PRACA POGLĄDOWA

Flow cytometric minimal residual disease assessment in multiple myeloma

Cytometryczna ocena minimalnej choroby resztkowej w szpiczaku plazmocytowym

Lucie Rihova1, Roman Hajek1, 2

1Department of Hematology, University Hospital Brno, Brno, Czech Republic

2Department of Hematooncology, University Hospital Ostrava, Ostrava, Czech Republic

Abstract

Minimal residual disease (MRD) assessment in light of the effectiveness of new multiple myeloma (MM) treatment modalities and related to it increasing ratios of achieved complete remissions (CR), becomes an important tool in recognition of the depth of the response. Multiparametric flow cytometry (MFC) is currently the most popular method for monitoring of MRD presence in bone marrow of MM patients; however, molecular techniques may also be used for MRD assessment. The choice of protocol utilized for MFC-MRD measurement can significantly affect the obtained results, nevertheless standardized and highly sensitive approach of next generation flow (NGF) is already available. The depth of response based on MRD assessment was shown to be an independent predictor of progression-free survival (PFS) and overall survival (OS). Furthermore, the MRD-negative status surpasses the prognostic value of CR achievement for PFS and OS. Thus, MRD status detected by highly sensitive and reproducible MFC is potentially a clinically applicable biomarker for evaluation of different treatment strategies efficacy potentially influencing treatment decisions, and acting as prognostic factor in MM patients.

Key words: multiple myeloma, minimal residual disease, flow cytometry

Hematologia 2017; 8, 3: 211–218

Streszczenie

W świetle wysokiej skuteczności nowych metod leczenia szpiczaka plazmocytowego oraz zwiększającego się odsetka osiąganych całkowitych remisji (CR) ocena minimalnej choroby resztkowej (MRD) jest obecnie istotną metodą określającą głębokość odpowiedzi. Wieloparametryczna cytometria przepływowa (MFC) jest obecnie najczęściej wykorzystywaną metodą monitorowania MRD w szpiku kostnym pacjentów ze szpiczakiem plazmocytowym, jednak w tym celu można również stosować metody molekularne. Rodzaj protokołu stosowanego przy badaniu MRD metodą MFC może istotnie wpływać na uzyskiwane wyniki, jednak obecnie jest już dostępny wystandaryzowany i wysoce czuły protokół cytometrii następnej generacji (NGF). Wykazano, że głębokość odpowiedzi oceniona na podstawie pomiaru MRD koreluje z przeżyciem wolnym od progresji (PFS) oraz przeżyciem całkowitym (OS) chorych na szpiczaka plazmocytowego. Ponadto ujemny wynik badania w kierunku MRD jest lepszym czynnikiem prognostycznym w odniesieniu do PFS oraz OS niż osiągnięcie CR. Z tych względów wynik oceny MRD, uzyskany wysoce czułą oraz powtarzalną metodą MFC, jest potencjalnie użytecznym klinicznie biomarkerem do oceny skuteczności różnych strategii leczniczych i może być podstawą do podejmowania decyzji terapeutycznej oraz użytecznym czynnikiem prognostycznym w szpiczaku plazmocytowym.

Słowa kluczowe: szpiczak plazmocytowy, minimalna choroba resztkowa, cytometria przepływowa

Hematologia 2017; 8, 3: 211–218

INTRODUCTION

Multiple myeloma (MM) is a hematologic malignancy characteristic by presence of clonal bone marrow plasma cells (PCs). Development of new therapies led to the significantly prolonged overall survival (OS) in newly diagnosed patients [1]. Effective treatment comes along with the need for more sensitive approaches to compare the efficacy of different treatment strategies, and implementation of individualized therapy monitoring strategies to prevent both under- and overtreatment [2]. There are still developed new therapeutics, but the transition to mainstream availability is much slower as randomized phase III clinical trials take years to show a benefit when using progression free survival (PFS) and OS as study endpoints [3]. Extensive data indicate that minimal residual disease (MRD) information can potentially be used as biomarker to evaluate the efficacy of different treatment strategies, help on treatment decisions, and act as surrogate for overall survival [2–4]. Confirmation of the elimination of myeloma residual cell clones resistant to the therapy should be the way to cure MM.

