Current perspectives on circulating tumor cells in newly diagnosed MM eligible for transplant

Introduction

Obtaining peripheral blood (PB) samples from patients for the assessment of circulating tumor plasma cells (CTPC) is a far less less-invasive technique than bone marrow (BM) biopsy or aspiration. However, CTC assessment still enables real-time estimation of tumor burden and genetic alterations in multiple myeloma (MM) cells and clonal lines. Precise assessment of CTCs can lead to an appropriate risk evaluation and improved disease management.

The MM Hub has previously published an article on the potential role of CTCs in the prognosis and management of MM, and reported on the utility of CTCs in the identification of high-risk MM patients.

Here, we present promising data around the measurement of CTCs in the investigation and management of newly diagnosed (ND) MM from recently published articles in Journal of Clinical Oncology by Garcés, et al.,¹ Bertamini, et al.,² and Chakraborty and Lentzsch.³

The role of CTC in staging patients with NDMM who are eligible for transplant¹

Study design

This study measured CTCs in the PB of 374 patients from the GEM2012MENOS65 (NCT01916252) and GEM2014MAIN (NCT02406144) trials. Next-generation flow cytometry was used to evaluate CTCs in PB at diagnosis and measurable residual disease (MRD) in BM throughout treatment. Patients who had no MRD assessment after consolidation were entered as MRD-positive if complete remission (CR) was not achieved and MRD-missing if in CR.

Subgroup analysis was performed according to CTC ranges, including 0.0001% to <0.001%, ≥0.001% to <0.01%, ≥0.01% to <0.1%, >0.1% to <1%, and ≥0.1%. Baseline features (age, International Staging System [ISS] stage, lactate dehydrogenase [LDH], cytogenetic abnormalities (CA), Revised-ISS (R-ISS) stage, plasmacytoma, and BM plasma cells [BM PCs]) and MRD response were also compared.

Results

• CTCs were detected in 92% of patients with NDMM, with a median of 0.017% CTCs.
• A modest correlation was found between CTCs and PCs in BM, as determined by morphology (correlation [ρ] = 0.41; probability p < 0.001) and flow cytometry (ρ = 0.46; p < 0.001 ).
• Increasing logarithmic percentages of CTCs were associated with inferior progression-free survival (PFS) (Table 1).

Table 1. Multivariable analysis of PFS and OS including the percentages of BM PCs*

BM, bone marrow; CI, confidence interval; CTC, circulating tumor cell; HR, hazard ratio; OS, overall survival; PC, plasma cell; PFS, progression-free survival. *Data from Garcés, et al.1
Biomarker, %	PFS		OS
	HR (95% CI)	p value	HR (95% CI)	p value
BM PCs	1.0 (1–1.0)	0.13	1.01 (1–1.0)	0.20
BM clonal PCs	1.0 (1–1.0)	0.23	0.99 (0.97–1.0)	0.47
CTC	1.1 (1.0–1.2)	0.01	1.14 (1.01–1.3)	0.03

• Patients with ISS III and II had significantly increased CTC levels compared with ISS I.
• CTC levels were increased in patients with elevated versus normal LDH levels or with high-risk versus standard-risk CA.
• High CTC levels were associated with an increased risk of relapse.
• Patients with undetectable CTCs had exceptional PFS regardless of CR and MRD status.
• Multivariable PFS analysis including ISS stage, LDH levels, and cytogenetics showed that the patients with ≥0.01% CTCs had inferior PFS.
• In all other subgroups, achieving undetectable MRD was significantly associated with a longer PFS (p ≤ 0.045).

PFS in patients grouped according to number of risk factors is summarized in Table 2.

Table 2. PFS and 5-year OS in patients according to the presence of risk factors from a proposed novel classification system for patients with NDMM*

NR, not reached; OS, overall survival; PFS, progression free survival. *Data from Garcés, et al.1 †Risk factors: albumin <3.5 g/dL, b-2-microglobulin ≥5.5 mg/L, elevated LDH levels, high-risk CA, and ≥0.01% CTCs.
Outcome	Risk factors†
	0	1–2	≥3
PFS, months	NR	73	32
5-year OS, %	88	85	57

Overall, the incorporation of CTC serum levels into the PB at diagnosis promises to enhance the sensitivity of current stratifying systems. CTC levels ≥0.01% improved the performance of current risk models incorporating ISS, LDH, and CA for transplant-eligible patients with NDMM. However, further research is needed to build on existing knowledge in this area through the validation and replication of these findings in large, prospective clinical MM trials.

The role of CTC in identifying aggressive disease in ASCT-eligible patients with NDMM²

Study design

This study performed CTC analysis on 401 patients recruited to the multicenter FORTE trial (NCT02203643), which has previously been discussed on the Multiple Myeloma Hub. CTC and MRD was assessed by second-generation multiparameter flow cytometry (sensitivity, 4 × 10^-5 and 10^-5, respectively) to predict PFS and OS in patients with NDMM, in the presence of other high-risk factors and MRD.

Hazard regression analysis was performed using three models:

1. ISS + CA + LDH or R-ISS (described as null model)
2. Null model with CTC
3. CTC alone

Results

• CTCs were detected in 67% of the patients at diagnosis.
• CTC levels were moderately correlated with BM PCs (r = 0.382; p < 0.01).
• The median follow-up was 50 months.

