Clinical Relevance of Interferon Regulatory Family-4 (IRF4) Expression in Newly Diagnosed Patients with Multiple Myeloma

Multiple myeloma (MM) is a malignant plasma cell neoplasm with complex biology and heterogenous course. Interferon regulatory factor 4 (IRF4) transcription factor, important key developmental stages of hematopoiesis, represents an excellent potential therapeutic target. The present work aimed to investigate the expression status of IRF4 in the diagnostic bone marrow biopsy (BMB) cores of MM patients. This prospective study included 62 newly diagnosed MM patients. The expression of IRF4 was assessed in the BMB by immunohistochemistry (IHC). The data were correlated to the patients’ clinico-pathological features, response to treatment and survival rates. IRF4 expression was observed in 50% of MM patients (31/62). IRF-4 positive patients were more frequently male patients (P = 0.018), have immunoglobulin heavy chain (IgH) translocations (P = 0.05) and tended to present with a higher platelets count (P = 0.07). Multiple myeloma patients presenting with urine M-protein had worse overall survival (OS) than negative cases (P = 0.012). Normocellular BM aspirate (BMA) was associated with better OS than hypercellular and hypocellular BMA (P = 0.006). Patchy distribution of plasma cells in BMB was associated with better disease-free survival (DFS) while diffuse infiltration had the worst (P = 0.019). Of note, after treatment, MM patients had significantly lower percentage of BMA plasma cells, platelet count, β2 microglobulin and creatinine levels (P = 0.037, < 0.001, 0.022 and 0.026, respectively). Had higher albumin level (P = 0.007), compared to initial investigations. No significant association was found between IRF4 expression and the patients’clinical outcomes. Patterns of plasma cells distribution in BMB, BMA cellularity and urine M-protein are prognostically relevant in MM. Supplementary Information The online version contains supplementary material available at 10.1007/s12288-023-01628-3.


Introduction
Multiple myeloma (MM) is an incurable hematologic cancer characterized by the abnormal proliferation of clonal BM plasma cells, with a 5-year OS of approximately 55%, despite the revolutionary novel therapies in the last decade [1,2].It is characterized by the secretion of a monoclonal immunoglobulins, affecting multiple organs like renal affection, bony osteolytic lesions and BM infiltration with plasma cells [3,4].
Multiple myeloma, accounting for 1% of all cancers and 10-15% of all hematologic malignancies, [5,6], also it is considered to be the second most frequent blood malignancy globally after lymphomas, causing 20-25% of deaths from hematological malignancies [6,7].Owing to the trend of global population aging, there was an 126% increase in the incidence of MM worldwide in recent years [8,9].
At the molecular level, Myeloma is not accounted to be a single disease but a heterogenous disease with numerous subtypes defined by specific gene expression profiles and recurrent chromosomal translocations [10][11][12].Many chromosomal abnormalities are frequently presented in myeloma patients like hyperdiploidy and IgH rearrangements [13].Fluorescent in-situ hybridization (FISH) studies of the BM revealed that about near half of MM patients are characterized by the presence of trisomies in the neoplastic plasma cells (trisomic MM), most of the rest have a translocation involving the IgH locus on chromosome 14q32 (IgH translocated MM), and a small part of patients have both trisomies and IgH translocations [14].Secondary cytogenetic abnormalities may arise along the disease course, including gain (1q), del(1p), del(17p), del (13), RAS mutations, and MYC translocations [15].Importantly, the prognostic impact of cytogenetic abnormalities on survival and treatment response in MM vary depending on the disease phase in which they are detected [16,17].
In pursuit for better prognostication with an better development of risk-adapted treatment, the current IMWG staging system included tumor burden and high-risk cytogenetics, in a new staging system known as (Revised International Staging System R-ISS) [18,19].
Transcription factor interferon regulatory factor 4 (IRF4), which is located at the 6p25.3locus, is an important factor of lymphocyte development, differentiation into Ig-secreting plasma cells [20,21], in addition to its major role in cell proliferation, apoptosis, oncogenic conversion susceptibility and T cell immune reaction [22].
Experimentally, it was found that the loss of function of IRF-4 gene have resulted in addiction of MM cells "to this abnormal gene expression program.Reduced IRF4 expression causes rapid and extended non-apoptotic cell death, irrespective of genetic etiology [26].It had been suggested that high expression of IRF4 in MM was associated with worse outcome and inferior survival [27].
In the present study, we investigated the characterization of IRF4 positive cases in newly diagnosed MM patients with special emphasis on the clinical features associated with those cases and their impact on the outcome of the patients.