There are available techniques on cellular (MFC, multiparametric flow cytometry; NGF, next generation flow) and/or molecular (qPCR, quantitative polymerase chain reaction; NGS, next generation sequencing) level including imaging methods which allowed to find that persistent MRD means worse survival in MM (Table 1) [2, 5–7]. MFC seems to be the most effective of existing approaches. Development of protocols for MFC-MRD followed technical progress of cytometry itself, availability of new antigens and fluorochromes together with standardization requirements [2, 8]. A novel validated NGF assay for high-sensitive, fast and standardized quantification of MRD in MM that overcomes previous limitations of conventional MFC-MRD approaches and improves prediction of patient outcome, is ready-to-use and well-suited for implementation in clinical trials to establish the diagnostic role of MRD in MM [7].

Table 1. Characteristics of available techniques to monitor minimal residual disease (MRD) in multiple myeloma (source [2])

Tabela 1. Charakterystyka dostępnych technik monitorowania minimalnej choroby resztkowej (MRD) w szpiczaku plazmocytowym (źródło [2])

NA — not available data

NEEDS FOR MRD DETECTION

The indicator of treatment effectivity is the number of residual clonal cells. Assessment of MRD is becoming standard diagnostic care for potentially curable neoplasms such as acute lymphoblastic leukaemia. In MM, the majority of patients will inevitably relapse despite achievement of progressively higher complete response (CR) rates, but new treatment approaches might further increase remission rates and potentially cure rates [2]. Interestingly, few of patients that reach suboptimal response (near CR and/or very good partial response) are relapse free at 10 years [9]. Paiva et al. [10] already in 2008 demonstrated the clinical importance of MRD evaluation by MFC and illustrated the need for further refinement of MM response criteria. Analysis of contribution of the serum free light chain ratio (sFLCr) or bone marrow (BM) clonality to the prognosis of MM revealed, that the sFLCr does not identify patients in CR at distinct risk; by contrast, flow cytometry revealed patients with a significantly inferior outcome. So the achieving of CR does not mean to achieve cure and new definition of CR is needed even as stringent CR (sCR) is not sufficiently informative in term of supposed PFS and/or OS [6]. However, the definition of clinical response criteria and clinical end points largely remained the same over the past 15 years. It was proved that MRD detection is sensitive and fast approach to acquire an independent predictor of PFS and OS [11, 12]. Even more, as was recently demonstrated, MRD-negative status surpasses the prognostic value of CR achievement for PFS and OS across the disease spectrum, regardless of the type of treatment or patient risk group. Thus MRD negativity should be considered as one of the most relevant end points for transplant-eligible and elderly fit patients with MM [13].

FLOW CYTOMETRIC ASSESSMENT OF MRD

Although flow cytometry is not a diagnostic tool in MM analyses (where morphology is widely available and still plays an irreplaceable role; on the other hand, the underestimation of PC number by flow cytometry is known), it provides important information about the presence and number of especially clonal PC. MFC is generally applicable to majority of myeloma patients with a sensitivity ranging from 10−4 to 10−6. The progress in MFC technology and wide availability of used antibodies allows MFC to be an integral part of laboratory investigations and the management of plasma-cell disorders (PCD) and can play an important part in the diagnosis, prognostic stratification, and monitoring of response to therapy via minimal residual disease detection, the understanding of the biology of disease progression, the study of the role of the tumour microenvironment in PCD and the identification of potential therapeutic targets on the malignant PC (Table 2) [2, 16–18]. Valid and/or even better standardized MRD detection will ensure superior uniform assessment of response and clinical prognostication.

