Study of Electrophoretic pattern in serum of Multiple Myeloma Patients

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1 Study of Electrophoretic pattern in serum of Multiple Myeloma Patients 1 Prakash I Shah, 2 Parloop A Bhatt, 3 P S patel, 1 Vijay L Ghori, 1 Pooja D. Vaghela 1 Shantilal Shah Pharmacy College, Bhavnagar University, Bhavnagar L.M.college of pharmacy, Ahmedabad, 3 Gujarat cancer and research institute of India, Civil hospital compound, Ahmedabad. Abstract: Multiple myeloma (MM) is a progressive and fatal disease characterised by clonal proliferation and accumulation of immunoglobulin producing cells that are terminally differentiated B cells. MM occurs when the plasma cells in the bone marrow are 10% or more than 10% population. The symptoms of MM include pain in the lower back region or in the ribs, recurrent infections, loss of appetite, nausea, thirst, fatigue, muscle weakness, restlessness etc.clinically, MM is recognised by the proliferation of malignant plasma cells in the bone marrow, osteolytic lesions seen in radiological examinations, monoclonal gammopathies,anemia, hypercalcaemia, and renal failure. The diagnosis of MM relies primarily on: Blood and urine tests, serum protein electrophoresis, non-invasive radiological examinations and invasive bone marrow biopsy. Electrophoresis separates proteins based on their physical properties, and the subsets of these proteins albumin, alpha-1globulin, alpha-2globulin, beta globulin, and gamma globulins are used in interpreting the results. In the study serum protein electrophoresis was carried out on 0.5% agarose gel by method of Laurell and Laurell, (1955) at biochemistry research division, GCRI, Ahmedabad. Healthy individuals were considered as control group (n = 25) and patients with multiple myeloma (n =28) to access the monoclonal gammopathies, diagnosis of MM and treatment monitoring in MM. In the present study out of twentyeight (n=28) patients; fifteen patients (n=15) were recently diagnosed and 13 were follow-up patients.out of 15 patients 53 % exhibited monoclonal band (n=8) and 47 % exhibited polyclonal band (n=7). In monoclonal band patients albumin levels were increased and attained values closer to higher limits of normal albumin level, while hypoalbuminaemia occurred in polyclonal band patients.all patients depicting either monoclonal or polyclonal band showed elevated alpha, beta, gamma and total protein levels. In the recently diagnosed 15 MM patients; 73 % suffered from anaemia, along with high ESR and Serum creatinine level was elevated in 86.7 % patients indicating renal damage.in the study, 13 patients were follow-up patients, whose serum protein electrophoresis was performed previously in biochemistry research division; G.C.R.I Ahmedabad.The data of 10 patients was available and was collected. Above patients were treated with standard anti-cancer chemotherapy comprising of combination of Vincristine, Melphelan, Cyclophosphamide, Prednisolone. Serum protein electrophoresis was performed for the above 10 patients following treatment. The results of post-treated patients were compared with pre treated patients. The results of serum protein electrophoresis showed increased albumin level in 50% patients with decrease in alpha fractions, decrease in gamma or beta globulin and increase in albumin / globulin (A/G) ratio in post-treated patients as compared to pre-treated patients. However, the decrease in gamma globulin level and change in A/G ratio were statistically insignificant in post treated patients as compared to pre treated patients, indicating poor response to the treatment. However, based on A/G ratio they were classified as responders and non-responders. Seventy percent patients responded to treatment, while thirty percent were non-responders to treatment as evident by A/G ratio. In the study, responders age range from years while non-responder age ranged from years, indicating that younger patients were more responsive to the treatment as compared to older patients, inferring the prognostic impact of age on survival seems to be better among younger patients than older patients. Key words:multiple Myeloma (MM), Serum protein electrophoresis (SPE), Gujarat cancer & research institute of India (GCRI), monoclonal gammopathy of unknown significance (MGUS) INTRODUCTION : Plasma cell neoplasms represent a spectrum of diseases including clinically benign conditions such as monoclonal gammopathy of unknown signifcance (MGUS).The more common malignant entity plasma cell myeloma or multiple myeloma (MM) is an incurable but treatable disease. Multiple myeloma is a progressive and fatal disease characterised by clonal proliferation and accumulation of immunoglobulin producing cells that are terminally differentiated B cells 1, 2. The marrow is located in spaces within the bones, especially within the sternum (breast bone), spine, skull, pelvic bones, and the 116

