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DETERMINATION OF CD20 PROTEIN EXPRESSION IN B-CELL LYMPHOMA CANCER & STUDY OF ANTI-CD20 THERAPY


Ram Darshiit*1, Dr. Kashyap Raval2

 1Department of Pharmaceutical Biotechnology, Noble Pharmacy College, Junagadh.
2Department of Quality Assurance, Noble Pharmacy College, Junagadh.

 ABSTRACT

Study is based on the incidence of CD20 in B-cell lymphoma and evaluation expression of CD20 protein on paraffin embedded tissue sections by Immunohistochemical analysis in B-cell lymphoma. Immunohistochemical analysis is advantageous because the antigen expression can be studied within the cancer cells. Correlate CD20 expression with established clinico-pathological parameters, and understanding antiCD20 therapy on B-cell lymphoma patients. It was assumed that data from the present study would provide better insight in to the role of CD20 in B-cell lymphoma patients, and further help in treatment planning of these patients.

 

Key words: lymphocytes, Immunohistochemical, B-cells, Chemotherapy

INTRODUCTION

Our body is composed of many millions of tiny cells, each a self-contained living unit. Normally, each cell coordinates with the others that compose tissues and organs of your body. Cancer develops when cells in a part of the body begin to grow out of control. Even though there are many kinds of cancer, they all start because of abnormal cells that grow out of control. Normal body cells grow, divide, and die in an orderly fashion. During the early years of a person’s life, normal cells divide more rapidly until the person becomes an adult. After that, cells in most parts of the body divide only to replace worn-out or dying cells and to repair injuries. Because cancer cells continue to grow and divide, they are different from normal cells. Instead of dying, they outlive normal cells & continue to form new abnormal cells. Lymphoma is a type of cancer involving cells of the immune system, called lymphocytes. Just as cancer represents many different diseases, lymphoma represents many different cancers of lymphocytes-about 35 different subtypes, in fact. Lymphoma is a group of cancers that affect the cells that play a role in the immune system, and primarily represents cells involved in the lymphatic system of the body. The lymphatic system is part of the immune system. It consists of a network of vessels that carry fluid called lymph, similar to the way that the networks of blood vessels carry blood throughout the body [1-4]. Lymph contains white blood cells called lymphocytes. Lymphocytes attack a variety of infectious agents as well as many cells in the precancerous stages of development. Lymph nodes are small collections of lymph tissue that occur throughout the body. The lymphatic system involves lymphatic channels that connect thousands of lymph nodes scattered throughout the body. Lymph flows through the lymph nodes, as well as through other lymphatic tissues including the spleen, the tonsils, the bone marrow, and the thymus gland.  People often confuse grade and stage [5-7]. Grade reflects the growth pattern of the lymphoma, whereas stage reflects the extent of the disease. Early Disease Stage (I), Locally Advanced Disease (II), Advanced Disease (III) & Wide Spread Disease (IV) The choice of treatment depends mainly on the type of non-Hodgkin lymphoma, its stage, how quickly the cancer is growing, age, whether you have other health problems: Chemotherapy, Combination of Chemotherapy Drugs, Chemotherapy Cycles, Radiation Therapy, Biological Treatment, Vaccine, Anti-Angiogenesis Therapies, Gene Therapy and Antisense Molecules etc… There are numerous Immunohistochemistry methods that may be used to localize antigens [8,9]. The selection of a suitable method should be based on parameters such as the type of specimen under investigation and the degree of sensitivity required. Direct Method, Two Step Indirect Methods, Soluble Enzyme Immune Complex Techniques (Strept) , Avidin-Biotin Technologies Etc…

 Anti CD-20 Therapy: CD stands for Cluster of Differentiation. CD markers are a group of special molecules on the surface of the cells in our body. There are more than 250 types of CD molecules [10-12]. All cells in our body have one or more of them, but they are most useful for classifying WBCs (white blood cells), a type of cell in the blood that protects us from infections.

