Evaluating murine double minute 2 status as a stratification tool for risk-adapted management in plasma cell neoplasms

Abstract

BACKGROUND

The E3 ubiquitin ligase murine double minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53 and has been implicated in the development of various cancers, including hematologic malignancies. In multiple myeloma (MM), increased MDM2 expression has been reported and may play a role in disease progression and resistance to therapy. Despite this, the prognostic implications of MDM2 detected through immunohistochemistry (IHC) remain insufficiently defined.

AIM

To evaluate the clinical, pathological, and prognostic significance of MDM2 expression in plasma cell neoplasms, with a focus on its potential utility as an early indicator of disease severity and therapeutic response.

METHODS

A retrospective analysis was conducted on 71 patients diagnosed with MM or related plasma cell disorders treated at the National Cancer Institute between 2018 and 2022. MDM2 protein expression was assessed using IHC on extramedullary lesion biopsy samples, employing the MDM2 (A.M.1) monoclonal antibody. Nuclear staining in at least 1% of plasma cells was used as the threshold for MDM2 positivity. Comparative analyses were performed between MDM2-positive and MDM2-negative groups, examining clinical characteristics, laboratory data, histopathological features, treatment responses at 12 weeks and 24 weeks, and survival outcomes, including relapse-free survival (RFS) and overall survival.

RESULTS

MDM2 expression was identified in 30% of patient samples. While no major differences were observed in baseline demographics, disease stage, or most laboratory values, serum albumin levels were significantly lower in MDM2-positive patients (P = 0.007). At 12 weeks, patients with MDM2-positive disease showed significantly poorer treatment responses based on International Myeloma Working Group criteria (P = 0.002), and early clinical response was moderately negatively correlated with MDM2 expression (Spearman’s P = 0.375, P = 0.001). This correlation was not observed at 24 weeks. Immunophenotypic analysis indicated that MDM2-positive plasma cells exhibited lower epithelial membrane antigen (P = 0.014) and higher CD45 expression (P = 0.039), suggesting altered differentiation. Kaplan-Meier survival analysis demonstrated a markedly shorter median RFS in the MDM2-positive group (22 months vs 68 months, P < 0.001), although no significant difference was found in overall survival.

CONCLUSION

IHC-detected MDM2 overexpression identifies a distinct subset of plasma cell neoplasms characterized by reduced early treatment responsiveness and significantly shorter RFS. These findings support the potential of MDM2 as a prognostic biomarker for early relapse risk in MM. Incorporating MDM2 assessment into diagnostic and prognostic workflows may enable more individualized treatment approaches. Further validation through prospective studies is recommended.

Key Words: Multiple myeloma; Murine double minute 2; Plasma cell neoplasms; Immunohistochemistry; Prognostic biomarker; Relapse-free survival; p53 pathway; Risk stratification; CD45; Epithelial membrane antigen

Core Tip: This research explores the prognostic significance of murine double minute 2 (MDM2) expression in plasma cell neoplasms, with particular attention to plasmacytomas. The findings reveal a distinct subset of patients with MDM2 positivity who demonstrate poorer early treatment responses and markedly shorter relapse-free survival intervals. Incorporating MDM2 expression into contemporary prognostic frameworks could enhance individualized treatment planning for those with multiple myeloma. Overall, the evidence indicates that MDM2 functions as both a marker of aggressive tumor biology and a promising indicator for early-stage risk stratification, supporting more vigilant follow-up and personalized therapeutic intervention to reduce relapse likelihood.

INTRODUCTION

The E3 ubiquitin ligase murine double minute 2 (MDM2) plays a central role in regulating the tumor suppressor protein p53 by binding to its transactivation domain and targeting it for ubiquitin-dependent degradation. Under normal physiological conditions, this autoregulatory feedback loop ensures that p53 levels remain low. However, in response to cellular stress, MDM2 activity is suppressed, allowing p53 to accumulate and activate its downstream tumor-suppressive functions[1,2]. In many cancers, MDM2 is frequently overexpressed or amplified, effectively disabling p53’s tumor-suppressive activity even in cases where the TP53 gene remains unmutated. Additionally, accumulating evidence highlights the p53-independent oncogenic functions of MDM2, including the stabilization of certain oncogenic mRNAs, such as c-Myc, and the facilitation of cell cycle progression[1,3,4]. In hematologic malignancies, dysregulated MDM2 expression has been noted in specific disease subsets and is often associated with aggressive clinical behavior. For instance, approximately 14% of diverse blood cancers exhibit MDM2 overexpression, which correlates with adverse cytogenetic profiles, resistance to therapy, and reduced survival[1,5].

