European Journal of Cardiovascular Medicine

Plasma cell myeloma is neoplastic proliferation of plasma cells with associated M proteins in serum or urine and end organ damage. Clinically the disease varies from asymptomatic to highly aggressive presentation. Plasma cell myeloma (MM) is the most common malignancy of plasma cells and accounts for about 1% of all malignant diseases and 10% of hematological malignancies. All plasma cells, including normal and malignant, express CD38 and CD138. CD38 catalyzes the synthesis and hydrolysis of cyclic ADP-ribose, playing main role maintenance of intracellular calcium levels.(1-2) CD138, aka syndecan-1, allows the plasma cell to attach to extracellular matrix proteins and acts as a co-receptor for epithelial growth factor (EGF), mainly context of multiple myeloma.(3,4) Apart from these two markers, distinguishing characteristics unique to malignant plasma cells is the loss of CD19 expression and the aberrant expression of CD56, a characteristic marker of natural killer (NK) cells. However, not all myeloma cells express CD56. (4,5).

 P53 protein is a TP53 gene product, which has a crucial role in cell death. Normally, the cell cycle of cells with damaged DNA is arrested at G1-S Checkpoint until the damage is repaired, but cells that lack P53 or contain a mutant form are not arrested at G1. Loss or mutation of TP53 is probably the most common single genetic change in malignancies when cells do not undergo apoptosis and so escape the control. Tumors that have not lost TP53, often have mutated versions of it. In Myeloma patients with mutated versions of TP53 produce aberrant P53 nuclear expression. Patients with mutated TP53 are diagnosed in advanced clinical and histological stages, as well as have significantly shorter overall survival than patients without this abnormality.

CD56, a neural cell adhesion molecule (NCAM), is a membrane glycoprotein of the immunoglobulin superfamily. Normal plasma cells do not express CD56. It is expressed by malignant plasma cells in approximately 65–80% of patients with myeloma (MM). CD56 expression in MM correlates with different clinicopathological behaviors and may be associated with osteolysis and a good prognosis. CD56 negativity may be associated with a poor prognosis, extramedullary involvement, plasmablastic morphology, and plasma-cell leukemic state.. Studies conducted by Mateo G, Montalbán MA, Vidriales MB, et al, Guo J, Su J, He Q, et al. and aiva B, Almeida J, Pérez-Andrés M, et al. CD56 absence leads to increased secretion of MMP-9, which promotes basement membrane degradation, invasion, and metastasis of myeloma cells, suggesting that patients with CD56-negative (CD56−) may have a worse prognosis.

The present study attempts to study expression of P53 and CD56 in Plasma cells of

Myeloma bone marrow biopsies and to study its correlation with prognostic markers

Plasma cell myeloma (MM) is the most common malignancy of plasma cells and accounts for about 1% of all malignant diseases and 10% of haematological malignancies. There is neoplastic proliferation of plasma cells with associated M proteins in serum or urine and end organ damage.

In recent years, with the development of novel agents such as immunosuppressants and proteasome inhibitors, the prognosis of patients with MM has improved. However, the disease has great heterogeneity and prognosis varies greatly among the different patients. Currently, laboratory indicators and cytogenetics are used by the international community to assess the risk of MM, excluding information on abnormal antigen expression. The immunophenotype of tumor cells differs from that of normal cells. CD56 is expressed in malignant PCs of MM patients. A recent retrospective study found that CD56 expression did not affect the prognosis of MM, but CD56 deficiency was significantly associated with several adverse prognostic factors (LDH, β2).

The TP53 gene, produces the p53 tumour suppressor protein. Its main biological function appears to involve the protection of the DNA integrity of the cell. p53 mutations were found to occur in fewer than 20% of patients with MM, which usually had advanced and aggressive forms of the disease including plasma cell leukaemia.

In the era of novel therapeutic agents, the prognostic significance of CD56 and P53 in MM patients remains controversial. To further investigate the clinical value of these markers, we explored the correlation between CD56 and P53 expression and clinicopathological features and patient sur Study setting- Dept. of Pathology, Govt. Medical College, Kozhikode Duration- January 1st 2017 – December 31st 2020, with 2 year follow up Sample size –100 cases of plasma cell myeloma tudy variables – Age, sex, haemoglobin, serum creatinine serum calcium bone lytic lesions, serum electrophoresis -presence of m band, percentage of plasma cells in bone marrow trephine biopsy, pattern of infiltration ,remission ,cd56 expression and p53 expression.