Patients
The present study included 62 newly diagnosed MM patients who were diagnosed during the period from May 2017 to December 2019.All patients were presented to the Outpatient Clinic of the Medical Oncology Department, National Cancer Institute (NCI), Cairo University, Egypt.Patients who received any previous chemotherapy outside NCI or with incomplete data were excluded from the study.All patients provided informed consent.The study was performed following the Declaration of Helsinki and was approved by the internal review board of the NCI.
The diagnosis of MM required 10% or more clonal plasma cells in the BM (and/or a biopsy proven plasmacytoma) plus any one or more myeloma defining events (MDE): endorgan damage (hypercalcemia, renal insufficiency, anemia, or bone lesions) attributable to the underlying plasma-cell disorder, BM clonal plasma cells ≥ 60%, serum involved to uninvolved free light chain (FLC) ratio ≥ 100 (provided involved FLC level is ≥ 100 mg/L), or more than 1 focal lesion (≥ 5 mm) on mMRI [5].The treatment responses were evaluated according to the International Myeloma Working Group Panel Consensus.

Immuno-histochemistry
Immunohistochemistry analysis was performed to evaluate the expression of IFR4.Specimens of pre-treatment BM trephine biopsy were transported and fixed in formalin, decalcified in Formic acid-Sodium Citrate and processed to paraffin-wax embedding.Representative sections (3-4 μm) of each case were cut.For each case, 3 slides were prepared: one for Haematoxylin and Eosin (H&E) staining, the second for IHC staining using anti-CD138 and the third IHC assessment of IRF4 using anti-IRF4 (rabbit monoclonal, IgG, Chongqing Biospes Genemed, Biotechnologies, INC), according to manufacturer's instructions.
Two senior hematopathologists independently scored the percentage of plasma cells and IRF-4 positive cells, using the staining of the endothelium as positive internal control in the tested slides by IRF4, and graded the immunostaining intensity as follows: plain: 0 point; shallow yellow: 1 point; pale yellow: 2 point; sepia: 3 point.For the scoring of positive results, a cut off of 10% was used.Based on the percentage of IRF4 positive cells, the patients were bichromatized into positive (≥ 10%) , and negative (< 10%).
Based on staining intensity and the percentage of cells staining positively, the proportion of cells expressing IRF4 were further sub-classified as follows: 0, < 10% of plasma cells or no staining intensity, 1+, ≥10% with light staining, 3+, ≥60% with strong staining intensity, and 2+: for cases not scored as 1+ nor 3+.

Cytogenetic Analysis
For conventional cytogenetic analysis, BM specimens were cultured for 24 and 72 h without mitogens, and stained by G-banding, following the standard techniques.In most of the cases, at least 20 metaphases were analyzed using an IKA-ROS imaging system (Metasystems, Altlussheim, Germany).

Management of the Patients
After diagnosis of MM, patients were divided into two main groups, transplant eligible and transplant ineligible.Transplant eligible patients with no marked co-morbidities and good performance status (PS 0-2) were offered bortezomib based regimen, namely VCD protocol (Cyclophosphamide 300 mg/m 2 , Bortezomib 1.5 mg/m2, Dexamethasone 40 mg on D1, 8,15,22).Patients were requested to repeat SPEP (with IF), sβ2M monthly and BMA after 12 weeks therapy.Monitor of renal and liver functions were requested before each cycle.Responders continued same regimen and were prepared for Autologous BMT.
Patients with poorer PS & ineligible for transplant were offered either VCD or Lenalidomide.Patients with refractory/progressive disease were offered 2nd line therapy versus best supportive care (BSC) according to their performance and comorbidities.Second line protocols included Lenalidomide/Dexamethasone, VAD and Melphalan and prednisone.All patients were offered supportive treatment including Zoledronic acid and palliative radiotherapy (if clinically/ radiologically indicated).