In the context of multiple myeloma (MM), elevated MDM2 expression has been consistently reported and appears to contribute to malignant plasma cell proliferation and survival. Prior research demonstrated abundant MDM2 protein levels in MM cell lines and plasma cell leukemia samples, contrasting with its low expression in normal bone marrow (BM) cells. Functional studies using antisense oligonucleotides to inhibit MDM2 in MM models resulted in cell cycle arrest at the G1 phase and induction of apoptosis, underscoring its role in promoting tumor cell viability[3,4]. Clinical gene expression datasets further reveal that MDM2 expression is significantly higher in MM patients compared to healthy individuals and tends to increase with more advanced stages according to the International Staging System (ISS) and revised ISS. Importantly, overexpression of MDM2 has been linked to disease progression in MM, with large-scale data indicating that patients with higher MDM2 levels experience more advanced disease at diagnosis, increased relapse rates, and shorter progression-free survival compared to those with lower MDM2 expression[3,4].

Given these findings, nuclear accumulation of MDM2 may serve as a potential biomarker for high-risk MM. However, the prognostic value of MDM2 protein expression detected by immunohistochemistry (IHC) in MM remains poorly defined. To explore this, we conducted a retrospective study involving 71 patients with MM or related plasma cell neoplasms treated at our National Cancer Institute between 2018 and 2022. Utilizing diagnostic criteria from the World Health Organization (WHO) and the International Myeloma Working Group (IMWG), we compared clinical features, treatment responses, and survival outcomes between patients whose BM plasma cells tested positive or negative for MDM2 by IHC. We hypothesized that MDM2 positivity would correlate with more aggressive disease behavior and inferior relapse-free and overall survival (OS).

MATERIALS AND METHODS

Study design and patient selection

This investigation was conducted as a retrospective study following approval from the Institutional Review Board of the National Cancer Institute. We retrospectively reviewed 71 patients diagnosed with MM or other plasma cell neoplasms, including solitary plasmacytoma and plasma cell leukemia, at the National Cancer Institute between January 2018 and December 2022. All diagnoses were established following the 5th edition WHO classification of hematolymphoid neoplasms and the IMWG criteria, which require ≥ 10% clonal plasma cells in the BM combined with hypercalcemia, renal impairment, anemia, bone disease features or myeloma-defining biomarkers. Clinical information was extracted from electronic health records, capturing patient demographics, Eastern Cooperative Oncology Group performance status, and disease staging using the ISS. Baseline laboratory parameters, including hemoglobin, serum calcium, creatinine, lactate dehydrogenase (LDH), β2-microglobulin, and monoclonal protein concentrations, were documented alongside BM pathology reports. Details regarding therapeutic interventions were recorded. Treatment responses were evaluated based on IMWG consensus criteria at weeks 12 and 24. Patients’ clinical follow-up was recorded with serial laboratory assessments, disease progression or relapse, and either death or last known follow-up.

Data quality control

Standardized case report forms were utilized in all data collection to ensure data integrity. The clinical data were abstracted by two independent reviewers and resolved in case of discrepancies by consensus review. The laboratory values were cross-checked with original laboratory reports, and the assessments of treatment response were validated with IMWG criteria. Missing data were documented and excluded from the relevant analysis. All pathological analyses were performed independently by MD-certified pathologists blinded to clinical outcomes to minimize bias. In instances of diagnostic disagreement, a final interpretation was established through collaborative evaluation and mutual agreement among the pathologists.

Immunohistochemical analysis

MDM2 expression was assessed on extramedullary lesions, which were biopsied for diagnosis and represent a more aggressive disease phenotype with distinct biological characteristics from BM-limited disease. Diagnostic tissue biopsy samples, previously preserved as formalin-fixed, paraffin-embedded blocks, were collected from the Oncologic Pathology Department for immunohistochemical analysis and sectioned at a thickness of 4 microns. Immunohistochemical staining for MDM2 protein was performed on the BenchMarkUltra system using a standardized avidin-biotin peroxidase method with a commercially sourced monoclonal antibody specific for MDM2 [MDM2 (A.M.1) monoclonal antibody]. The ≥ 1% threshold for MDM2 positivity was established using established precedent from prior research that analyzed oncogenic protein expression in neoplasms[6-8], where percentage cut-offs have been of clinical importance in MDM2 testing. Nuclear intensity of staining was analyzed by four blinded pathologists in a consensus fashion, and good concordance among pathologists in marking positive vs negative cases was obtained. The percentage of positively stained nuclei was documented, and cases were categorized into positive or negative groups for comparative analyses. To ensure the specificity of MDM2 expression, 10 control patients with normal BM samples free of hematologic malignancy were analyzed, and there was little nuclear staining for MDM2 in normal plasma cells (0%-1% positivity), validating the ≥ 1% threshold of abnormal overexpression.