Data collection procedure and technique-Fixed decalcified bone marrow trephine biopsies were stained with Haematoxylin and eosin and assessed for the presence of plasma cell infiltrates, its precursors, binucleated forms and patterns of infiltration for the cases of multiple myeloma in department of pathology, GMC, Calicut will  studied from 1st January 2017 to 31st January 2020. WHO criteria for diagnosis of multiple myeloma were followed. The details of patients with variables like age, sex, Hb, serum calcium, Serum creatinine, lytic bone lesions, serum electrophoresis were collected from case records and directly from patients. Written informed consent were obtained from each patient in local language. The synopsis was sent to institutional ethics committee for approval.

Procedure for immunohistochemistry

5 micrometre sections made from the paraffin blocks are incubated at room temperature overnight and then at 600 Celsius for 1 hour. Section were deparaffinised by dipping thrice in xylene solution for 5 minutes each. Hydration was done by immersing the section in descending concentrations of isopropanol of 100 %, 95 %, 70 % and 50 % for 5 minutes each. The sections were washed with distilled water, two changes of 2 minutes each. Antigen retrieval was done in citrate buffer, at a pH of 6.5 for 15-20 minutes by using MERS technique by heating at a sub boiling temperature of 900 Celsius. Washed the sections again in distilled water, two changes of 2 minutes each. The sections were washed in TBST (Tris buffered saline tween) for 2 minutes. (TBST contains Tris base 2.42g, Sodium chloride 8g in 1 litre of water. The pH is adjusted to 7.6 with concentrated HCl and stored at 40 Celsius. At the time of use Tween 20 is added). Endogenous peroxidase blocking is done (20 minutes) by using 3% hydrogen peroxide solution in methanol. Then it was rinsed with distilled water and TBST twice. Primary antibodies were added to the prepared section and kept in room temperature for 45 minutes. Then rinsed with distilled water and TBST twice. Poly Excel Target Binder was added and kept for 20-30 minutes at room temperature (to enhance the reaction), and rinsed with distilled water and TBST.DAB (Diamino benzidine) working solution was added and kept for 5 minutes at room temperature in a dark room and rinsed with TBST. Then the slides were counterstained with haematoxylin for 30 seconds. Dehydration is done by using ascending concentrations of isopropanol 50%, 75%, 95% and 100% and in Xylene for 5 minutes, two changes and mounted with DPX.

Interpretation of reults -Grading of P53 and CD56

Percentage of p53-positive tumor nuclei in all major foci of bone marrow were used for  p53 immunohistochemical scoring system. The percent of p53 immunoreactive tumor cells was scored as 0 to 3+ in p53 positive regions.

Cases were considered as CD56 “positive (+)” when a positive membranous expression with or without cytoplasmic staining in 10% or more of neoplastic cells was present.

Data analysis

Data was entered in Microsoft Excel and analyzed using SPSS software. statistical tests were done

Age in our studied population ranged from 32 to 85 years. The maximum number of patients were in 51-60 years age group (35%) followed by 61-70 years age group (30%).

Table:1  Age distribution

The maximum number of patients were in 51-60 years age group (35%) followed by 61-70 years age group (30%).

Gender-Out of 100 cases 54 were male and 46 were female.

Table: 2 Gender distribution

Haemoglobin: Normal level of haemoglobin were taken for male as 12.6-17.5 g/dl and for females as 11.8 – 16.1 g/dl.

Table: 3 Haemoglobin

 Serum creatinine -Normal level of serum creatinine was taken as 0.6-1.2 mg/dL in adult males and 0.5-1.1 mg/dL in adult female. In diagnostic criteria for multiple myeloma, for an end organ damage attributable to plasma cell proliferative disorder, S.Creatinine should be more than 2mg/dL.

Table: 4 Serum creatinine

In our study 60 cases had serum creatinine >1.2mg/dL and 26 cases had serum creatinine more than 2mg/dL.