2 long bone of the thigh. Bone marrow is a very active tissue that is responsible for producing the different cells that circulate in blood. These include red blood cells, platelets, and many types of white blood cells. Plasma cells are responsible for helping the body to fight against infections caused by microbes. They produce substances called antibodies, also called as immunoglobulins. There are five major classes of immunoglobulins including IgG, IgA, IgM, IgD and IgE 3 which are synthesised by normal B cells and plasma cells. A tremendous amount of work has gone into the search for the cause of MM; to date no cause for this disease has been identified. However, the search for a cause has suggested possible associations between myeloma and a decline in the immune system, genetic factors 4 certain occupations, certain viruses, exposure to chemicals 5 and radiation 6. MM occurs when the plasma cells in bone-marrow are 10% or more population 7, 8. The symptoms of MM include pain in the lower back or in the ribs, recurrent infections 1, loss of appetite, nausea, thirst, fatigue, muscle weakness, and restlessness.clinically, MM is recognised by the proliferation of malignant plasma cells in the bone marrow, lytic bone lesions seen in radiological examinations, monoclonal gammopathies,anemia, hypercalcaemia and renal failure 9. The diagnosis of MM relies primarily on: non-invasive radiological examinations and invasive bone marrow biopsy. The conventional histopathology based on light microscopy, however, has recently been complemented with ultra structure, immuno histochemistry and molecular diagnostics. Molecular medicine has lead to the discovery on light microscopy and application of molecular tumor markers; which make histology more accurate and additionally help to prognosticate MM. Tumor markers are bio- chemical substances produced by tumors and secreted into blood, urine or other body fluid and tissues of MM patients in higher than normal amounts. The measurement of tumor marker levels; when used along with other diagnostic test can be useful in the diagnosis and treatment monitoring of MM patients. Serum protein electrophoresis (SPE) is a laboratory examination that is commonly used to diagnose MM patients. It is a less invasive method as compared to bone marrow biopsy. Serum protein form an extra ordinary complex mixture with varying functional attributes; such as nutritive, osmotic pressure regulator, transport agent protective and enzymatic substance 10. Serum contains two major types of proteins; Albumin and Globulin. Albumin is the major protein component ; which is produced by the liver under normal physiological condition. The reduction in albumin concentration is the most common change in the serum proteins in neoplastic patient 10. however,the reasons are not understood. Globulins comprise of much smaller fraction of the total serum protein content. The subsets of these proteins such as albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin fractions, and their relative quantity are the primary focus of the interpretation of serum protein electrophoresis 11. Electrophoresis is a method of separating proteins based on their physical properties. Serum is placed on a specific medium, and a charge is applied. The net charge (positive or negative) and the size and shape of the protein commonly are used in differentiating various serum proteins 11. Plasma protein level display reasonably predictable changes in response to acute inflammation, malignancy, necrosis and chemical injury

3 Albumin lies toward the positive portion of the gel; and globulin moves towards the negative portion of the gel. Much of the clinical interest is focused on the gamma region of the serum protein electrophoresis because; immunoglobulins migrate to this region 11. A homogenous spike like peak in the focal region of the gamma globulin zone indicates monoclonal gammopathy 9. Monoclonal gammopathies are associated with a clonal process that is malignant or potentially malignant, including MM, Walden strom s macroglobulinaemia, solitary plasmacytoma, heavy chain disease and amyloidosis 12. The elevated gamma globulin (M- Protein) and reduced albumin affects decrease in albumin: globulin (A: G) ratio; which can help to diagnose and monitoring treatment of MM patient. The M-protein concentration can also help to diagnose the MM patient; and monitoring the treatment outcomes in MM patients. Any reactive or inflammatory process may cause polyclonal gammopathies, and they are usually associated with nonmalignant conditions. Polyclonal gammopathy is characterized by a broad diffuse band with one or more heavy chains and kappa and lambda light chains 12. If monoclonal gammopathy is identified by serum protein electrophoresis, MM can be differentiated from other causes of this type of gammopathies by the quantity of M- Protein (abnormal protein), the results of bone marrow biopsy, lytic bone lesions and hypercalcaemia and other clinical symptoms 12. The treatment of MM includes melphalan and prednisone. They were the first successful combination chemotherapy for myeloma and in subsequent years various other single agents and combinations have been investigated and reported to have significant antimyeloma activity. Various chemotherapeutic combinations have been investigated in myeloma, including vincristine (V), cyclophosphamide (C), carmustine, melphalan (M), doxorubicin, Adriamycin (A), and prednisone (P), Commonly used combinations include VBMCP or VMCP/VBAP 13,14,15. Radiation therapy was considered the mainstay of the treatment for myeloma before availability of chemotherapeutic options. However, with more effective chemotherapy, especially high-dose chemotherapy, the role of radiation has now been limited 16. Considering its potential clinical utility; the study was undertaken to assess the role of agarose gel serum protein electrophoresis in clinical practice: As a prognostic marker for diagnosis and monitoring the treatment outcomes of multiple myeloma patient. MATERIALS AND METHODS : SUBJECTS: The Study included 53 blood samples from two groups of each as followings: Group-I: Multiple Myeloma Patients Twenty-eight patients were selected from the outdoor/indoor patients of Gujarat Cancer and research institute; Ahmedabad with clinically and radio- logically diagnosed multiple myeloma. The disease was diagnosed based on their clinical, radiological and histopathological examinations. Group-II: Healthy controls Twenty five healthy controls were included in the study, who had no major illness in the recent past year (1 Year); to define the normal levels of markers. Controls were selected from patient s relative or hospital staff members. The details of the subjects as shown in Table