Statistical analysis

Patients were divided into MDM2-positive and MDM2-negative cohorts for comparative evaluation. Continuous variables were reported as medians or means and compared using either the independent t-test or the Mann-Whitney U test, depending on data distribution. Categorical variables were summarized as n (%) and compared using χ² or Fisher’s exact tests, as appropriate. The timing of remission was defined as achieving either complete remission or very good partial remission. Relapse-free survival (RFS) was defined as the interval from the time of best response, either complete or partial remission, to the first documented disease progression or relapse, with patients remaining in remission censored at last follow-up. OS was measured from the date of diagnosis to death from any cause or last clinical contact. Kaplan-Meier survival curves were constructed for both RFS and OS, with comparisons between MDM2-positive and MDM2-negative groups assessed via the log-rank test. The non-parametric correlation analyses were conducted using Kendall’s tau-b and Spearman’s rho coefficients. All statistical tests were two-tailed, and a P-value of less than 0.05 was considered statistically significant. Statistical analyses were conducted using SPSS version 28 software (IBM Corp., Armonk, NY, United States).

RESULTS

Patient demographics and baseline characteristics

A total of 71 patients diagnosed with MM or plasma cell neoplasms were included in this retrospective analysis. Among these, 50 patients (70%) were classified as MDM2-negative, while 21 patients (30%) were MDM2-positive. Table 1 provides an overview of clinical and laboratory characteristics recorded at both baseline and after 12 weeks of therapy, stratified by patients’ MDM2 expression status.

Table 1 Descriptive data of age and laboratory parameters by murine double minute 2 status, mean ± SD.

Parameter	MDM2-negative (n = 50)	MDM2-positive (n = 21)	Total (n = 71)
At diagnosis
Age (years)	57 (34-89) (59.1 ± 11.5)	58 (40-79) (58.5 ± 10.3)	58 (34-89) (58.9 ± 11.1)
Calcium (mg/dL)	10.6 (7.9-13.5) (10.9 ± 1.4)	10.4 (8.0-12.9) (10.6 ± 1.2)	10.5 (7.9-13.5) (10.8 ± 1.4)
Creatinine (mg/dL)	1.1 (0.5-3.5) (1.5 ± 0.8)	1.3 (0.7-4.0) (1.5 ± 0.8)	1.2 (0.5-4.0) (1.5 ± 0.8)
β2-microglobulin (mg/L)	4.9 (1.9-22.8) (6.6 ± 4.5)	4.7 (2.1-15.3) (5.9 ± 3.4)	4.9 (1.9-22.8) (6.4 ± 4.2)
LDH (U/L)	278 (149-790) (321.3 ± 159.5)	275 (188-450) (286.1 ± 85.4)	276 (149-790) (310.9 ± 142.0)
Total protein (g/dL)	10.2 (6.3-13.4) (10.4 ± 1.4)	10.5 (7.0-12.9) (10.3 ± 1.4)	10.4 (6.3-13.4) (10.4 ± 1.4)
Albumin (g/dL)	4.1 (2.0-5.9) (3.9 ± 1.2)	2.9 (2.0-5.3) (3.1 ± 0.9)	3.7 (2.0-5.9) (3.7 ± 1.2)
BM plasma cells (%)	20.5 (0-87) (28.2 ± 21.8)	26 (7-80) (34.2 ± 23.2)	22 (0-87) (30.0 ± 22.2)
Hemoglobin (g/dL)	8.4 (5.0-12.0) (8.2 ± 1.8)	8.0 (5.0-10.2) (8.1 ± 1.4)	8.3 (5.0-12.0) (8.2 ± 1.7)
At 12 weeks
Hemoglobin (g/dL)	10.0 (6.4-14.0) (9.7 ± 1.8)	9.4 (5.4-11.5) (9.3 ± 1.4)	10.0 (5.4-14.0) (9.6 ± 1.7)
β2-microglobulin (mg/L)	2.3 (0.5-9.3) (2.9 ± 1.9)	2.4 (0.9-9.6) (2.9 ± 1.8)	2.3 (0.5-9.6) (2.9 ± 1.9)
Creatinine (mg/dL)	8.9 (7.2-11.8) (8.9 ± 0.9)	8.9 (6.3-10.1) (8.8 ± 0.8)	8.9 (6.3-11.8) (8.9 ± 0.9)
Calcium (mg/dL)	1.1 (0.6-5.6) (1.3 ± 0.8)	1.3 (0.5-2.5) (1.3 ± 0.5)	1.1 (0.5-5.6) (1.3 ± 0.7)
Total protein (g/dL)	8.0 (5.1-12.8) (8.3 ± 1.6)	8.0 (5.7-11.9) (8.4 ± 1.8)	8.0 (5.1-12.8) (8.3 ± 1.6)

MDM2: Murine double minute 2; BM: Bone marrow; LDH: Lactate dehydrogenase.