LYTIC LESIONS

≥1 bone lytic lesions in skeletal radiography, CT or PET is needed for diagnosis of end organ damage attributable to MM.

Table: 5 Lytic lesions

Serum calcium - for end organ damage attributable to plasma cell proliferative disorder S.calcium should be 11mg/ dL.

Table: 6 Serum calcium

Serum protein electrophoresis : Patients with MM have either an intact immunoglobulin or a free light can be detected by protein electrophoresis either in serum or urine. We have done serum electrophoresis and out of 100 cases 68 cases had M band.

          Percentage of plasma cells - Percentage of plasma cells in affected bone marrow in our study population ranged from 10% to >80% with 65% cases have 20-50% plasma cells in their marrow.

Table: 7 Percentage of plasma cells

PATTERN OF INFILTRATION

Neoplastic plasma cells showed multiple patterns of infiltration like focal clusters, sheets, diffuse, intestitial and paratrabecular. 1% case had plasmablastic morphology.

Table: 8 Pattern of infiltration

REMISSION

Our study population was 100 cases of multiple myeloma from 2017 to 2020 and we followed up the patients for 2 year duration (upto December 2022). Any patients after treatment who presented with relapse of the disease were taken as patient not in remission.

Table:9 Remission

In our study, 11% cases were reported not in remission and 89% cases in remission.

CD56 EXPRESSION- Out of 100 cases 62% cases were CD56 positive, 38% cases were negative.

Only 18.2 percent of the 62 - CD56 positive cases are not in remission, representing a remission rate of 67%. 81.8 percent of the 38 CD56 negative cases were not in remission, while 32.6 percent were in remission. The p value is less than 0.05, and hence the relationship between CD56 expression and remission status is statistically significant

Table: 10 CD56 expression and remission status

Table:11 CD 56 expression and haemoglobin, serum creatinine, lytic lesions, serum calcium and M band

The correlation between CD56 and Bone lytic lesions and presence of M band is statistically significant.

P53 EXPRESSION

Out of 100 cases 9% cases were P53 positive and 91% cases were P53 negative.

Table:12 P53 expression

Table:13 P53 and Remission

Out of the 9 P53+ve cases, 72.7% cases were not in remission. Therefore relationship between P53 and status of remission is statistically significant.  

Table:14 P53 and Heamoglobin, S.Creainine, Lytic lesions, S.Calcium, M band

The correlation between P53 and Bone lytic lesions, presence of M band and S. Creatinine is statistically significant.

Histological examination combined with immunohistochemistry plays an important role in MM management. Besides being the gold standard for diagnosis, it provides prognostic information such as degree of bone marrow infiltration, grade of tumour cell atypia and immunophenotype. Experimental treatment based on immunohistochemical findings was recently introduced for refractory MM, for ex-ample, by stimulation of p53-related apoptosis and antibody based treatment with anti-CD56 drugs (Severine et al.,2003; Teoh et al., 2014; Lonial et al., 2016). (75,76,77)The current tendency of individually tailored treatment implies that in the future, the immunophenotype of each MM patient will have to be taken into consideration, since genetic and immunohistochemical characteristics are very heterogeneous.

In the current study, we retrospectively analysed plasma cell antigenic biomarkers CD56, and p53 in 100 trephine bone marrow biopsies and evaluated their relation to serological, clinical and laboratory parameters in a cohort of primary MM patients.

In our analysis, patients were primarily distributed in the 51–60 age range (35 percent), followed by 61–70 age (30 percent), 71–80 age (21 percent), and 41–50 age (11 percent ).Similar findings were found in Jurijs and Regîna's study, which had a median age range of 41–81. The male to female ratio was 1:0.85 and the predominant pattern of involvement being diffuse. The percentage of plasma cells ranged from 10 to more than 80%. These results are consistent with the earlier research.

It is known that overall survival of MM patients is correlated with the creatinine level and GFR (Durie and Salmon, 1975; Hartmut et al., 2001; Greipp et al., 2005).(78,79,80) Since previous studies (Sailer et al.,1995; Greipp et al., 2005; Lonial et al., 2016)(81,80,77) demonstrated prognostic significance of the immunohistochemically detected phenotype of tumour cells in MM, new data on relations between biochemical and histological prognostic markers could be of particular interest.