Table 2. List of the most useful antigens allowing normal and abnormal plasma cells (PC) discrimination (sources [14, 15])

Tabela 2. Lista najbardziej użytecznych antygenów do różnicowania prawidłowych i nieprawidłowych komórek plazmatycznych (PC) (źródła [14, 15])

*Own results

MFC in MRD definition

The use of MFC in the detection of MRD in BM was demonstrated by several studies from 2002. The sensitivity of the flow cytometry assay was highlighted by the presence of detectable PC in nearly a third of the patients with negative immunofixation (IFx–) results and patients who were MRD-positive (MRD+) had a worse outcome [19, 20]. Flow cytometry was mentioned for the first time in stringent CR (sCR) definition, where absence of BM clonal cells by immunohistochemistry or immunofluorescence was required [21]. Presence/absence of clonal cells was based upon the κ/λ ratio which required a minimum of 100 PC for analysis, an abnormal ratio reflecting presence of an abnormal clone was κ/λ of > 4:1 or < 1:2 [21]. Then the term MFC remission was used and patients were considered to be in MFC remission when MM-PC were undetectable in the BM sample at the MFC sensitivity limit of 10–4 (ie, 1 MM-PC in 104 N-PC). Only 4-colour MFC was used and a minimum of 3 × 105 BM cells was acquired [10]. Later, the updated International Myeloma Working Group (IMWG) response criteria in 2011 incorporated some new designations to traditional CR definitions and immunophenotypic CR (iCR, sCR+) was defined as mentioned in previous sentence [10, 22]. Recently published IMWG MRD criteria defines flow MRD-negative (MRD–) sample as an absence of phenotypically aberrant clonal plasma cells by NGF on BM aspirates using the EuroFlow standard operation procedure for MRD detection in MM (or validated equivalent method) with a minimum sensitivity of 1 in 105 nucleated cells or higher [18].

Highly sensitive MFC and standardisation

MFC seems to be the most perspective approach for detection of MRD in MM in terms of speed, price and availability of method. Applicability of MFC is over 90% of MM patient regardless of the knowledge of the phenotype in time of diagnosis. The initial lower sensitivity of the method (when used 4-colour flow cytometry) was increased by simultaneous detection of 8 markers/fluorochromes and acquisition of sufficient number of leukocytes in 1 run up to 10–7, thus MFC is comparable with techniques based on molecular level [7]. The technique has been modified to include an initial bulk lysis step to consistently measure more than 5 × 106 leucocytes per tube. Transition to the quantitative assessment of residual clonal PC will improve predictive potential of analyses as higher logarithms of clonal PC depletion significantly improved the length of OS [12]. But harmonisation and/or standardisation of MFC in MM are still relatively open as majority of clinical labs use their own protocols which are not inter-laboratory comparable (Figure 1) and often not sufficient enough in a sensitivity manner [4].

Figure 1A–H. Different approaches of multiparametric flow cytometry of minimal residual disease (MFC-MRD) analysis. Comparison of “home-made” (A–D) and 2nd generation of Euroflow PCD (E–H) protocol was done. Almost similar position of polyclonal (n-PCs, blue dots) and clonal (a-PCs, red dots) plasma cells (PCs) are visualised. Data acquired on BD FACSCanto II with Diva SW (BD Biosciences) and reanalysed by Infinicyt SW (Cytognos)

Rycina 1A–H. Odmienne podejścia do analizy minimalnej choroby resztkowej (MRD) metodą wieloparametrycznej cytometrii przepływowej (MFC). Przeprowadzono porównanie analizy za pomocą własnego protokołu typu home-made (A–D) i protokołu Euroflow PCD drugiej generacji (E–H). Zwraca uwagę podobne położenie poliklonalnych (n-PCs, niebieskie punkty) i klonalnych (a-PC, czerwone punkty) plazmocytów (PC). Akwizycję danych przeprowadzono na cytometrze przepływowym BD FACSCanto II z oprogramowaniem Diva SW (BD Bioscience); ponownej analizy danych dokonano przy użyciu oprogramowania Infinicyt SW (Cytognos)