Treatment protocols

Initial treatment modalities (Table 2) included chemotherapy (CTH), radiotherapy, and combined CTH and radiotherapy. CTH regimens predominantly consisted of cyclophosphamide, bortezomib, and dexamethasone (CyBorD) and vincristine, adriamycin, and dexamethasone (VAD). Additionally, daratumumab was administered in certain cases (Table 2). Maintenance therapy was most commonly with lenalidomide (67%) or bortezomib (33%) in the transplant-eligible population. Treatment response was evaluated by IMWG criteria at 12 weeks and 24 weeks, with most patients demonstrating at least a partial response. This is critical to interpreting our resistance findings, since patients who were MDM2 positive had less favorable responses even when they received optimal, effective regimens. Out of the fifty eligible patients, seven patients underwent autologous stem cell transplantation after induction.

Table 2 Treatment protocols among patients.

Initial treatment type	Frequency	Percent (%)	Initial chemotherapy type	Frequency	Percent (%)
CTH	62	87.3	CyBorD	37	52.1
CTH and RTH	8	11.3	VAD	29	40.8
RTH	1	1.4	Daratumumab	4	5.6

CTH: Chemotherapy; RTH: Radiotherapy; CyBorD: Cyclophosphamide, bortezomib, and dexamethasone; VAD: Vincristine, adriamycin (doxorubicin), and dexamethasone.

Associations between MDM2 expression status and baseline clinical, laboratory, and BM characteristics

No statistically significant difference between the two groups regarding age (P = 0.83). gender distribution (P = 0.74), family history of malignancy (P = 0.251), and comorbidities (P = 0.143). The prevalence of comorbidities, including diabetes, hypertension, or both, was also similar between groups (P = 0.12). Additionally, no statistically significant associations were identified for MDM2 status and pathological fractures (P = 0.605), paraplegia (P = 0.521), plasmacytoma classification (P = 0.444), extramedullary plasmacytoma location (P = 0.538), Eastern Cooperative Oncology Group performance status (P = 0.423), M-protein type by immunofixation (P = 0.478), circulating clonal plasma cells (P = 0.518). According to the ISS, stage 3 disease predominated (41%) across the entire cohort, with a nonsignificant distribution between MDM2 categories (P = 0.118). Regarding other recognized high-risk factors, no statistical association between MDM2 expression and M-protein quantitation in serum (P = 0.159), elevated levels of β2-microglobulin (P = 0.97), elevated LDH (P = 0.88), or renal insufficiency (P = 0.47). The sole finding of statistical significance was association with low serum albumin (P = 0.007). BM assessment, including BM plasma cell percentage in aspirates and biopsy, overall marrow cellularity, and reticulin fibrosis grade as per WHO classification, did not reveal any significant differences between MDM2-positive and MDM2-negative cases. There were no substantial differences in the 12 and 24-week laboratory parameters between the MDM2-negative and MDM2-positive groups. These included renal function, calcium levels, total protein, and hemoglobin values.

MDM2 correlation with laboratory parameters

A significant inverse correlation was observed between MDM2 positivity and serum albumin levels at presentation (r = -0.319,P = 0.007), suggesting that lower serum albumin concentrations were more frequent in patients expressing MDM2. No other significant correlations were detected between MDM2 status and the remaining continuous parameters, including age, calcium, creatinine, β2-microglobulin, LDH, total protein, BM plasma cell burden, hemoglobin, or their respective 12-week values, with all P-values exceeding 0.05.

MDM2 and treatment response and disease control

At the 12-week evaluation, a statistically significant association was found between MDM2 status and IMWG response categories [Pearson’s χ² (2) = 12.530, P = 0.002]. This result was supported by the likelihood ratio test [χ² (2) = 13.541, P = 0.001] and the linear-by-linear association test, which revealed a significant linear trend across the ordered response categories [χ² (1) = 10.897, P = 0.001]. Collectively, these analyses indicate that MDM2 positivity was associated with poorer clinical outcomes at this early stage of treatment. At the 24-week timepoint, although testing the association between MDM2 status and IMWG response categories did not reach conventional levels of statistical significance [χ² (2) = 5.482, P = 0.065], it hinted at a potential trend. The likelihood ratio test approached significance [χ² (2) = 6.140, P = 0.046], while the linear-by-linear association test did not indicate a significant trend [χ² (1) = 0.473, P = 0.492]. These findings suggest that the prognostic impact of MDM2 status on response outcomes diminishes as treatment progresses. In addition, the association between MDM2 status and the timing of remission, defined as achieving either complete remission or very good partial remission, was evaluated using χ² tests. No significant association was observed in this analysis [Pearson’s χ² (64) = 68.600, P = 0.324], although the likelihood ratio test trended toward, but did not achieve, significance [χ² (64) = 83.456, P = 0.052].