4 Table-1 S. No 1 2 Characteristics of subjects Controls(healthy individuals) Multiple Myeloma patients Male Female Total No. of sub. (n) Age range in years Median age in years minutes; and sera were separated, aliquot coded and stored in cryovials at c until analyzed. TOTAL SERUM PROTEIN ESTIMATION 17 : The tubes were incubated in water bath at 37 c for 10 minutes. The absorbance was measured at 540 nm (Green filter). The method is standardized and factor is multiplied by optical density. Factor: multiply Absorbance Table-2 S.No. Reagents Blank tube 1 Distilled water 3.0ml Test sample 2.9ml 2 Serum sample ml 3 Biuret reagent 5.0ml 5.0ml Table-3 Total 8.0ml 8.0ml S.No. Parameter Method 1 Haemoglobin Sinum 2 Erythrocyte sedimentation rate Western green Arsenazo III 3 Serum calcium 4 Serum creatinine 5 Blood urea nitrogen(bun) Ca-Arsenazo complex Vajppres Reaction Enzymatic COLLECTION OF SAMPLE: Blood samples were collected from controls and Multiple myeloma patients by Venipuncture between 9 am and 12 noons on every occasion. The blood samples were allowed to co-agulate at room temperature for 1 hour and then centrifuged at 2200 rpm for SERUM PROTEIN ELECTROPHORESIS 18 The method was modified at Biochemistry research division; Gujarat Cancer and research institute, Ahmedabad. This method is less invasive as compared to bone marrow biopsy. METHOD OF SERUM PROTEIN ELECTROPHORESIS: Method of spreading: 1. Spreading: One ml. of fresh gel was spreaded evenly on microscopic slide; kept on a flat surface with the help of 1ml of glass pipette. 2. Drying: The microscopic slide was dried with the help of the drier. 3. Loading: Electrophoretic chamber was filled with barbitone buffer. Serum sample was loaded on a 1mm to 5mm whatmann filter paper wicks placed at the 1/3 rd distance of the slide length towards cathodic end. A constant current supply of 100mv voltage was applied and run for 66 minutes. 4. Fixation: After completion of run; slide was dipped in fixative solution methanol and finally dried. 119

5 Table-4: Age distribution in Multiple Myeloma (MM) Patients having Monoclonal and polyclonal band pattern: Particulars MM with Monoclonal Band MM with Polyclonal Band Male No. Female No. Total No. Age range in Years Male Female Total Median age in yrs Table-5: Mean values of routine biochemical parameters in serum of monoclonal band and Polyclonal band patients. Parameters Reference values Patients with Monoclonal band (n=8) Patients with Polyclonal band (n=7) Haemoglobin Male: (gm %) Female: Serum calcium (gms/dl) Serum creatinine (gms/dl) Blood Urea Nitrogen (gms/dl) Indicates significantly different from healthy controls (P 0.05) Table-6: Mean values of different serum protein components in controls, monoclonal and polyclonal band patients with multiple myeloma: Serum Protein Components Control (25) Mean + SE Patients with Monoclonal Band Mean + SE (8) Patients with Polyclonal Band Mean + SE (7) Albumin Alpha Alpha Beta Gamma A/G Ratio Total Protein Indicates significantly different from healthy controls (P 0.05) 120

6 Table-7: Age distribution in Multiple myeloma (MM) follow up patients Particulars MM with Monoclonal Band MM with Polyclonal Band Male No. Female No. Total No. Age range in Years Male Female Total Median age in yrs Table-8: Mean values of routine biochemical parameters in serum of treated Patients. Parameters Reference values Post treated patients Haemoglobin (gm %) Male: Female: ± Serum calcium (gms /dl) ± Serum creatinine (gms /dl) ± Blood urea nitrogen(gms /dl) ± Table-9: Mean values of serum protein components in pre and post treated patients. Serum protein Components Control (25) Mean ± SE Pre-treatment (10) Mean ±SE Post-treatment (10) Mean ±SE Albumin 4.101± ± ± Alpha Alpha Beta ± ± ± Gamma ± ± ± A/G ratio ± ± ± Total protein ± ± ± Indicates significantly different from healthy controls (P 0.05) 121