• A cut-off of 0.07% CTCs was selected as the level providing the highest prognostic value for the prediction of PFS and OS.
•  Two populations were identified based on the CTC cut-off: CTC-high (>0.07%) and CTC-low (≤0.07%).

• • Over 50% of the CTC-low population (n = 132) had undetectable CTC.
  • Adverse prognostic factors were more prevalent in CTC-high patients (Table 3).

Table 3. Prevalence of adverse prognostic features in CTC-high versus CTC-low patients*

CR, complete response; CTC, circulating tumor cells; MRD, minimal residual disease. *Adapted from Bertamini, et al.2
Feature/response, %	CTC-high	CTC-low	p value
Total	68	32
International Staging System			<0.001
I	61	29
II	27	40
III	13	31
Cytogenetic abnormalities			<0.001
Low risk	74	53
High risk	26	47
High lactate dehydrogenase	8	25	<0.001
Revised International Staging System
I	40	12
II	55	60
III	5	19
MRD negativity at consolidation	59	42	0.001
Premaintenance ≥CR	54	43	0.055

PFS and OS were significantly lower in CTC-high patients compared with CTC-low or CTC-undetectable patients (Figure 1), with no difference observed between CTC-undetectable and CTC-low patients. Both PFS (hazard ratio [HR], 2.61; 95% confidence interval [CI], 1.49–2.97, p < 0.001) and OS (HR, 2.16; 95% CI, 1.49–4.56) were reduced in CTC-high patients relative to those with low CTC (Figure 1).

OS, overall survival; PFS, progression free survival.
*Adapted from Bertamini, et al.² 

MRD negativity after induction nullified the inferior influence of CTC-high in this patient group (interaction P = 0.039). In patients with any level of CTC, negative MRD was significantly associated with an increase in PFS (p ≤ 0.045).

Overall, high levels of CTC (≥0.07%) robustly identifies a high-risk population and MRD negativity is the most important factor in removing the negative prognostic influence of high CTC.

Are we ready to incorporate CTC assessment into routine clinical evaluation and treatment of patients with MM?³

Two cut-off values of CTC (≥0.01% and ≥0.07%) were identified as a prognostic factor in NDMM, using three groups of randomized controlled trial data (Table 4). Despite different cut-off values in each trial, there was consistent evidence that CTC burden is a strong, negative, and independent prognostic factor for transplant-eligible patients with NDMM. Thus, CTC assessments using next-generation flow cytometry at baseline could be used for risk stratification in this population.

Table 4. Characteristics of studies assessing CTCs in patients with NDMM*

BM PC, bone marrow plasma cells; CTC, circulating tumor cell; MFC, multiparameter flow cytometry; ND, newly diagnosed; MM, multiple myeloma; n/a, not applicable; NGF, next-generation flow cytometry; PCL, plasma cell leukemia. *Adapted from Chakraborty and Lentzsch.3 †The survival cohort consisted of individual patient-level data from eight studies. ‡Spearman rho or r reflects the strength of association between two variables, ranging from –1.0 (perfect negative correlation) to 11.0 (perfect positive correlation). A value between 0.30 to 0.49 reflects moderate positive correlation. §Adjusted R2 determines the extent of variance in the dependent variable, which can be explained by the independent variable. The values can range from 0 (indicating that the variables cannot be explained by each other) to 1 (indicating that the response variable can be perfectly explained by the predictor variable).
Characteristic	GEM2012MENOS65 and GEM2014MAIN	FORTE	EMN12/HO129, CASSIOPEIA, and HO143
Median follow-up, years	5	4.2	4.8 in the pooled survival cohort†
Methodology used for CTC detection	MFC	MFC	MFC
Sensitivity (limit of detection), cells	2 × 10-6 (NGF)	4 × 10-5	2 × 10-6 (NGF)
ND patients with CTC, %	92	67	87
Correlation between CTC and BM PC burden	ρ = 0.41‡ (p < 0.001)	ρ = 0.382‡ (p < 0.1)	Adjusted R2 = 0.16§ (p < 0.001)
CTC cut off for risk stratification, %	≥0.01	≥0.07	n/a

Unmet needs for future MM studies on CTC burden

• It is necessary to use high CTC burden as an inclusion criterion for study participants in the absence of clinical plasma cell leukemia but with >5% CTC on morphology at first diagnosis.
• At diagnosis, almost all MM cells express CD38. The effects of anti-CD38 monoclonal antibody-based regimens on the outcomes of patients who are CTC-high at baseline needs investigating.
• To determine the impact of novel immunotherapies such as T-cell engagers and chimeric antigen receptor T-cell therapies in CTC-high MM.
• To determine the utility of CTC for the prognostication of PCL-like disease

Conclusion

High levels of CTCs, above an optimal cut-off (≥0.01%), represent a robust and independent high-risk factor in patients with NDMM. CTC-burden evaluation can increase the sensitivity of existing risk-stratification criteria and holds potential for the identification of high-risk disease and risk-adapted treatment approaches. However, before incorporation of high CTC burden into routine clinical practice, further research is needed in transplant-eligible and transplant-ineligible patients receiving similar therapeutic regimens.