To further assess the relationship between MDM2 status and treatment response, non-parametric correlation analyses were conducted using Kendall’s tau-b and Spearman’s rho coefficients. At 12 weeks, a moderate, positive, and statistically significant correlation was observed (Kendall’s tau-b = 0.368, P = 0.002; Spearman’s P = 0.375, P = 0.001), indicating that patients with positive MDM2 status were more likely to experience less favorable responses, reflected by assignment to higher IMWG response categories. By 24 weeks, however, this relationship had substantially weakened and was no longer statistically significant (Kendall’s tau-b = 0.051, P = 0.653; Spearman’s P = 0.054, P = 0.657). Of particular importance, a strong, positive, and highly significant correlation persisted between IMWG response categories at 12 weeks and 24 weeks (Kendall’s tau-b = 0.627, P < 0.001; Spearman’s P = 0.678, P < 0.001), demonstrating that early treatment responses strongly predicted subsequent outcomes throughout the course of therapy. These findings highlight MDM2 status as a valuable early prognostic biomarker in MM. Its assessment appears to be particularly useful for predicting early treatment response, while the consistency of initial response categories provides a robust indicator of long-term therapeutic trajectories.

Immunophenotypic and histopathologic features

Interestingly, epithelial membrane antigen (EMA) expression differed markedly: 45% of MDM2-negative patients were EMA-positive, whereas none of the MDM2-positive patients exhibited EMA positivity, a difference that reached statistical significance (P = 0.014). Additionally, leukocyte common antigen expression patterns varied, with 36% of MDM2-positive cases demonstrating focal expression compared to 5.6% in MDM2-negative patients (P = 0.039). The observed EMA negativity and altered leukocyte common antigen (CD45) expression patterns in the MDM2-positive group reflect disrupted plasma cell differentiation and maturation programs with significant biological implications. EMA loss indicates progression toward terminal plasma cell differentiation. More critically, CD45 expression patterns serve as surrogate markers for plasma cell proliferative activity and disease aggressiveness. While CD45-positive plasma cells represent earlier, more proliferative populations with enhanced interleukin-6 responsiveness, patients with ≥ 20% CD45-positive clonal plasma cells paradoxically experience significantly shorter OS and reduced time to next therapy[9-11]. The correlation between MDM2 positivity and specific EMA/CD45 expression patterns suggests that MDM2 overexpression may mark plasma cells with altered differentiation trajectories that maintain proliferative capacity while acquiring aggressive biological features. This dual pattern of EMA loss (terminal differentiation) coupled with CD45 positivity (proliferative activity) may identify a subset of plasma cells undergoing dysregulated maturation, potentially explaining the adverse prognosis associated with MDM2 positivity in this study.

Among cases for which there was accessible CD56 IHC (n = 60), CD56-negative cases showed a trend towards greater MDM2 positivity (42% vs 23%, P = 0.08), consistent with the known relationship between CD56 negativity and aggressive MM biology. Other studies incorporating comprehensive flow cytometry panels and measurement of MRD would provide valuable confirmation of MDM2’s prognostic significance relative to that of established biomarkers.

Survival outcomes concerning MDM2 status

In this study, patients were followed for a period ranging from 1.64 months to 81.50 months, with an average follow-up duration of 25.54 ± 20.89 months. To investigate the prognostic relevance of MDM2 status, a Kaplan-Meier analysis assessed RFS in a cohort of 71 patients stratified according to MDM2 status (Table 3). The results demonstrated a clear and significant difference in outcomes between the groups. Patients without MDM2 expression achieved a notably higher median RFS compared to those with positive MDM2. The mean RFS values also favoured the MDM2-negative group. This difference was statistically significant. Additionally, a higher censoring rate was observed in the MDM2-negative group (88%), reflecting a greater proportion of patients remaining relapse-free or alive at last follow-up. These data collectively indicate that MDM2 status is a strong prognostic indicator for RFS in patients with MDM2 positivity, linked to significantly poorer outcomes.

Table 3 Comparison of relapse-free survival and overall survival between murine double minute 2-negative and murine double minute 2-positive patient groups.

Parameter	MDM2-negative (n = 50)	MDM2-positive (n = 21)	P value
Median RFS (months)	68	22	< 0.001
Mean RFS (months)	62.93	21.98
Median OS (months)	Not reached1	35 (95%CI: 23-47)	0.401
Mean OS (months)	54.8 (95%CI: 42-67.6)	46.2 (95%CI: 33.9-58.5)

¹Median overall survival for the murine double minute 2-negative group was not reached.

RFS: Relapse-free survival; OS: Overall survival; CI: Confidence interval; MDM2: Murine double minute 2.

Subsequently, the effect of MDM2 status on OS was examined through a separate Kaplan-Meier analysis within the same patient cohort. Among the 50 MDM2-negative patients, 10 deaths (20%) occurred, while 40 cases (80%) were censored. In comparison, 21 patients with MDM2 included 11 deaths (52.4%) and 10 censored cases (47.6%). The mean OS was higher in the MDM2-negative group than in the MDM2-positive group. However, the difference between the MDM2-positive and MDM2-negative groups did not reach statistical significance. These findings suggest that while MDM2 status is a significant prognostic factor for RFS in this setting, its effect on OS is less evident.