7 Table-10: Serum protein fractions in pre-treated patients and post-treated patients. A/G Albumin Alpha-1 Alpha-2 Beta Gamma Classification Ratio R/NR NR R NR R R R R R R NR R= respondents & NR= Non-respondents Fig-1.: Representative patterns of Serum protein agarose gel electrophoresis Normal Monoclonal Polycl 122

8 Figure-2: Comparison of serum protein components in MM patients having monoclonal band as compared to healthy controls L /d 8 m g ALBUMIN TOTAL PROTEI N 5 4 L3 /d m g2 1 0 ALPHA-1 ALPHA-2 BETA GAMMA A/G RATIO CONTROL(n=25) PATIENTS(n=8) CONTROL PATIENTS Indicates significantly different from healthy controls (P 0.05) Figure-3:Comparison of serum protein components in MM patients having polyclonal band as compared to healthy controls L 6 /d m g CONTROL(n=25) ALBUMIN PATIENTS(n=7) TOTAL PROTEIN gm/dl ALPHA-1 ALPHA-2 BETA GAMMA A/G RATIO CONTROL(n=25) PATIENTS(n=7) Indicates significantly different from healthy controls (P 0.05) 123

9 Fig-4: Comparison of serum protein components in pre and post treated MM Patients L 8 /d m6 g ALBUMIN control(n=25) pretreatment(n=10) posttreatment(n=10) TOTAL PROTEIN L /d 2 m g ALPHA-1 ALPHA-2 BETA GAMMA A/G RATIO control(n=25) pretreatment(n=10) posttreatment(n=10) Indicates significantly different from healthy controls (P 0.05) 5. Staining: Slide was stained with amidoblack Slide was destained with 3% acetic acid 6. Quantitation of proteins: Scanning and quantitation of individual protein band was done densitometrically with biored gel documentation system. BIOCHEMICAL PARAMETERS : Biochemical parameters were analyzed by auto analyzer as per table-3: Statistical analysis: Diagnosis of Multiple Myeloma: Results were analyzed statistically using students t test to compare mean levels of bio markers in healthy controls and Multiple Myeloma patients. Differences were considered significant only when P Treatment of Multiple Myeloma: Results were analyzed statistically using students t test to compare mean levels of bio markers in control, untreated and treated MM patients. Differences were considered significant only when P RESULTS: In the study electrophoretic protein estimation in serum of control and multiple myeloma patients revealed multiple protein bands, which were further categorized into Albumin, Alpha-1, Alpha-2, Beta and Gamma fractions. The following figure depicts serum protein fractions on agarose gel, differentiated by electrophoresis. Serum of 28 subjects was electrophoreted for serum protein fractions. Results of study are divided into two parts. Part-I: Includes diagnosis of multiple myeloma where number of patients is 15(n=15). 124

10 Part II: Includes the treatment of multiple myeloma (MM) patients where number of patients is 13(n = 13). Part I: Diagnosis : (I) General features Out of 15 patients, 8 had monoclonal bands while 7 depicted polyclonal bands. In patients with monoclonal bands, the age of male patients ranged from years, while female ranged between years. Hence the mean age for monoclonal band patients was52 years. In patients with polyclonal band, the age of male patient s ranged from years, while female ranged between years. Hence mean age for polyclonal band patients was 51 years. (II) Bio-chemical parameters Bio-chemical parameters viz. haemoglobin, serum calcium, serum creatinine and blood urea nitrogen were analysed. The mean values of haemoglobin, serum calcium, serum creatinine and blood urea nitrogen were as depicted in following table. There was a significant decrease in mean value of haemoglobin in monoclonal and polyclonal band patients as compared to reference standard values. Patients with mono and polyclonal bands were anaemic as evident by their low Hb values. Serum calcium, creatinine and blood urea nitrogen values were normal in monoclonal band patients as compared to reference standard value; while in polyclonal band patients serum calcium, creatinine and blood urea nitrogen values were increased as compared to reference standard values. (III) Serum protein levels Serum protein fractions were analyzed as albumin, alpha-1, alpha-2, beta, and gamma by electrophoresis method, total protein was also estimated in monoclonal,polyclonal and healthy controls while Albumin/Globulin (A/G) ratio was calculated. There was a significant elevation in mean values of serum components alpha-1, alpha-2 and significant decrease in Albumin/Globulin (A/G) ratio in monoclonal and polyclonal band patients as compared to healthy controls. In monoclonal band patients there was significant elevation in albumin, total protein, beta globulin and gamma globulin components, while in polyclonal band patients there was a decrease in albumin and increase in beta globulin, gamma globulin and total protein levels as compared to healthy controls. Part II: Treatment monitoring (I) General features Treatment, a combination of vincristine (2mg. /day intravenously continuous infusion for 4 days), cyclophosphamide (600mg/m 2 intravenously daily for 4 days), melphalan (0.15mg/kg. daily for 7 days orally), prednisolone (20mg. t.i.d. orally), were given to 13 patients and serum protein electrophoresis was performed for 13 patients. Pre-treatment data for 10 patients out of 13 patients was available; it was also performed at G.C.R.I. Ahmedabad. Post treatment data of serum protein electrophoresis was compared with pretreatment data. There was no change in band patterns in pre and post-treated patients. Post treatment data of serum protein electrophoresis results showed 8 patients with monoclonal band and 2 patients with Polyclonal band. The age range of patients (all males) with monoclonal band was between years, mean age being 52.6 years, while age range for polyclonal band patients (all patients) was between years. Their mean age being 43.5 years. 125