Project EuroFlow offers standardized process of sample preparation and data acquisition when defined validated panels of selected and verified markers are used for every type of haematological malignancy. Also innovative software Infinicyt for data analysis is used [23]. Original EuroFlow panel designed for PCD was not sufficient for MRD detection so 2nd generation of panel was developed (Table 3). This can identify clonal PC on a background of normal regenerating BM (Figure 1) [8]. The novel NGF-MRD approach takes advantage of innovative tools and procedures recently developed by the EuroFlow Consortium for sample preparation, antibody panel construction (including choice of type of antibody and fluorochrome), and automatic identification of PC against reference databases of normal and patient BM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ≥ 107 cells/sample. Prospective validation of the whole procedure at two distinct centers confirmed its robustness and significantly greater sensitivity vs. conventional 8-color MRD approaches, comparable to current NGS methods, with an improved prediction of patient outcome [7]. On the other hand, semi-standardised approach is available from Beckman Coulter Company as premixed dry tubes combining 8 surface markers (CD38/CD45/CD81/CD27/CD19/CD200/CD138/CD56) for effective detection of clonal PC without clonality assessment (Figure 2).

Table 3. Development of Euroflow PCD panel (sources [7, 23])

Tabela 3. Opracowanie panelu Euroflow PCD (źródła [7, 23])

PB — pacific blue; PO — pacific orange; FITC — fluoresceinisothiocyanate; PE — phycoerythrin; PerCP-Cy5.5 — perridin chlorophyll-cyanine 5.5; PC7 — phycoerythrin-cyanine 7; APC — allophycocyanin; APC-H7 — allophycocyanin-H7; BV421 — brilliant violet 421; BV510 — Brilliant Violet 510; APC-C750 — allophycocyanin-C750

Figure 2A–H. Polychromatic analysis of plasma cells (PCs) in daratumumab treated multiple myeloma. Comparison of “home-made” (A–D) and Duraclone RE PC (E–H) protocol was done. Phenotype of clonal PCs (red dots) is similar CD19–CD56+CD27– (B, C, F, G) with high CD138 expression (A, E) in both approaches. Clonality assessment confirmed cytoplasmic expression of lambda light Ig chain (D), low CD81 and higher CD200 expression is relevant to pathological PCs as well (H). Data acquired on BD FACSCanto II with Diva SW (BD Biosciences) and reanalysed by Infinicyt SW (Cytognos)

Rycina 2A–H. Wielokolorowa analiza komórek plazmatycznych (PC) chorego na szpiczaka plazmocytowego leczonego daratumumabem. Przeprowadzono porównanie analizy za pomocą własnego protokołu typu home-made (A–D) i protokołu Duraclone RE PC (E–H). W obu podejściach fenotyp PC jest podobny: CD19–CD56+CD27– (B, C, F, G) z wysoką ekspresją CD138 (A, E). Ocena klonalności potwierdziła cytoplazmatyczną ekspresję lekkiego łańcucha Ig lambda (D); niska ekspresja CD81 i wyższa Cd200 jest również typowa dla patologicznych plazmocytów (H). Akwizycję danych przeprowadzono na cytometrze przepływowym BD FACSCanto II z oprogramowaniem Diva SW (BD Bioscience); ponownej analizy danych dokonano przy użyciu oprogramowania Infinicyt SW (Cytognos)

Preanalytical rules

It is very important to obtain BM sample not diluted by peripheral blood, where marrow elements must be present, for obtaining a high quality results. There is a preference of EDTA anticoagulants as Heparin decrease CD138 intensity and PC should be less recognisable in context of whole leukocytes. When transportation of sample is needed, only the room temperature must be used. Analysis must be done until 36 hours from sample acquisition [4, 24].

Trouble shooting

Because of reduced number of PC after treatment, the procedure for cell concentration must be used (bulk lysis), to allow MRD assessment without loss of cell subpopulation, and to acquire at least limit of detection (LOD) and better limit of quantification (LOQ) of method [25]. Using of targeted therapies as anti-CD38 (daratumumab) may complicate analysis by home-made protocols, but replacement of CD38 by CD229 and/or using multiepitope CD38 resolve that problem [26]. The therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PC show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles [27]. Although characteristic phenotype profile of clonal PC is already known (Table 2), their detection should be impossible in small part of patients with atypical and/or changed profile (diagnostic vs. MRD antigenic profile), where different spectrum of antibodies should be used. Analysis of listmodes without SW assistance requires well educated operator and it is relatively time consuming.

Results report

The reporting of results to the clinicians should be descriptive and clear including % of plasma cells from leukocytes, % of clonal PC from whole PC population, phenotype of clonal cells, and sensitivity of analysis. Unsuitable and/or not representative samples should be reported.

CLINICAL RELEVANCY OF MFC-MRD ASSESSMENT

Previous approaches to measurement of MRD levels were based on morphological assessment of BM, analysis of the paraprotein levels, or polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain variable-diversity-joining (VDJ) region. Historically, first results demonstrated the clinical importance of MFC in MRD detection, whichwere published in 2002, when Rawstron at al. shown that analysis of normal and neoplastic PC levels is more sensitive than IFx [19]. Patients with detected neoplastic PC had a significantly shorter PFS than those with no detectable disease (median 20 months vs. > 35 months, p = 0.003). Neoplastic PC were detectable in 27% (9 of 33) of IFx(–) CR patients. These patients had a significantly shorter PFS than IFx(–) patients with no detectable neoplastic PC (p = 0.04) [19]. Similarly San Miguel et al. [20] found out that treatment-induced changes in the PC compartment correlated with disease outcome and patients in whom at least 30% of gated PC had a normal phenotype after treatment with a significantly longer PFS (60 ± 6 vs. 34 ± 12 months, p = 0.02). Detailed analysis of newly diagnosed MM patients treated in Spanish the GEM2000 protocol showed that PFS (median 71 vs. 37 months, p < 0.001) and OS (median not reached vs. 89 months, p = 0.002) were longer in patients who were MRD(–) versus MRD(+) at day 100 after autologous stem cell transplantation (ASCT). Similar prognostic differentiation was seen in patients who achieved IFx(–) complete response after ASCT. Moreover, MRD(–)IFx(–) patients and MRD(–)IFx(+) patients had significantly longer PFS than MRD(+)IFx(–) patients. Multivariate analysis identified MRD status by MFC at day 100 after ASCT as the most important independent prognostic factor for PFS (hazard ratio [HR] = 3.64, p = 0.002) and OS (HR = 2.02, p = 0.02) [10]. Results from Medical Research Council (MRC) Myeloma IX trial has shown that absence of MRD at day 100 after ASCT was highly predictive of a favorable outcome (PFS, p < 0.001; OS, p = 0.0183). This outcome advantage was demonstrable in patients with favorable and adverse cytogenetics (PFS, p = 0.014 and p < 0.001, respectively) and in patients achieving IFx(–) CR (PFS, p < 0.0068). The effect of maintenance thalidomide was assessed, with the shortest PFS demonstrable in those MRD(+) patients who did not receive maintenance and longest in those who were MRD(–) and did receive thalidomide (p < 0.001). Further analysis demonstrated that 28% of MRD(+) patients who received maintenance thalidomide became MRD(–). MRD assessment after induction therapy in the non-intensive-pathway patients did not seem to be predictive of outcome (PFS, p = 0.1) [28]. Also was demonstrated that the prognostic impact of MRD following ASCT is independent of the induction therapy received [29]. Surprisingly, MFC-MRD monitoring has the prognostic value also in relapsed MM and is one of the most relevant prognostic factors in elderly MM patients, irrespectively of age or cytogenetic risk [11, 30]. Using NGF in multicentre evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs. conventional 8-color MFC-MRD with MRD(+) rate of 47 vs. 34% (p = 0.003). Thus, 25% of patients classified as MRD(–) by conventional 8-color MFC were MRD(+) by NGF, translating into a significantly longer PFS for MRD(–) vs. MRD(+) CR patients by NGF (75% PFS not reached vs. 7 months, p = 0.02) [7]. Very recent publication showed that achievement of CR in the absence of MRD negativity was not associated with prolonged PFS and OS compared with near-CR or partial response (median PFS 27, 27, and 29 months, respectively; median OS, 59, 64, and 65 months, respectively). MRD(–) status was strongly associated with prolonged PFS (median 63 months, p < 0.001) and OS (median not reached, p < 0.001) overall and in subgroups defined by prior transplantation, disease stage, and cytogenetics, with prognostic superiority of MRD negativity versus CR particularly evident in patients with high-risk cytogenetics [13].

CONCLUSION

As treatment strategies for MM become more effective and PFS becomes longer, assessing the treatment efficacy according to MRD levels becomes increasingly important. Different approaches for MFC-MRD monitoring are available. Implementation of highly sensitive automated MFC-MRD assessment by NGF should confirm a new biomarker for treatment effectivity assessment and replace obsolete indicators defining clinical response and prediction of OS in MM. The prerequisite for that is standardisation of sample processing, sample and data analysis and verification of this approach in clinical studies.

ACKNOWLEDGEMENT

Supported by Ministry of Health of the Czech Republic, grant nr. 17-30089A. All rights reserved.

Adres do korespondencji: Lucie Rihova, Department of Clinical Haematology, University Hospital Brno, Jihlavska 20, 625 00 Brno, Czech Republic, tel. +420 532 232 873, fax +420 532 233 613, e-mail: lucie.rihova@fnbrno.cz

PIŚMIENNICTWO

1. Mateos MV, Ocio EM, Paiva B, et al. Treatment for patients with newly diagnosed multiple myeloma in 2015. Blood Rev. 2015; 29(6): 387–403, doi: 10.1016/j.blre.2015.06.001, indexed in Pubmed: 26094881.

2. Paiva B, van Dongen JJM, Orfao A. New criteria for response assessment: role of minimal residual disease in multiple myeloma. Blood. 2015; 125(20): 3059–3068, doi: 10.1182/blood-2014-11-568907, indexed in Pubmed: 25838346.

3. Oldaker TA, Wallace PK, Barnett D. Flow cytometry quality requirements for monitoring of minimal disease in plasma cell myeloma. Cytometry B Clin Cytom. 2016; 90(1): 40–46, doi: 10.1002/cyto.b.21276, indexed in Pubmed: 26201282.

4. Rawstron AC, Paiva B, Stetler-Stevenson M. Assessment of minimal residual disease in myeloma and the need for a consensus approach. Cytometry B Clin Cytom. 2016; 90(1): 21–25, doi: 10.1002/cyto.b.21272, indexed in Pubmed: 26202864.

5. Puig N, Sarasquete ME, Balanzategui A, et al. Critical evaluation of ASO RQ-PCR for minimal residual disease evaluation in multiple myeloma. A comparative analysis with flow cytometry. Leukemia. 2014; 28(2): 391–397, doi: 10.1038/leu.2013.217, indexed in Pubmed: 23860448.

6. Martínez-López J, Paiva B, López-Anglada L, et al. Spanish Multiple Myeloma Group / Program for the Study of Malignant Blood Diseases Therapeutics (GEM/PETHEMA) Cooperative Study Group. Critical analysis of the stringent complete response in multiple myeloma: contribution of sFLC and bone marrow clonality. Blood. 2015; 126(7): 858–862, doi: 10.1182/blood-2015-04-638742, indexed in Pubmed: 26089396.