We detected an aberrant CD56 expression in myeloma cells in 62% of cases, using immunohistochemical method. According to literature data, patients that are CD56-negative (detected by flow cytometry) have poorer prognosis (Hagueet et al.; Korsrneyer et al.). Among our CD56- negative cases 81.8% were not in remission which matches with the study. In our study CD56 expression was found to have statistically significant association with serum bone lytic lesions, and presence of M band by serum protein electrophoresis. Jurijs and Regîna in their study among 120 MM chorot observed similar results. (82).

p53 was detected in 9% of examined cases using the immunohistochemical method. In our study; among p53 positive cases, 72.7% cases found to be not in remission, compared to 98.9% p53 negative cases in remission. Therefor p53 expression was correlated with poor remission status, which is similar to study by Jurijs and Regîna. In our study, p53 expression in myeloma cells within a group of patients was also correlated with many prognostic clinical and laboratory findings, such as renal insufficiency indicated by elevated creatinine level, bone lytic lesions and presence of M band. In the aforementioned study, p53, haemoglobin, and creatinine were also found to be significantly correlated. 

We discovered a statistically significant association between the expression of CD56 and p53 in myeloma plasma cells and a number of clinical and laboratory features of MM. Poor remission status in MM was correlated with positive p53 and CD56 antigen status in myeloma plasma cells, and also associated with presence of lytic lesions in skeletal survey and presence of M band in electrophoresis. Expression of p53 and the absence of CD56 antigen in neoplastic plasma cells were related to the advancement of chronic renal failure, which was also linked to a worse prognosis in MM patients, as these patients had higher blood levels of creatinine.

Statistical analysis of our study results showed that expression of p53 and CD56 was not correlated with age, gender, percentage of neoplastic plasma cells in the marrow, pattern of infiltration, haemoglobin or serum calcium.

• p53 antigen positivity and CD56 negativity in myeloma plasma cells, as well as the prevalence of lytic lesions in skeletal surveys and the presence of the M band in electrophoresis, were all associated with poor remission status in MM.
• Expression of p53 and the absence of the CD56 antigen in malignant plasma cells were associated with the progression of chronic renal failure, which was also associated with a worse prognosis in MM patients due to their higher blood creatinine levels.
• Age, gender, the proportion of neoplastic plasma cells in the bone marrow, the pattern of infiltration, haemoglobin, or serum calcium did not correlate with the expression of p53 and CD56.

SUMMARY

In the current study, we retrospectively analysed plasma cell antigenic biomarkers CD56, and p53 in 100 trephine bone marrow biopsies and evaluated their relation to serological, clinical and laboratory parameters in a cohort of primary MM patients.

In our analysis, patients were primarily distributed in the 51–60 age range (35 percent), the male to female ratio was 1:0.85 and the predominant pattern of involvement being diffuse. The percentage of plasma cells ranged from 10 to more than 80%.

We detected an aberrant CD56 expression in myeloma cells in 62% of cases, using immunohistochemical method. Among our CD56- negative cases 81.8% were not in remission. In our study CD56 expression was found to have statistically significant association with serum bone lytic lesions, and presence of M band by serum protein electrophoresis.

Using the immunohistochemistry technique, p53 was found in 9% of the cases that were studied. In our study, 72.7 percent of p53 positive individuals were determined to not be in remission, compared to 98.9 percent of p53 negative cases who were. Therefor p53 expression was correlated with poor remission status. In our study, p53 expression in myeloma cells within a group of patients was also correlated with many prognostic clinical and laboratory findings, such as renal insufficiency indicated by elevated creatinine level, bone lytic lesions and presence of M band.

LIMITATION OF THE STUDY

This study was mainly limited to multiple myeloma cases, an elaborate study including all the plasma cell neoplasms would help to identify significance of theses markers in other cases. Additional factors including GFR, macroglobulin, albumin, thrombocyte count, and clinical staging would have resulted in a complex investigation for the relationship between these indicators and prognostic factors. Due to ongoing SARS Corona Virus disease crisis during the study period, potential difficulties were encountered in sample collection, other technical aspects and patient follow up.

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