11 (II) Bio-chemical parameters Bio-chemical parameters such as haemoglobin, serum calcium, and serum creatinine and blood urea nitrogen in posttreated patients were analyzed. The mean values of haemoglobin, serum calcium, and serum creatinine and blood urea nitrogen are as depicted in table. There was a decrease in mean value of haemoglobin in post treated patients as compared to reference standard values; while serum calcium, serum creatinine and blood urea values were normal to reference standard in post-treated patients. (III) Serum protein levels Serum protein levels in pre treated and post-treated MM patients were analyzed by electrophoresis method. The mean values of serum protein components in control, pretreated and post treated patients are as depicted in table-6. Pre-treatment data was compared with control data of serum protein electrophoresis. Our result showed significant increase in gamma, total protein and A/G ratio in pretreated patients as compared to healthy controls. There was an increase in albumin, alpha-1, alpha-2, beta, and total protein level and decrease in gamma and A/G ratio in post-treated patients as compared to pre treated patients. However the change was statistically insignificant. (IV) Classification of treated patients Results of post-treated patients when compared with pretreated patients were statistically insignificant. Since the study is clinical, due significance is given to clinical signs and symptoms and irrespective of statistical analysis, our results suggest that disease is incurable however, the treatment is effective. Hence subjects were further categorized as responders and nonresponders. Serum protein components in treatment group patients (n=10) are as depicted in table-7. Based on Albumin/ Globulin (A/G) ratio if patient depicts increase in A/G ratio following treatment; they are classified as responders. If patient depicts decrease in Albumin/Globulin (A/G) ratio then they are classified as nonresponders. DISCUSSION: Patients with Multiple myeloma (MM) present a variety of symptoms such as presence of tumor, para-proteinemia (abnormal protein), osteolytic lesions, renal dysfunction 9, recurrent infections 1 and haematological abnormalities, which are not specific to the disease, thus delaying the diagnosis.the emergence of techniques likes bone marrow aspiration or biopsy (invasive); and serum protein electrophoresis (less invasive) facilitates the diagnosis and monitoring the treatment response in MM patients. Occasionally, plasma cells do not secrete any para protein (abnormal protein); in such conditions the results of bone marrow biopsy, radiological examinations (non invasive) and other clinical symptoms interpret MM. In MM patient, hypoalbuminemia is observed 10. This reduced albumin is often accompanied by an elevation in the alpha globulins 11. The changes in serum protein fractions reflect chiefly the reactions of the host to the presence of tumor. Diagnosis of multiple myeloma: We observed overall changes in the electrophoretic pattern of serum protein profile. The study included 28 multiple myeloma patients. The diagnostic profile comprised of 15 patients, while treatment profile 126