Notably, MDM2 positivity was also not associated with other recognized high-risk features like advanced ISS stage, elevated β2-microglobulin, or renal insufficiency. MDM2 expression ≥ 1% emerges as a highly significant independent risk factor for early relapse in MM patients. The adjusted odds ratio of 370.73 indicates an extraordinarily strong association, with patients expressing any level of MDM2 having approximately 371 times higher odds of experiencing relapse compared to those with no MDM2 expression. This suggests that MDM2’s prognostic impact on RFS represents an independent biological pathway and is not a marker of global severity of disease.

Figures 1-3 provide illustrative examples of the histopathological and immunophenotypic characteristics commonly observed in MM and related plasma cell disorders. Figure 1A-C displays a series of hematoxylin and eosin-stained sections at increasing magnifications, capturing extramedullary tissue infiltration by malignant plasma cells. Figure 1D focuses on BM involvement, showing dense intertrabecular infiltration by atypical plasma cells with hallmark morphological features. In Figure 2, peripheral blood smears reveal circulating neoplastic plasma cells alongside classic hematologic findings such as rouleaux formation. Figure 3 showcase immunohistochemical profiles of critical diagnostic markers, CD56, CD117, kappa and lambda light chains, EMA, CD45, and CD138, highlighting both lineage specificity and aberrant antigen expression patterns typical of plasma cell neoplasia. Figure 4 illustrates nuclear MDM2 expression with variable staining intensity among malignant plasma cells, underscoring its potential relevance as a prognostic marker. Figure 5 depicts Kaplan-Meier curves demonstrating a marked reduction in RFS among patients exhibiting MDM2 positivity.

Figure 1 Hematoxylin and eosin. A-C: Hematoxylin and eosin-stained tissue sections illustrating extramedullary involvement by malignant plasma cells, characteristic of multiple myeloma, shown at progressively increasing magnifications. At low magnification (× 100), the tissue reveals widespread infiltration by densely clustered abnormal plasma cells arranged in continuous sheets, effectively displacing the normal tissue architecture (A). At intermediate magnification (× 200), the neoplastic plasma cells display significant nuclear pleomorphism, frequent binucleation, and scattered mitotic activity, with occasional foci of necrosis observed within the densely cellular environment (B). At high magnification (× 400), individual malignant plasma cells are clearly visualized, showing eccentrically positioned nuclei with a vesicular appearance, prominent nucleoli, distinct perinuclear clearing (Hof), and eosinophilic cytoplasm. Marked nuclear atypia and frequent mitotic figures are evident at this level (C); D: Hematoxylin and eosin-stained bone marrow biopsy sections demonstrate infiltration by myeloma cells in a patient with multiple myeloma with plasma cell leukemia. The marrow notably shows malignant plasma cells that display characteristic morphological features, including eccentrically located nuclei, moderately to abundantly basophilic cytoplasm, and a distinct perinuclear clearing (Hof). Occasional binucleated forms and mitotic figures are observed. Original magnification × 400.

Figure 2 Peripheral blood smears from a patient diagnosed with multiple myeloma with plasma cell leukemia exhibit distinct morphological hallmarks. A prominent feature is the presence of rouleaux formation, where red blood cells appear stacked, typically reflecting elevated serum protein levels. Notably, several abnormal plasma cells are observed, characterized by eccentrically positioned nuclei, deeply basophilic cytoplasm, and a perinuclear clearing (Hof). Staining technique: Wright-Giemsa. Magnification: 1000 × under oil immersion. These hematologic findings are consistent with multiple myeloma and align with the presence of circulating malignant plasma cells and blood hyperviscosity commonly seen in advanced stages of the disease.