Statistical Analysis
Data management and analysis were performed using SPSS, version 22 (IBM, Armonk, Ny, USA).Qualitative data were presented as numbers and percentages, while the quantitative data were presented as median and interquartile ranges (IQR) or mean and standard deviation (SD), according to the appropriate normality test.The comparison between groups were performed using Chi-square test and/or Fisher exact test which appropriate.Mann-Whitney test was used for comparing numerical variables between two groups.
Survival analysis was done using the Kaplan-Meier test, and comparison between survival curves was done using the log-rank test.All tests of hypotheses had been conducted at the alpha level of 0.05, with a 95% confidence interval.All tests were two-tailed.The OS was calculated from the date of diagnosis till the date of last follow-up or death due to any cause while the DFS was calculated from the date of the diagnosis until the date of relapse, death during induction, death during CR, or second malignant neoplasms.

Patients Characteristics
Among the 62 patients with MM, 39 (62.9%) patients were male while 23 patients (37.1%) were female, with a median age of 55 years at diagnosis.By skeletal survey, all the patients presented with bony lesions, of which 50/62 (80.6%) patients had single or multiple osteolytic lesions while 12/62 patients (19.4%) presented with pathological fractures.Out of 62 patients, 22 (35.5%)presented with soft tissue plasma cells.Baseline characteristics are shown in Table 1.
M Protein was found in the serum of all patients, while it was found in the urine of 8/62 (12.9%) of patients.Monoclonal M-band was found in 60/62 (96.7%) wile biclonal bands were present in 2/62 (3.3%) patients.

Association of IRF4 with MM Patients' Characteristics
By IHC, there was positive expression of IRF4 in 31/62 (50%) patients.as shown in Fig. 1.Upon scoring of IRF-4 expression, depending on the intensity and distribution, 19/62 (30.6%) were classified as +, 5/62 (8.1%) as ++ and 7/62 (11.3%) as +++.The relations between IRF4 staining with scoring system and different laboratory parameters are shown in the supplementary table S1.IRF-4 was expressed in plasma cells nuclei and not observed in neutrophils, lymphocytes nor macrophages.Out of 31 IRF-4 positive cases, 24 were males compared to 7 patients were females (61.5% vs. 30.4%,P = 0.018).There was a trend of IRF4-positive cases to have higher platelet count than negative cases with a P value of 0.081.In the same context, there was a non-significant trend of patients with 1+ to have higher platelet count than 2+ and 3+ (P = 0.07).
Interestingly, rearrangements of IgH were more prevalent in IRF4-positive cases than negative cases (68.4% vs. 31.6%)with a P value of 0.054.No other significant relation between IRF4 expression and patient characteristics as illustrated in Table 3 and Fig. 2.

Impact of IRF4 and Other Prognostic Factors on the Clinical Outcome of MM Patients
The median follow up of OS was 20.8 months with a range of 0.1-43.4months and a mean (± SD) of 18.5 (± 13.1) months.The known prognostic factors had been investigated and revealed improved OS for MM patients with higher Hb level at diagnosis (P = 0.047), and non-significant better survival among patients with lower calcium level (P = 0.07).In addition, patients with Urine M-protein had worse OS than negative cases (2 years-cumulative survival % of 25% compared to 66.7% of negative cases) with a P value of 0.012.Normocellular BMA was associated with better survival than hypercellular and hypocellular marrow (P = 0.006) as illustrated in Fig. 3.After a median (range) follow up period of 12.5 (0.8-34.3) months and a mean (± SD) of 15.4 (± 10.4) months, all prognostic factors had no significant relation with DFS except for the pattern of BMB infiltration as patchy distribution was associated with better DFS while diffuse infiltration had the worst survival (P = 0.019) as shown in Fig. 4. No significant association between IRF4 expression and DFS nor OS.Although the median OS in ISS stage I was longer than that of stage II and stage III (23.18 vs 19.1 and 11.12 months, respectively), the relation failed to show statistical significance (0.266).Relations between different prognostic factors with OS and DFS are illustrated in Table 4.