12 comprised of 13 patients. In the study 8 patients (53%) exhibited monoclonal band pattern, while 7 patients (47%) depicted Polyclonal band. The electro-phoretic pattern of serum protein profile revealed increase in levels of alpha-1, alpha-2, beta, gamma and total protein levels and decrease in A/G ratio in both monoclonal and polyclonal band patients as compared to healthy controls. Albumin levels were decreased in polyclonal band patients while increased in monoclonal band patients as compared to healthy controls. Albumin band represents the largest protein component of human serum 11,19. Albumin level is decreased, when there is less production of the protein by liver or increased loss of albumin. The reduction in albumin concentration is the most common change in the serum of MM patients 10. In the study serum protein electrophoresis results depicted albumin levels a higher limit of normal albumin range in monoclonal band patients; and decreased albumin levels in polyclonal band patients as compared to healthy controls. Severe hypo-albuminemia occurs in polyclonal band MM patients. Statistical analysis for monoclonal band is significant while for polyclonal band patient s results are statistically insignificant. Alpha fraction lies toward the negative portion of the gel 11, 19. Alpha peak involves alpha-1 and alpha-2 components. Alpha 1 fraction comprise of alpha 1 antitrypsin, thyroid binding globulin, and transcortin. Alpha-1 globulin and Alpha-2 globulin levels are increased in MM patients 11, 19. Our results confirm that alpha-1 and alpha-2 globulin levels are significantly increased in monoclonal and polyclonal band patients as compared to healthy controls. Beta fraction has two peaks labelled as beta-1 and beta Beta-1 is mainly composed of transferrin, and beta 2 contains beta lipoprotein. IgA, IgM, and sometimes IgG, along with complement proteins can be identified in the beta region. Beta globulin levels are increased significantly in MM patients 12, 19. Our results also showed significant increase in beta globulin levels in monoclonal band patients while in polyclonal band patient s beta globulin levels were increased but statistically the results were insignificant. Immunoglobulins migrate to the gamma region of the serum protein spectrum.c reactive protein (CRP) is located in the area between the beta and gamma components 11. In multiple myeloma the gamma level is increased 12, 19. Our results showed significant elevated volume of gamma globulin fraction in 87% of monoclonal band patients. Although many conditions can cause an increase in the gamma region, several disease status causes a homogeneous spike like peak in the focal region of the gamma globulin zone. These so called monoclonal gammopathies constitute a group of disorder that is characterized by proliferation of a single clone of plasma cells that produced a homogeneous M Protein 9. The M protein spike is usually greater than 3-gm/dL in-patients with MM, up to 1/5 th of patients with this tumor may have M protein spike of less than 1 gm/dl 20. In the study our results showed that in polyclonal band patients, M protein spike is less than 3 gm/dl signifying monoclonal gammopathies of undetermined significance (MGUS). However, results of bone marrow biopsy, osteolytic lesions in radiological examination and clinical symptoms confirm MM from MGUS. Overall globulin fractions were increased in monoclonal and polyclonal band MM patients 12. Our results confirm that globulin fractions (alpha, beta, and gamma) were increased in monoclonal and polyclonal band patients. Albumin / Globulin ratio (A/G ratio) was significantly decreased in monoclonal and polyclonal band MM patients, in association 127

13 with decreased albumin levels and increased globulin level. Total protein levels were increased in monoclonal and polyclonal band patients, however statistical analysis was significant for monoclonal band patients while insignificant for polyclonal band patients. In both, monoclonal and polyclonal band patients, 73% patients suffered from anaemia, and elevated erythrocyte sedimentation rate (ESR). Serum creatinine and blood urea nitrogen levels were higher in polyclonal band patients then monoclonal band patients, indicating renal damage occurs progressively faster in polyclonal band patients as compared to monoclonal band patients. The median age of onset in current study was 52.8 years in males and 48.6 years in female depicting increase in incidence of MM with advancing age. Treatment of multiple myeloma: In the treatment profile study, 13 patients diagnosed with MM were treated for it. Treatment comprised of standard, chemotherapy viz. combination of vincristine (2mg.in a 4 days continuous infusion), cyclophosphamide (600mg/m 2 intravenously daily for 4 days), melphalan (0.15mg/kg. daily for 7 days orally), prednisolone (20mg. 3 times a days orally), 13, 14. The role of radiation is highly limited in MM 16.The pre- treatment data of serum protein electrophoresis for the above same 13 patients were performed at the biochemistry research division, G.C.R.I. Ahmedabad earlier. Out of the 13 patients data for 10 patients were available, which was collected. Post treatment analysis of serum protein electrophoresis was later performed for the same 10 patients. The results were compared before treatment (untreated) and after treatment (treated) for the MM patients. The reduction in serum albumin concentration is the most common change in MM patients 10. The standard therapy did not alter albumin levels. Alpha fraction (alpha-1, alpha-2) lies towards cathode of the gel. Alpha subsets are elevated in MM patients 11,19. In the present study alpha-1 and alpha-2 levels were increased in 50% of treated patients as compared to pre treated patients. Beta globulin levels were increased significantly in MM patients 12, 19. Our results confirmed that beta globulin levels were significantly elevated in post-treated patients as compared to pre treated patients. Much of the clinical importance is focused on the subset gamma region of the Globulins; because, predominantly immunoglobulin migrate to this region 11.In multiple myeloma the gamma level is increased 11, 19. In the present study, following treatment Gamma globulin levels were decreased however, normal levels in post treated patients were not attained. In the present study,two patients unusually depicted decrease in elevated gamma globulin level and elevated normal beta globulin level following treatment rendering them as non- responders based on A/G ratio. In the study Albumin / Globulin (A/G) ratio was increased following treatment, however, normal levels were not attained. Based on the result of gamma globulin levels and A/G ratio normal levels were not attained, suggesting that the therapy (Combination of vincristine, melphalan, cyclophosphamide, and prednisolone) is not curative however, it can be suggested to be suppressive. Hence, if the patient showed increase in A/G ratio following treatment; they were classified as responders and if the patient showed decrease in Albumin/Globulin (A/G) ratio 128