Figure 3 Immunohistochemical staining. A and B: Immunohistochemical staining for CD56 (neural cell adhesion molecule) using 3,3’-diaminobenzidine (DAB) chromogen highlights the expression of this marker in neoplastic plasma cells associated with multiple myeloma, both in extramedullary tissue and bone marrow samples. In the extramedullary tissue section, there is diffuse and intense CD56 staining observed on the membranes and within the cytoplasm of atypical plasma cells arranged in cohesive sheets (DAB chromogen; original magnification × 400) (A). The bone marrow biopsy shows scattered clusters and interstitial infiltrates of malignant plasma cells with strong membranous CD56 expression (DAB chromogen; original magnification × 200). Residual normal hematopoietic cells and adipocytes are also visible in the background (B); C and D: Immunohistochemical analysis of CD117 (c-Kit) expression in a bone marrow biopsy from a patient diagnosed with multiple myeloma, visualized using DAB chromogen and hematoxylin counterstain. At low magnification (× 200), scattered CD117-positive plasma cells are observed within the bone marrow, distributed among adipocytes and other marrow components (C). High magnification (× 400) reveals clusters of malignant plasma cells exhibiting strong membranous and cytoplasmic CD117 immunoreactivity, highlighting the abnormal expression pattern of c-Kit in neoplastic cells (D); E and F: Immunohistochemical staining of a bone marrow biopsy from a patient with multiple myeloma, illustrating light chain restriction using DAB chromogen with hematoxylin counterstaining. Staining for kappa light chains demonstrates strong, diffuse cytoplasmic positivity in a large population of plasma cells, indicating a predominance of kappa-restricted neoplastic cells (original magnification × 100) (E). In contrast, lambda light chain staining shows little to no expression, confirming light chain restriction to kappa (original magnification × 200) (F); G and H: Immunohistochemical analysis of a tissue section from a patient with multiple myeloma, demonstrating differential expression of cellular markers using DAB chromogen and hematoxylin counterstain. Staining for epithelial membrane antigen reveals scattered immunoreactive cells, suggestive of plasmacytoid differentiation or the presence of an epithelial component (original magnification × 200) (G). Leukocyte common antigen (CD45) staining shows focal membranous positivity in isolated lymphoid cells, consistent with hematopoietic lineage (original magnification × 400) (H); I and J: Immunohistochemical analysis of bone marrow biopsy sections from a patient diagnosed with multiple myeloma, stained for CD138. Panels (I) and (J) depict separate microscopic fields demonstrating strong membranous and cytoplasmic CD138 immunoreactivity (brown coloration, DAB chromogen), clearly delineating aggregates and sheets of neoplastic plasma cells dispersed among adipocyte-rich marrow. These findings are indicative of marrow involvement by plasma cell neoplasia. Original magnification: I (× 200), J (× 400).

Figure 4 Immunohistochemical evaluation of murine double minute 2 nuclear expression in bone marrow samples of patients with multiple myeloma. Immunohistochemical analysis illustrating murine double minute 2 (MDM2) expression obtained from extramedullary lesions of patients diagnosed with multiple myeloma. Detection was performed using the DAB chromogen, with positively stained nuclei exhibiting a characteristic brown hue. The intensity and extent of nuclear MDM2 expression varied among cases. A-C: Images depict mild to focal nuclear MDM2 staining confined to scattered malignant plasma cells; D-F: Fields demonstrate a higher frequency and intensity of nuclear MDM2 expression, with numerous plasma cells exhibiting moderate to strong positivity. Original magnification: A-D (× 200), E and F (× 400).

Figure 5 Kaplan-Meier curves depicting relapse-free survival in months according to murine double minute 2 status in patients with plasma cell neoplasms. MDM2: Murine double minute 2.

DISCUSSION

In this 71-patient cohort of MM, we determined that nuclear MDM2 expression in ≥ 1% of myeloma cells was associated with significantly reduced RFS. Our findings demonstrate that MDM2-positive patients had significantly inferior median RFS of 22 months compared with MDM2-negative patients at 68 months (P < 0.001), as well as significantly inferior early treatment response at 12 weeks (P = 0.002). These results concur with new data from a series of recent works indicating the prognostic significance of the p53-MDM2 pathway in plasma cell malignancies. Our findings concur with previous works demonstrating the adverse prognosis of dysregulation of the p53 pathway in MM. Pruneri et al[12] had previously indicated that p53 nuclear accumulation, determined by IHC in BM biopsies, was significantly correlated with reduced survival, and its prognostic value held even after multivariate analysis. This is consistent with our finding that MDM2 overexpression, which functionally inactivates p53, is associated with unfavorable clinical outcomes. In the same vein, the systematic review by Flynt et al[1] also highlighted that TP53 dysregulation, specifically MDM2 overexpression, is a key mechanism of high-risk disease biology in MM and that patients have significantly reduced progression-free and OS[1]. The molecular basis for our results is underpinned by recent genomic analyses. Lv et al[13] revealed that MDM2 is a key member of p53-linked gene signatures implicated with adverse prognosis in MM and that overexpression of MDM2 has been linked with poorer OS in large patient cohorts. Moreover, studies have proven that overexpression of the MDM2 protein enhances the proliferative capability of MM cells by binding E2F-1, p53, and p21 to circumvent normal cell cycle checkpoints and promote cancer growth. Our observation that MDM2-positive patients had clear-cut immunophenotypic characteristics - i.e., diminished EMA expression (P = 0.014) and augmented CD45 expression (P = 0.039) - suggests that overexpression of MDM2 may be associated with less differentiated, more malignant plasma cell clones. This phenotypic pattern is consistent with the prediction that disruption of the p53 pathway underlies cancellation of normal differentiation programs and enhanced stem cell-like behavior, conferring resistance to therapy and early relapse. Our findings must be viewed against conflicting evidence regarding the prognostic function of MDM2 in hematologic malignancies. Conflicting evidence comes from the work of Elnenaei et al[14], in which they compared the amplification of the MDM2 gene and trisomy 12 in 48 patients with MM using fluorescent in situ hybridization. Contrary to our observations, they concluded that although MDM2 gene amplification was observed in 8% of the cases, it was not correlated with poor prognosis, response to therapy, survival, or event-free survival. The authors made the conclusion that “the presence of amplification or trisomy 12 did not appear to be related to an unfavorable prognosis”. This discrepancy highlights a key methodological observation: Our research quantified MDM2 protein expression by IHC, while Elnenaei et al[14] quantified gene amplification. The inability of gene amplification to correlate with clinical outcomes in their research suggests that MDM2 overexpression can be the result of mechanisms other than gene amplification, including more stable mRNA, increased protein translation, or reduced protein degradation. Actually, their study found that “the mechanism of this overexpression, as well as the role of MDM2 in MM, is not well established”. The broader MDM2 literature as a prognostic biomarker is more complex[14]. Onel and Cordon-Cardo’s comprehensive review[15] emphasized that “while overexpression of MDM2 is common in cancer, it can be a good or bad predictor of outcome in different tumors, and its utility as a biomarker remains contentious”. Such variability may be attributed to variation in tumor biology, MDM2 regulatory mechanisms, p53 mutational status, and study methods among studies.