7. Flores-Montero J, Sanoja-Flores L, Paiva B, et al. Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Leukemia. 2017; 31(10): 2094–2103, doi: 10.1038/leu.2017.29, indexed in Pubmed: 28104919.

8. Flores-Montero J, de Tute R, Paiva B, et al. Immunophenotype of normal vs. myeloma plasma cells: Toward antibody panel specifications for MRD detection in multiple myeloma. Cytometry B Clin Cytom. 2016; 90(1): 61–72, doi: 10.1002/cyto.b.21265, indexed in Pubmed: 26100534.

9. Martinez-Lopez J, Blade J, Mateos MV, et al. Grupo Español de MM, Programa para el Estudio de la Terapé utica en Hemopatía Maligna. Long-term prognostic significance of response in multiple myeloma after stem cell transplantation. Blood. 2011; 118(3): 529–534, doi: 10.1182/blood-2011-01-332320, indexed in Pubmed: 21482708.

10. Paiva B, Vidriales MB, Cerveró J, et al. GEM (Grupo Español de MM)/PETHEMA (Programa para el Estudio de la Terapéutica en Hemopatías Malignas) Cooperative Study Groups. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation. Blood. 2008; 112(10): 4017–4023, doi: 10.1182/blood-2008-05-159624, indexed in Pubmed: 18669875.

11. Paiva B, Chandia M, Puig N, et al. The prognostic value of multiparameter flow cytometry minimal residual disease assessment in relapsed multiple myeloma. Haematologica. 2015; 100(2): e53–e55, doi: 10.3324/haematol.2014.115162, indexed in Pubmed: 25381128.

12. Rawstron AC, Gregory WM, de Tute RM, et al. Minimal residual disease in myeloma by flow cytometry: independent prediction of survival benefit per log reduction. Blood. 2015; 125(12): 1932–1935, doi: 10.1182/blood-2014-07-590166, indexed in Pubmed: 25645353.

13. Lahuerta JJ, Paiva B, Vidriales MB, et al. GEM (Grupo Español de Mieloma)/PETHEMA (Programa para el Estudio de la Terapéutica en Hemopatías Malignas) Cooperative Study Group. Depth of Response in Multiple Myeloma: A Pooled Analysis of Three PETHEMA/GEM Clinical Trials. J Clin Oncol. 2017; 35(25): 2900–2910, doi: 10.1200/JCO.2016.69.2517, indexed in Pubmed: 28498784.

14. Rawstron AC, Orfao A, Beksac M, et al. European Myeloma Network. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica. 2008; 93(3): 431–438, doi: 10.3324/haematol.11080, indexed in Pubmed: 18268286.

15. Rihova L, Muthu Raja KR, Calheiros Leite LA. immunophenotyping in multiple myeloma and others monoclonal gammopathies. In. Intech, 2013.

16. Paiva B, Almeida J, Pérez-Andrés M, et al. Utility of flow cytometry immunophenotyping in multiple myeloma and other clonal plasma cell-related disorders. Cytometry B Clin Cytom. 2010; 78(4): 239–252, doi: 10.1002/cyto.b.20512, indexed in Pubmed: 20155853.

17. Raja KRM, Kovarova L, Hajek R. Review of phenotypic markers used in flow cytometric analysis of MGUS and MM, and applicability of flow cytometry in other plasma cell disorders. Br J Haematol. 2010; 149(3): 334–351, doi: 10.1111/j.1365-2141.2010.08121.x, indexed in Pubmed: 20201947.

18. Kumar S, Paiva B, Anderson KC, et al. International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma. Lancet Oncol. 2016; 17(8): e328–e346, doi: 10.1016/S1470-2045(16)30206-6, indexed in Pubmed: 27511158.

19. Rawstron AC, Davies FE, DasGupta R, et al. Flow cytometric disease monitoring in multiple myeloma: the relationship between normal and neoplastic plasma cells predicts outcome after transplantation. Blood. 2002; 100(9): 3095–3100, doi: 10.1182/blood-2001-12-0297, indexed in Pubmed: 12384404.

20. San Miguel JF, Almeida J, Mateo G, et al. Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: a tool for comparing the efficacy of different treatment strategies and predicting outcome. Blood. 2002; 99(5): 1853–1856, doi: 10.1182/blood.v99.5.1853, indexed in Pubmed: 11861305.

21. Durie BGM, Harousseau JL, Miguel JS, et al. International Myeloma Working Group. International uniform response criteria for multiple myeloma. Leukemia. 2006; 20(9): 1467–1473, doi: 10.1038/sj.leu.2404284, indexed in Pubmed: 16855634.

22. Rajkumar SV, Harousseau JL, Durie B, et al. International Myeloma Workshop Consensus Panel 1. Consensus recommendations for the uniform reporting of clinical trials: report of the International Myeloma Workshop Consensus Panel 1. Blood. 2011; 117(18): 4691–4695, doi: 10.1182/blood-2010-10-299487, indexed in Pubmed: 21292775.

23. van Dongen JJM, Lhermitte L, Böttcher S, et al. EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708). EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. 2012; 26(9): 1908–1975, doi: 10.1038/leu.2012.120, indexed in Pubmed: 22552007.

24. Stetler-Stevenson M, Paiva B, Stoolman L, et al. Consensus guidelines for myeloma minimal residual disease sample staining and data acquisition. Cytometry B Clin Cytom. 2016; 90(1): 26–30, doi: 10.1002/cyto.b.21249, indexed in Pubmed: 25907102.

25. Arroz M, Came N, Lin P, et al. Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting. Cytometry B Clin Cytom. 2016; 90(1): 31–39, doi: 10.1002/cyto.b.21228, indexed in Pubmed: 25619868.

26. Pojero F, Flores-Montero J, Sanoja L, et al. EuroFlow group. Utility of CD54, CD229, and CD319 for the identification of plasma cells in patients with clonal plasma cell diseases. Cytometry B Clin Cytom. 2016; 90(1): 91–100, doi: 10.1002/cyto.b.21269, indexed in Pubmed: 26130131.

27. Paiva B, Corchete LA, Vidriales MB, et al. Spanish Myeloma Group/Program for the Study of Malignant Blood Diseases Therapeutics (GEM/PETHEMA) Cooperative Study Groups. Phenotypic and genomic analysis of multiple myeloma minimal residual disease tumor cells: a new model to understand chemoresistance. Blood. 2016; 127(15): 1896–1906, doi: 10.1182/blood-2015-08-665679, indexed in Pubmed: 26755711.

28. Rawstron AC, Child JA, de Tute RM, et al. Minimal residual disease assessed by multiparameter flow cytometry in multiple myeloma: impact on outcome in the Medical Research Council Myeloma IX Study. J Clin Oncol. 2013; 31(20): 2540–2547, doi: 10.1200/JCO.2012.46.2119, indexed in Pubmed: 23733781.

29. de Tute RM, Rawstron AC, Gregory WM, et al. Minimal residual disease following autologous stem cell transplant in myeloma: impact on outcome is independent of induction regimen. Haematologica. 2016; 101(2): e69–e71, doi: 10.3324/haematol.2015.128215, indexed in Pubmed: 26471484.

30. Paiva B, Cedena MT, Puig N, et al. Grupo Español de Mieloma/Programa para el Estudio de la Terapéutica en Hemopatías Malignas (GEM/PETHEMA) Cooperative Study Groups. Minimal residual disease monitoring and immune profiling in multiple myeloma in elderly patients. Blood. 2016; 127(25): 3165–3174, doi: 10.1182/blood-2016-03-705319, indexed in Pubmed: 27118453.