14 then classified as non-responders. In present study 70% patients (n=7) were responders while, 30% patients (n=3) were nonresponders. Out of 10 patients; 9 patients exhibited monoclonal band while 1 patient depicted polyclonal band. In the study responders (n=7) following treatment depicted increased albumin levels along with decreased alpha fractions in 50% of patients. Responders showed increased Albumin / Globulin (A/G) ratio. Beta globulin levels in responders (n=7) were decreased in 3 patients (43%) after the treatment; In 2 patients (28%) beta globulin levels were increased and in 2 patient (29%) no change was observed. Gamma globulin levels in responders (n=7) depicted decreased gamma globulin fractions; indicating positive response to the treatment. In the study the non-responders (n=3) following treatment depicted increased albumin levels along with decreased alpha fractions in 50% of patients. The nonresponders (n=3) depicted significantly elevated beta fractions in 67% patients (n=2) and decreased beta levels in 33% patient (n=1). Gamma globulin levels were increased in 2 patients but 1 patient showed decreased gamma globulin level with significantly elevated beta globulin levels.in the study non-responders showed decreased A/G ratio following treatment patients as compared to pre treated patients. Overall analysis showed that in responders the A/G ratio and gamma globulin levels were affected following treatment, the changes were not statistically significant suggesting the treatment to be suppressive rather than curative. All the 10 patients showed high ESR, even after treatment. From the responders following treatment 56% suffered from anaemia, while from the non-responder 66% suffered from anaemia. In the study, responder s age range from years while non-responders age ranged from years, indicating that younger patients were more responsive to the treatment as compared to older patients, inferring the prognostic impact of age on survival seems to be better among younger patients than older patients. The study of Electrophoretic Pattern in Serum of Multiple Myeloma was performed at Biochemistry Research Division, GCRI, Ahmedabad. The present study included 28 patients, out of which 15 patients were recent inclusions while 13 patients were follow-up patients. Serum protein electrophoresis is used to identify patients with multiple myeloma (MM). The analysis was performed on 0.5% agrarose gel and the protein fractions were estimated in terms of albumin, alpha, beta, and gamma globulins. Total protein was estimated by biuret method. The following conclusions were summarised from the present study. Of the recently diagnosed patients 53% exhibited monoclonal protein bands (n=8) while 47% depicted polyclonal protein bands (n=7). Patients exhibiting monoclonal band showed significant increase in alpha globulins, beta globulins, gamma globulins and total protein levels while, albumin levels were closer to higher limit of normal albumin range as compared to healthy controls. Albumin/Globulin (A/G) ratio was significantly decreased in monoclonal band patients as compared to healthy controls. Patients exhibiting polyclonal band suffered from hypoalbuminemia with 129