Our findings have therapeutic implications for the clinical context in addition to prognostication and provide potential for therapeutic application. The p53-MDM2 pathway has been an encouraging target for therapy, with MDM2 antagonists such as nutlin-3 and more recently identified inhibitors showing preclinical efficacy. However, it has been suggested by Flynt et al[1] that such agents are optimally active in wild-type p53 contexts but of diminished use in del17p or TP53-mutated MM. Our research suggests that immunohistochemical analysis of MDM2 has the potential to identify patients who will be candidates for MDM2-targeting therapies, provided they have wild-type p53 activity. Recent clinical breakthroughs lend evidence to the therapeutic efficacy of MDM2 targeting in MM. A first-in-model first-in-human phase I clinical trial of the novel MDM2/p53 inhibitor alrizomadlin evidenced promising early evidence of activity in solid tumors, with a recommended phase II dose of 100 mg every other day[16]. While this trial did not specifically treat patients with MM, the results suggest that MDM2 inhibition remains an effective therapeutic strategy in tumor types retaining intact p53 signaling.

The absence of relevant OS differences despite shorter RFS in MDM2-positive patients should be accounted for by considering some underlying causes. The majority of patients in our series received effective salvage treatments in the setting of post-relapse, such as new drugs and autologous stem cell transplantation, which potentially counteracted survival differences. Furthermore, our median follow-up time of 25.54 months may have been too brief to detect long-term survival differences. This trend suggests that MDM2 would be involved primarily in early resistance to treatment and relapse kinetics, but later treatment may well overcome the initial biological disadvantage.

There are some limitations of the present study that bear mentioning. First, the single-center, retrospective study design limits generalizability and introduces potential selection bias. Second, our patient sample of 71 patients, while adequate for the aims of initial biomarker evaluation, must be validated in larger multicenter cohorts. Third, MDM2 assessment was performed on extramedullary lesions rather than BM samples and thus may not optimally represent the BM disease biology. Fourth, our follow-up time, while long enough to demonstrate significant differences in RFS, may not uncover the ultimate effect on OS, particularly given the enhanced survival benefit with more contemporary MM therapies. Fifth, we lacked complete information on some established prognostic markers, such as cytogenetic abnormalities and minimal residual disease status. Sixth, we did not obtain TP53 mutation status through screening, which could influence the functional importance of MDM2 overexpression. Finally, the observational nature of the study design precludes causal inferences between MDM2 expression and clinical outcomes.

CONCLUSION

This study demonstrates that overexpression of MDM2 protein in MM is associated with significantly reduced RFS, indicative of a more aggressive disease course. Immunohistochemical detection of MDM2 may offer prognostic value in identifying patients at higher risk of early relapse. Clinically, this biomarker could help guide risk-adapted management strategies, including closer monitoring or consideration of novel targeted therapies in MDM2-positive individuals. While our study shows the potential utility of MDM2 as a prognostic biomarker, validation by large, multicenter prospective trials is required before clinical use. Subsequent studies will need to specifically examine MDM2’s use in risk stratification models and its utility to inform therapeutic choices in risk-adapted therapies.

ACKNOWLEDGEMENTS

The authors sincerely acknowledge Cairo University for its institutional support, with special appreciation extended to the National Cancer Institute and the Department of Oncologic Pathology. Their expert insight, collaborative spirit, and access to critical research facilities significantly contributed to the successful completion of this work.