatment strategies. It was observed that a higher S-phase% (i.e., S phase% ≥2) at diagnosis was associated with an inferior PFS and overall survival (OS) in multivariate analysis containing conventional prognostic features [10].

In this study, we aim to investigate whether the S-phase% of the residual plasma cells in the bone marrow at the time of ASCT retains its prognostic value in predicting for a shorter PFS and OS outcomes after ASCT. We seek to identify a subset where the benefit of ASCT is so limited that alternative strategies should be considered in the first line.

Methods

This retrospective study included patients diagnosed with MM between January 1, 2013, and August 31, 2024, who underwent ASCT within one year of diagnosis at the Mayo Clinic in Rochester, MN. The study was approved by the institutional review board (IRB #24-009552). As shown in (Fig. 1), of 2,348 patients identified, 1,157 met the inclusion criteria. After excluding 21 patients without available S-phase data, 1,136 patients were included in the final analysis.

Fig. 1: CONSORT diagram illustrating patient selection for analysis.
figure 1

From a total of 2348 patients with multiple myeloma, exclusions were made for those not undergoing ASCT within 1 year of diagnosis (n = 1191) and those lacking S-phase data (n = 21). The final cohort of 1136 was categorized as S-phase <2% (n = 372), S-phase ≥2% (n =  142), and S-phase not assessable (n = 622). ASCT autologous stem cell transplantation.

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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) as previously described [9]. The plasma cell clonality was determined through demonstrating CD38 and CD138 positivity, absence of CD19/CD45 expression, immunoglobulin light chain restriction, and/or ploidy difference by DAPI staining. The percentage of clonal plasma cells in S-phase was determined by measuring the proportion of cells with DNA content between the G0/G1 and G2/M peaks.

A minimum of 300 abnormal plasma cells is required for reliable S-phase assessment using the PCPRO assay; samples with fewer events were considered non-assessable [11], which usually reflects patients with a CR or better after induction therapy.

Following previously established cutoffs [12], patients were stratified into three groups: S-phase <2%, S-phase ≥2%, and non-assessable. A secondary analysis further divided assessable cases into four subgroups: <2%, 2–4.9%, 5–9.9%, and ≥10%.

High-risk cytogenetic abnormalities (HRCA) by FISH included del(17p), t(4;14), t(14;16), t(14;20), and 1q21 gain/amplification. PFS was defined from the date of transplant to relapse, progression [13], or death, OS from transplant to death from any cause, with censoring at the date of last follow-up.

Categorical variables were compared using the chi-square or Fisher’s exact test, and continuous variables using the Wilcoxon rank-sum test. PFS and OS were estimated using the Kaplan–Meier method and compared using log-rank tests. Univariable and multivariable Cox proportional hazards models were used to assess the prognostic impact of S-phase%, while adjusting for age, ISS stage, serum creatinine, high-risk cytogenetics, depth of response at ASCT, and post-transplant maintenance therapy. All analyses were performed using JMP software (version 10; SAS Institute) and R software (version 4.2.2; R Foundation for Statistical Computing).

Results

A total of 1136 patients who underwent ASCT within one year of MM diagnosis were identified. Baseline patient characteristics are summarized in Table 1. Compared to patients with an S-phase <2%, those with an S-phase ≥2% were more likely to have high-risk cytogenetic abnormalities (59.8% vs 31.9%, P < 0.0001), ISS stage III disease (31.6% vs 19.3%, P = 0.0048), and creatinine ≥2 mg/dL (18.3% vs 8.6%, P = 0.0044). Additionally, patients with an S-phase ≥2% had a higher rate of achieving very good partial response (VGPR) or better at the time of ASCT (58.4% vs 36.8%, P < 0.0001) with no difference in the likelihood to receive maintenance therapy post-transplant (13.4% vs 11.3, P = 0.5339).

Table 1 Demographic and clinical features—all patients.
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At a median follow-up of 41 months (interquartile range [IQR], 21–63); the median PFS was 26 months (95% CI: 17–35) in the S-phase ≥2% group compared to 47 months (95% CI: 39–58) in the S-phase <2% group (HR 1.7; 95% CI:1.3-2.3) (P < 0.0001) (Fig. 2A). The median OS was 57 months (95% CI: 42–NR) in the S-phase ≥2% group, while it was NR (95% CI: 110–NR) in the S-phase <2% group (HR 3.3; 95% CI: 2.3–4.8) (P < 0.0001) (Fig. 2B). In multivariate analysis, adjusting for age, ISS stage, serum creatinine level, high-risk FISH, response at ASCT, and maintenance therapy, S-phase ≥2% remained an independent predictor of inferior PFS (HR, 1.4; 95% CI: 1.0–2.3; P = 0.0266) (Table 2). Similarly; OS remained inferior in the group with S-phase ≥2% (HR, 1.8; 95% CI: 1.1–2.9; P = 0.0191) in multivariate analysis (Table 3).

Fig. 2: Kaplan–Meier survival analysis in patients with multiple myeloma who underwent ASCT within one year of diagnosis, stratified by S-phase percentage at the time of transplantation <2% and S-phase ≥2%.
figure 2

A PFS. B OS. ASCT autologous stem cell transplantation, MM multiple myeloma, PFS progression-free survival, OS overall survival, NR not reached.

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Table 2 Univariable and multivariable analyses for progression-free survival (PFS).
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Table 3 Univariable and multivariable analyses for overall survival (OS).
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We also compared outcomes between patients with an S-phase <2%, ≥2% and those with non-assessable S-phase. Median PFS was 26 months (95% CI: 17–35) for patients with S-phase ≥2%, 47 months (95% CI: 39–58) for patients with S-phase <2%, and 104 months (95% CI: 95–NR) for patients with non-assessable S-phase (P < 0.0001). The median OS was 57 months (95% CI: 42–NR) for patients with S-phase ≥2%, NR with a lower bound of 110 months (95% CI: 110–NR) for patients with S-phase <2%, and NR (95% CI: NR–NR) for patients with non-assessable S-phase (P < 0.0001) (Supplementary Figs. 1 and 2).

After subcategorizing patients with assessable S-phase results into groups with S-phase <2% (n = 372), 2–4.9% (n = 105), 5–9.9% (n = 27), and ≥10% (n = 10), PFS and OS were most inferior in the subgroup with an S-phase% of ≥10%. The median PFS and OS were 13 months (95% CI: 7–35) and 34 months (95% CI: 13–NR) respectively in the group with a S-phase% of 5–9.9% (Fig. 3A, B). The group with an S-phase ≥10% had a median PFS and OS of 3.5 months (95% CI: 1–6) and 9.5 months (95% CI: 3–26), respectively.

Fig. 3: Kaplan–Meier survival analysis in patients with multiple myeloma who underwent ASCT within one year of diagnosis, stratified by S-phase percentage at the time of transplantation (<2%, 2–4.9%, 4.9–9.9%, and ≥10%).
figure 3

A PFS. B OS. ASCT autologous stem cell transplantation, MM multiple myeloma, PFS progression-free survival, OS overall survival, NR not reached.

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Discussion

Under normal physiological conditions, polyclonal plasma cells are terminally differentiated and non-proliferative [14]. However, in MM, clonal plasma cells may re-acquire proliferative capacity through molecular alterations, leading to more aggressive disease behavior [15]. Increased proliferative activity, as captured by the S-phase% measurement, has been associated with worse clinical outcomes [16]. We previously showed that S-phase% assessment by PCPRO test at MM diagnosis carries prognostic significance in this population [10]. Our study extended the utility of assessing the S-phase% of monoclonal plasma cells measured by the PCPRO test at the time of ASCT as a prognostic tool to predict post-ASCT outcomes in MM patients, as we demonstrated inferior PFS and OS outcomes in patients who had high S-phase% at the time of ASCT. These findings align with an earlier study utilizing the PCLI test, a slide-based, labor-intensive method for estimating plasma cell proliferation, which also demonstrated prognostic relevance at the time of ASCT [8]. Importantly, the PCPRO utilized to measure S-phase% offers a more accessible and scalable alternative to PCLI [17,18,19]. Our results reinforce the clinical value of S-phase analysis in the modern therapeutic landscape, which has been transformed by the integration of triplet and quadruplet induction regimens and the widespread use of CD38-targeted monoclonal antibodies [20,21,22]. Also, it shows its significance in the current understanding of the prognostic impact of cytogenetic abnormalities [23].

While the PCPRO assay is not yet widely adopted in general practice, its flow-based methodology makes it considerably more practical than older techniques such as PCLI. This highlights the importance of incorporating S-phase assessment into prospective studies, which could support broader use in routine pre-transplant evaluations.

In the modern era, despite the ongoing and rapidly evolving treatment options, the role of ASCT in upfront consolidation after induction therapy remains the standard of care irrespective of baseline poor prognostic risk factors for early relapse. However, the most striking and clinically relevant finding of our study is that patients with an S-phase ≥5%, and especially those with ≥10%, experienced dramatically inferior outcomes after ASCT, with median PFS of only 13 and 3.5 months, respectively. This identifies a functionally high-risk group that may derive little or no benefit from standard ASCT consolidation. Recognizing such patients before transplant could profoundly impact treatment planning. Many patients confronted with median PFS < 1 year may elect to forgo ASCT, concluding that the impact on quality of life and geographic relocation to a transplant center for up to 6 weeks would not be justified for this short period of PFS. In the context of expanding options, including quadruplet induction regimens and novel immunotherapies, these findings support considering alternative frontline strategies or enrollment in clinical trials for patients identified with highly proliferative disease at the time of ASCT evaluation.

Indeed, chimeric antigen receptor (CAR) T-cell therapy and T-cell engagers are being studied at the frontline treatment of patients with newly diagnosed MM (NCT05257083, NCT05695508) and could potentially be standard options for these difficult to treat subgroups of patients.

A major strength of our study is the large patient cohort. Although a significant proportion of patients did not have assessable S-phase at the time of ASCT, primarily due to an insufficient number of abnormal plasma cells (fewer than 300 events required for PCPRO analysis at Mayo Clinic), we addressed this subset of patient by comparing their clinical outcomes to those with both low ( < 2%) and high ( ≥ 2%) S-phase fractions. Outcomes in these patients with no assessable S-Phase% were the most favorable, supporting the notion that they represent a biologically favorable group. This would be expected given the fact that fewer than 300 plasma cells by flowcytometry reflects the deepest response to anti-myeloma induction therapy and one would anticipate a better outcome than patients with greater than 300 plasma cells presenting for consideration for ASCT.

This study’s limitations include its retrospective nature. Additionally, the relatively small number of patients with extremely high S-phase values (≥10%) limited the statistical power for some subgroup analyses.

In conclusion, incorporating flow-based S-phase% assessment of residual clonal plasma cells in the bone marrow at the time of ASCT into routine pre-transplant evaluation could enhance individualized treatment planning. Notably, identifying patients with S-phase ≥5% — who had exceptionally poor outcomes and represent a functionally high-risk group — could directly inform decisions to consider alternative upfront strategies, deferring stem cell transplantation given the short duration of PFS. Future prospective studies are warranted to validate these observations and to define optimal management approaches for patients with highly proliferative disease.