15 increase in beta globulins, gamma globulins, and total protein levels as compared to healthy controls; however, statistically the results were insignificant. Alpha globulin fractions were significantly elevated and Albumin/Globulin (A/G) ratios were significantly decreased in polyclonal band patients as compared to healthy controls. The above multiple myeloma patients having monoclonal or polyclonal band suffered from anemia and depicted high erythrocyte sedimentation rate (ESR). Patients with polyclonal band exhibited elevated serum creatinine and blood urea nitrogen levels as compared to monoclonal band patients indicating more prominent renal damage in polyclonal band patients as compared to monoclonal band patients. Patients with multiple myeloma having polyclonal band also showed clinical symptoms of MM in conjunction with noninvasive radiological examination and results of bone-marrow biopsy supported our inference of multiple myeloma. Our results also highlight that the median age of onset in current study was 52.8 years in males and 48.6 years in females and the incidence of MM increases with advancement in age. Serum protein electrophoresis is used to monitor the treatment in multiple myeloma patients. The study included 13 patients for monitoring the treatment in multiple myeloma patients. Pre- treatment data of serum protein electrophoresis was available for 10 patients. Treatment to the above 10 patients comprised of standard chemotherapy viz. Combination of vincristine (2mg./day intravenously continuous infusion), cyclophosphamide (600mg/m 2 intravenously daily for 4 days), melphalan (0.15mg/kg. daily for 7 days orally), prednisolone (20mg. ti.d. orally) alongwith radiotherapy. Following treatment, serum protein electrophoresis was performed for the same 10 patients. Out of 10 patients; 8 patients depicted monoclonal while 2 patients depicted polyclonal band. The pre and post treatment data of 10 patients were statistically compared. Results were insignificant for all serum protein components. Cancer (MM) is not curable but treatable disease, hence based on albumin/globulin (A/G) ratio they were classified as; responders (if A/G ratio was increased), and non-responders (if A/G ratio was decreased). In study responders (n=7) depicted increased albumin levels, A/G ratio and decreased fractions of alpha globulins, beta globulins or gamma globulins, while the non-responders (n=3) showed increased albumin, significantly elevated beta globulins or gamma globulins and decreased alpha globulins. Of the treated group patients (n=10); 69% suffered from anaemia and depicted high ESR. From the above results we summarize that gamma globulin levels were not decreased statistically significantly and A/G ratio were not statistically significantly increased in post- treated patients as compared to pre-treated patients; confirming that the present anticancer chemotherapy comprising of (combination of vincristine, melphalan, cyclophosphamide, prednisolone) in MM patients is not curative however, suppressive, and prognostic impact of age on survival seems to be better among younger patients than older patients. CONCLUSION: We suggest that serum protein electrophoresis on agarose gel is less invasive and a critical diagnostic test along with radiological examination (non- 130

16 invasive) and invasive bone-marrow biopsy to diagnose multiple myeloma from other haematological cancers.serum protein electrophoresis estimation exhibited increase in alpha globulins, beta globulins gamma globulins and decrease in A/G ratio in MM patients having monoclonal or polyclonal band. Hypoalbuminaemia was observed in polyclonal band patients, and albumin levels were attained closer to higher normal values in monoclonal band patients. Serum creatinine and blood urea nitrogen values were elevated in polyclonal band (female) patients indicating renal damage occurs progressively faster in polyclonal band patients as compared to monoclonal band patients. Our results also confirm the incidence of MM increases with advancing age.in treated group patients, serum protein electrophoretic estimation exhibited increased in albumin, and A/G ratio; while decrease in alpha globulins, beta globulins, and gamma globulins. Above results was not statistically significant indicating that present anticancer chemotherapy (combination of vincristine, melphalan, cyclophosphamide, prednisolone) is not curative, however, it could be considered as suppressive. Based on increase in A/G ratio patients were labelled as responders and decrease in A/G ratio they were labeled as non-responders. Our results also suggest that the prognostic impact of age on survival seems to be better among younger patients than older patients. BIBLIOGRAPHY: 1. Munshi, NC, Hematol Oncol Clin. North Am 1997; 11: Tricot,GJ., Naucke, S., Curr Top Microbiol Immunol 1996; 210: Hobbs, JR., Advances in clinical chemistry, edited Bodansky, O. and Latner, A.L., Academic Press,1971; 14: Brown, LM., Linet, MS., Greenberg, RS., et al., 1999; 85:2385.cancer Res.; 64: Bergsagel, DE. Wong, O., Bergsagel, PL., et al., Blood 1999; 94: Riedel, DA., Pottern, LM, Hematol Oncol Clin. North Am 1992; 6: Barlogie, B., Gale, RP. Leuk Lymphoma 1991; 26: Bartl, R., Frisch, B., Pathol Biol (Paris) 1999; 47: Kyle, RA., Arch Pathol Lab Med 1999; 123: Petermann, ML, Med clin North AM, 1961; 9: Jacoby, RF., Cole, CE, Molecular diagnostic methods in cancer genetics. In: Abeloff MD, et al; eds. Clinical oncology. 2 nd ed. Newyork: churchill Livingstone, 2000: George, ED., Sadovsky, R., Multiple myeloma: recognition and management. Am Fam Physician 1999; 59: Boccadoro, M., Marmont, F., Tribalto, M., et al., J Clin Oncol 1991; 9: Gregory, WM., Richards, MA, Malpas, JS, J.Clin Oncol 1992; 10: Salmon, SE, Shadduck, RK, Schilling, Cancer Chemother Rep 1967; 51: Hu, K., Yahalom, J., Oncology, 2000; 14: Reinhold, JG., in standard methods of clinical chemistry, edited M.Reiner Academic press, New York and London 1953;1: Laurell, CB, and Laurell, Lancet 1955, 2: Ravel, R., Clinical laboratory medicine: clinical application of laboratory data 6 th ed. St.Louis: Mosby, 1995: , Alexanian, R., Weber, D., Liu, F., Arch Pathol Lab Med 1999; 123: