Hematopoietic Score for the Prognostic Evaluation of Multiple Myeloma Patients Undergoing First-Line Treatment With A Bortezomib-Based Regimen

Background: It is very important to evaluate the prognosis of multiple myeloma (MM) patients before starting treatment. Although hematopoietic status may have a signicant impact on patient survival, it has not received sucient attention in current clinical practice. Methods: This was a retrospective study of 328 newly diagnosed MM patients received rst-line treatment with a bortezomib-based regimen. The effects of hematopoietic factors, including hemoglobin (Hgb) levels, mean corpuscular volume (MCV), and platelet count (Plt) on the prognosis of the patients were analysed. Results: Hgb<100 g/L, MCV>99.1fL and Plt<150×10 9 /L signicantly affect the progression-free survival (PFS) and overall survival (OS) of MM patients, each of the above factors was assigned a value of 1 to generate a hematopoietic score. According to the results, 93 (28.4%), 103 (31.4%), 90 (27.4%) and 42 (12.8%) patients had a score of 0, 1, 2, 3, respectively. The median PFS were 38.7, 55.9, 23.9 and 16.7 months, respectively (P<0.001), and the median OS were not reached (NR), 68.4months, 53.6 and 33.2 months, respectively (P<0.001). Multivariable analysis showed that hematopoietic score (2-3 vs 0-1, HR, 1.64) and bone marrow plasma cell percentage (>30%, HR, 1.54) are independent prognostic predictors for PFS; age ( ≥ 70 years, HR, 1.67), hematopoietic score (2-3 vs 0-1, HR, 1.60), serum creatinine level (>177umol/L, HR, 2.15) and bone marrow plasma cell percentage (>30%, HR, 1.81) are independent prognostic predictors of OS. Conclusions: This study suggests that the hematopoietic score used Abbreviations: Staging R-ISS, Revised International Staging System; Hgb, hemoglobin; Plt, platelet; WBC, white blood cell; MCV, mean corpuscular volume; LDH, lactate dehydrogenase; Cr, creatinine; PD, bortezomib, dexamethasone; PCD, bortezomib, dexamethasone, cyclophosphamide; PAD, bortezomib, dexamethasone, adriamycin; PTD, bortezomib, dexamethasone thalidomide; ASCT: Autologous hematopoietic stem cell transplantation. MM drug

Currently, the proteasome inhibitor bortezomib and the immunoregulatory agents thalidomide and lenalidomide are the most important and commonly used drugs for the treatment of MM in China. Since February 2006, bortezomib has been used in our centers for the rst-line treatment of Chinese MM patients (4)(5)(6), with an overall response rate (ORR, partial remission and better) of 90%; over 60% of patients achieve very good partial remission (VGPR) or better.
MM is a heterogeneous disease, and it is important to assess the prognosis of patients before the disease is treated. The Durie-Salmon staging system was once the most important staging system, and included the following factors: Hgb level, serum calcium level, extent of bone lesions, M protein level, and renal function status (6). However, this staging system is closely related to the tumor burden of MM, and its prognostic signi cance is often questioned in the era of new drugs. Currently, the International Staging System (ISS) and the latest Revised International Staging System (R-ISS) are commonly applied in patients undergoing rst-line therapy. However, the former contains only two indicators, namely, the serum albumin and β2-microglobulin levels, while the latter combines those with the serum lactate dehydrogenase (LDH) level and/or uorescence in situ hybridization (FISH)-based cytogenetic abnormalities (7,8), which have limited prognostic evaluation in MM patients.
Anemia is very common clinical symptom in MM patients for a variety of reasons, for example, the in ltration of plasma cells into the bone marrow inhibits erythropoiesis, and the bone marrow microenvironment is altered; there is also a decline in the level of in erythropoietin due to renal insu ciency and anemia owing to the release of various in ammatory factors that affect normal hematopoiesis. The role of Hgb in the prognosis of MM patients is clearly de ned in the Durie-Salmon staging system and 100 g/L is used as one of the cut-off values for an abnormal Hgb level (6). In addition, abnormal red blood cell size, indicated by abnormal red blood cell distribution width (RDW) or average red blood cell volume values, and Plt counts are important MM prognostic indicators (2,6,(9)(10)(11)(12)(13).
However, these indicators have not received enough attention in current clinical practice.
Therefore, this study retrospectively analyzed the effect of the "hematopoietic score" including hemoglobin (Hgb), mean corpuscular volume (MCV) and platelet (plt) count on the prognosis of MM patients treated with bortezomib as rst-line therapy, in the hope that this score could be served as an important supplement to the prognostic evaluation of newly diagnosed MM patients in the new drug era.

Patients
This study was a retrospective analysis and was approved by the Ethics Committee of the First A liated Hospital of the Medical School of Zhejiang University. From May 2013 to June 2019, patients who were newly diagnosed according to the World Health Organization (WHO) or International Myeloma Working Group (IMWG) diagnostic criteria in our center were included if they had symptomatic MM with detectable M protein in their blood and/or urine and received rst-line treatment with bortezomib-based regimen. From the beginning of treatment to the end of follow-up, all patients were informed of their condition and survival status through inpatient services, outpatient services or telephone contact.

Treatment regimen and e cacy evaluation
All patients received a bortezomib-based regimen either as: a two-drug combined PD regimen (bortezomib combined with dexamethasone) or a three-drug combined regimen, with a third drug, such as cyclophosphamide (PCD), adriamycin (PAD) or thalidomide (PTD), added to the PD regimen. The speci c administration method is detailed elsewhere (5). If indicated by the patient's age, physical condition and willingness and if partial remission (PR) was obtained after at least 3-4 courses of induction treatment, autologous hematopoietic stem cell transplantation (ASCT) performed. After induction therapy with or without ASCT, patients received maintenance therapy based on bortezomib, lenalidomide, or thalidomide.
The IMWG e cacy evaluation standard was used to assess the e cacy of treatment, including complete remission (CR), VGPR, PR, stable disease (SD) and progressive disease (PD) (15). Progression-free survival (PFS) was de ned as the time from the beginning of the patients' rst course of treatment to disease progression, death or the nal follow-up visit. Overall survival (OS) was calculated from the time of the rst course of treatment to death or the nal follow-up visit.

Data acquisition
All patients were hospitalized during the initial diagnosis and treatment, and data regarding routine blood examinations, hepatic and renal function indicators, LDH levels, and bone marrow examination results were obtained from the hospital information system. All patients were evaluated based on the Durie-Salmon staging system and the ISS at the time of diagnosis. Using uorescence in situ hybridization (FISH), bone marrow cells were examined for speci c chromosomal abnormalities, including del-(17p13), 1q21 gain, del(13q14), and 14q32 rearrangement, and speci c translocations, including t (4;14), t (11;14), and t (14;16), were detected in a small number of patients with 14q32 rearrangement. Retrospective assessment of the patients was based on the R-ISS, combining the patients' ISS stages, serum LDH levels, and FISH results, of which 1q ampli cation was also considered a genetic abnormality indicative of a poor prognosis (14). The data obtained included the WBC count, Hgb level, MCV, Plt count, serum creatinine level, serum LDH level, bone marrow plasma cell ratio and FISH examination results of bone marrow cells.

Statistical analysis
All patients were followed up until June 30, 2020, and the e cacy of treatment was evaluated after completing each course. The cut-off values for the Hgb level (<100 g/L); Plt count (<150×10 9 /L) were based on the literature (2) The upper limit of normal for the MCV in our center is 99.1fL, which was selected as the threshold. The boundary value of other data is based on the normal boundary value of our center or the ROC curve. Chi-square test was employed for comparisons of classi cation data, the Kaplan-Meier method was used to generate survival curves and the log-rank test was used to compare differences in patient survival. Univariate analysis using the Cox proportional hazards model was performed for age; Durie-Salmon stage; ISS, R-ISS; bone marrow plasma cell ratio; blood creatinine level; blood LDH level; peripheral blood WBC count, Hgb level, MCV, Plt count and the hematopoietic score to evaluate their impact on patient survival. All statistical tests were two-sided and P-values <0.05 were considered to be statistically signi cant. Only factors with P-values <0.1 were included in the multivariable analysis. The hazard ratio (HR) and 95% con dence intervals (CI) were reported. The statistical analysis was performed using SPSS for Windows 20.0.

Hgb level, MCV and platelet count and patient characteristics
A total of 328 newly diagnosed MM patients were enrolled in this study, of whom 180 were males and 148 were females; the median age was 63 years (range from 31 to 84). Table 1 shows the baseline characteristics and treatment speci cs of the patients. According to the results of the routine peripheral blood examinations at the time of diagnosis, 72 patients (22.0%) had WBC counts lower than normal (4×10 9 /L), 197 patients (60.1%) had an Hgb level lower than 100 g/L, 84 patients (25.6%) had a MCV higher than normal (99.1fL) and 128 patients (39.0%) had a Plt count lower than 150×10 9 /L.   Based on univariable analysis, MCV and platelet counts signi cantly affect the patient's PFS, while Hgb, MCV and platelet counts signi cantly affect the patient's OS (Tables 2, 3, and Supplementary Table 2).
The median PFS was 38.7 months for patients with Hgb levels ≥ 100 g/L and 26.5 months for Hgb levels < 100 g/L (P = 0.173), and the median OS was 64.8 and 56.8 months, respectively (P = 0.018 Additionally, the median PFS was 42.8 months for patients with Plt counts ≥ 150×10 9 /L and 24.1 months for patients with Plt counts < 150×10 9 /L (P = 0.002), and the median OS times were not reach (NR) vs 51.1 months(P = 0.001), respectively. Each of the above three indicators was assigned a score of 1 to generate the hematopoietic score. The integral values were 0, 1, 2, and 3, and the score signi cantly affected both PFS and OS (P < 0.001) ( Tables 2, 3 and Fig. 1A, 1B). Overall, 93 (28.4%), 103 (31.4%),90 (27.4%) and 42 (12.8%) patients had scores of 0, 1, 2 and 3, respectively; the median PFS times were 38.7 months, 55.9 months, 23.9 months and 16.7 months, respectively (P < 0.001), and the median OS times were NR, 64.8 months, 53.6 and 33.2 months, respectively (P < 0.001). The median PFS was 43.1 months in patients who had a hematopoietic score from 0 to 1, and the estimated 3-year and 5-year PFS rates were 54.5% and 37.6%, respectively. In patients with a score of 2-3, the median PFS was only 23.5 months, with estimated 3-year and 5-year PFS rates of 37.3% and 20.6%, respectively (P < 0.001) ( Fig. 2A, Table 1, Supplementary Table 2). The median OS was NR for patients who had a hematopoietic score from 0 to 1, and the estimated 3-year and 5-year OS rates were 78.8% and 56.6%, respectively. In contrast, the median OS of patients who had a score of 2 to 3 was only 31.4 months, with estimated 3-year and 5year OS rates of 58.0% and 40.7%, respectively (P < 0.001) (Fig. 2B, Table 2, Supplementary Table 2).

Discussion
MM is an extremely heterogeneous disease, and survival can range from months to long-term stable disease. Therefore, it is very important to evaluate the prognosis of patients at the time of initial diagnosis. Study has con rmed that the occurrence and development of MM depends on the tumor microenvironment in the bone marrow, and the interaction between myeloma cells and the bone marrow microenvironment is very complicated. In addition to occupying the niches where hematopoietic cells can grow, the proliferation of MM cells can also result in the secretion of a large amount of hematopoietic inhibitory factors, which affect normal hematopoiesis (16,17), regarding megakaryocytic and erythroid precursor cells in particular, both the number and function are signi cantly inhibited, including clone formation ability and self-renewal ability (17). Therefore, low Hgb levels and Plt counts and an increased red blood cell volume may indicate the presence of more plasma cells in the bone marrow. Moreover, normal hematopoietic function is inhibited by various mechanisms, which indicates that the disease is more invasive.
In an early Mayo clinic study (with patients enrolled from 1985 to 1988), it was found that a Hgb level < 100 g/L and a Plt count < 150×10 9 /L were predictive of relatively worse outcomes based on univariate analysis (2). In 2020, clinical data of MM patients treated at the Mayo Clinic in the United States for more than 10 years were retrospectively analysed (11), and propose the in uence of hematopoietic score composed of Hgb level, MCV and plt count on the prognosis of MM patients, which con rmed that patients with high hematopoietic scores tend to have a worse prognosis. However, those data were obtained for patients in the previous era of traditional chemotherapy, less than 45% of the patients in this group were treated with a proteasome inhibitor and/or immunomodulator-based regimen; therefore, the prognostic signi cance of the hematopoietic score needs further con rmation in the era of new drugs.
In this study, we evaluated the prognostic effect of hematopoietic score in MM patients treated with a bortezomib-based regimen, and con rmed that hematopoietic score is an independent prognostic predictor for PFS and OS. Our data suggest that Hgb < 100g/L indicates worse OS, which is an important prognostic indicator for newly diagnosed MM patients; an increased MCV similarly suggests worse PFS and OS and these occur more often in patients with Hgb level < 100 g/L and bone marrow plasma cell percentage > 30%. An increased red blood cell volume is often an indication of folic acid or vitamin B12 de ciency; however, it is regrettable that only a few patients in our group were tested for serum vitamin levels, with no de ciency found. According to the literature, vitamin B12 de ciency has been reported in approximately 13-20% of patients with plasma cell disease (11,18,19). Regardless, this may not be related to macrocytosis in these patients, and some patients with vitamin B12 de ciency do not show an increase in their MCV (11,18). It is believed that increased an erythrocyte volume is more common in elderly patients, which may be related to the shortened erythrocyte life span and the emergence of more new erythrocytes (reticulocytes) to replace the lost red blood cells, resulting in a slightly larger volume (20) and an increased peripheral blood red blood cell distribution width (9,21).
Our study showed that the peripheral blood Hgb level, MCV, and Plt counts all affected PFS and/or OS in MM patients, while the WBC count did not. We also found that among patients older than 70 years old, with high-risk ISS and R-ISS stages, high bone marrow cell percentages and high serum LDH levels were more likely to occur when the Hgb levels were less than 100 g/L, the MCV was above the normal level and/or the Plt count was less than 150×10 9 /L. The Hgb level, MCV, and Plt count are each assigned a score of 1 when they deviate from the given parameters, generating the hematopoietic score, which adequately predicts the survival of patients undergoing rst-line treatment with a bortezomib-based regimen.
The hematopoietic score is simple and easy to obtain. Indeed, all three indicators can be obtained very readily from routine blood examinations. Furthermore, the score has signi cant prognostic value in patients receiving new drug treatments, indicating that it can be used as an important supplement to the R-ISS stage, especially when FISH cannot be routinely performed. However, this study was retrospective, and the number of cases was not large. Furthermore, blood vitamin B12 and folic acid levels were not detected. We intend to further expand the number of cases and re-evaluate the value of the hematopoietic score for prognostic prediction in MM patients in a more standardized prospective study and to combine cytogenetic abnormalities with the ISS stage; by doing so, we aim to establish a new prognostic system for MM patients that is applicable in the era of new drug therapy. Progression-free survival (1A) and overall survival (1B) based on the hematopoietic score (0, 1, 2, 3

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