Discussion
Multiple myeloma (MM) is an incurable clonal plasma cell malignancy characterized by multiple remissions and relapses.Despite the improvement of MM treatment by the use of proteasome inhibitors and immunomodulatory agents, it remains an incurable disease as almost all the patients who survive initial treatment will eventually relapse, requiring further therapy [29].Given that MM cells depend on IRF4 for survival [30,31], deletion of IRF4 gene results in apoptosis of plasma cells as reported by Low et al. [32].Blocking IRF4 expression or interfering with its transcriptional network provide an attractive and applicable therapeutic option for the many subtypes of MM [26].
Compared to other cohorts [19,31,[33][34][35][36][37], our patients were younger (median of 55 years vs. 65 years), presented more commonly with osteolytic lesions (90% vs 35%) and had more frequently higher levels of LDH (35% vs. 12%).In line with other series [33], combined abnormal cytogenetics analyses were detected in 61.3% of our patients and 22.6% had hyperdiploidy.The frequency of IgH rearrangements was lower than that reported by other cohorts [36,37], this discrepancy could be explained by the fact that FISH was performed on unsorted plasma cells in our study.
Our frequency of IRF4 positivity of 50% was comparable to that of a previous study reporting IRF4 positivity in 65% of MM patients, as assessed by IHC [22].IRF-4 expression was more frequently found in male patients.Other than that, no association was found between IRF-4 expression and the clinical parameters, such as age, pathological fractures, sCr, ALB, and LDH, in agreement with Bai et al.Consistent with other reports, IRF4 expression was positively associated with IgH rearrangements.This is consistent by the fact that IRF4 overexpression may result of activating mutations or recurrent translocations involving the 6p25 IRF4 locus, of  which IgH is one of the potential partner genes in patients with t(6;14) (p25;q32) [24,25].
Previous studies [22,38] suggested that the high expression of IRF4 is correlated with poor survival and dismal outcome.However, our current study did not support this conclusion as we didn't find a statistical association between IRF4 expression and patients' OS nor DFS.This could be explained by the small number of patients in our study in comparison to others or the presence of underlying genetic or factors related to patients' clinical characteristics that necessitates more comprehensive study on larger cohort.
The known prognostic factors had been investigated and revealed improved OS for MM patients with higher Hb level at diagnosis, and non-significant better survival among patients with lower calcium level, in line with other studies.In the same context, MM patients presented with urine M-protein had an inferior OS compared to patients lacking urine M-protein, previous study comparing the progression of MGUS and smoldering myeloma with severe myeloma regarding urinary proteins [39].
Of note, there was a statistical association between pattern of infiltration of plasma cells in the biopsy sections and DFS, where patchy infiltration was associated with longer DFS compared to interstitial and diffuse infiltration, which could be explained by the lesser load of plasma cells in the patchy pattern of infiltration, this finding was in agreement with Rajkumar et al. [40].
We found in our study like others [41], that plasma cell loads in the BM was not statistically associated to patients survival.However, this finding was in contrast to Wei et al. who considered that the initial high plasma cells found in the marrow was a remarkable predictor of patients ability to relapse [42].In the same context, we noted a statistical significant decline of plasma cell burden after treatment, followed by positive infiltration of BM which we could explain this observation like Tandon et al. who found increased sensitivity of myeloma patients to treatment but with decreased long term response to those treatments and reduced outcomes [43].Although about two thirds of our patients cohort achieved CR after first line chemotherapy, no significant correlation with improved OS or DFS could be found, which was in contrast to others reporting there was an ultimate association between patients achieving CR from the first line chemotherapy and improved OS and DFS [44][45][46].In conclusion, MM is a mix of different disease entities.Hemoglobulin levels, patterns of plasma cells distribution in BMB, BMA cellularity and urine M-protein are prognostically relevant in MM.No significant association was found between IRF4 expression and survival rates of the patients.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Declarations
Conflict of interest All authors declare that there is no possible conflict of interest.

Fig. 2
Fig. 2 Cytogenetic presentations in myeloma patients in relation to IRF4 expression

Fig. 3
Fig. 3 Overall survival of A IRF±, B urine M-protein and C BMA cellularity

Fig. 4
Fig. 4 Disease free survival of A IRF±, B BMB pattern of infiltration

Table 1
Patient characteristics at diagnosisData are shown as a number (percentage) or a *median (interquartile range) ALB albumin, BMA bone marrow aspirate, BMB bone marrow biopsy, CR complete remission, Hb hemoglobin, IF immunofixation, ISS international staging system, LDH lactate dehydrogenase, sβ2M serum β2microglobulin, sCr serum creatinine, TLC total leucocytic count

Table 2
Relation of initial and follow up laboratory investigations Fig. 1 A 10 × magnification, B 40 × magnification Leica Microscope, BMB IHC with IRF4 positive Myeloma cells

Table 4
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