The CD nomenclature was proposed and established in the 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), which was held in Parisin 1982. This system was intended for the classification of the many monoclonal antibodies (mAbs), generated by different laboratories around the world, against various surface molecules (antigens) on leukocytes (white blood cells) [13-14]. Since then, the use has expanded to many other cell types, and more than 250 CD clusters and subclusters have been identified. CD20 is a CD marker – a molecule on the cell surface that can be used to identify and type a particular cell in the body [15].

CD20 is a marker for B-cells, a type of white blood cell (WBC) that protects the body from infections. There are two types of WBCs – B-cells and T-cells. CD20 is present on the surface of B-cells but not T-cells[16]. Testing for CD20 using a special technique called Immunohistochemistry (IHC) is a special method of determining whether an abnormal cancerous WBC is a B-cell or T-cell.

MATERIALS AND METHODS

A series of 110 patients with B-cell Lymphoma disease were enrolled in this study. The age range of the subjects was from 17 to 80 years. These subjects were diagnosed and treated at the one of Institute. A detailed clinical history (age, gender, types of disease, grade, stage, treatment given, abuse, disease status, general condition of subjects during follow-up)  of all the subjects was obtained from the case files maintained at the institute. The surgical specimens were transported from operation theatre to histopathological department. Tumour part was selected by a pathologist and fixed in formalin for 24 hours, routinely processed and paraffin embedded. Immunohistochemical staining was performed on BENCHMARK XT(IHC/ISH ) by VENTANA the CD20 was studied using the indirect immunohistochemical approach. The clone and dilution of the antibody used are mentioned below.

Antibody used Antigen detected Clone Dilution Company
CD20 CD20 L26 1:100 Neo marker

Method

  • 3-4 µm thick sections were cut from paraffin embedded tissue block on microtome (Shandon Hypercut) and fixed in egg albumin coated slide.
  • Place the slides on hot plate 40ºC for 30 min. (for better fixation of tissue on slides).
  • Then deep the slides in milk for 30 min. (for better fixation of tissue on slides).
  • Kept overnight at 60ºC in hot air oven for tissue fixation.

Protocol for CD20 in BENCHMARK  XT(IHC/ISH ) by VENTANA is as below

  • Paraffin
  • Deparaffinization
  • Cell conditioning: Conditioner #1 (Short-8 min. conditioning), Mild CC1 (mild-30 min. conditioning), Standard CC1  (standard-60 min. conditioning)
  • Ab incubation temperatures
  • Disable heat for ab incubation
  • Titration: Primary antibody, Medium incubation time, 32 min.
  • Counterstain: (Hematoxylin): Incubation time, 8 min
  • Post Counterstain: (Bluing Reagent): Incubation time, 4 min
  • Run time 3 hours
  • Slides rinsed in tap water for 5 min.
  • One dip in acid alcohol (30-40 sec.) to remove of excessive stain and rinsed in tap water.
  • Kept in absolute alcohol for 30 sec. followed by xylene for 15 min.
  • Slides were mounted with DPX.

Reagents

HEMATOXYLIN (<= 48%), BLUING REAGENT (-0.1 M, Li2Co3, o.5 M, Na2Co3), Ultra view UNIVERSAL DAB inhibitor, Ultra view UNIVERSAL DAB multimer, Ultra view UNIVERSAL DAB chromogen, Ultra view UNIVERSAL DAB H2O2, Ultra view UNIVERSAL DAB copper

Buffers

EZ prep (for deparaffinization), LCA (for liquid cover slip), 2x SSC, Reaction Buffer (for washing), CC1 (for antigen retrival), CC2 (for antigen rertrival), Option.

Statistical analysis

The data were statistically analyzed using the SPSS statistical software. Two tailed χ2 test was used to assess the association between two parameters. The P values less than 0.05 were considered significant.

RESULT AND DISCUSSION

In this study 110 patients of B-cell Lymphoma were enrolled, among them 27% were in the age group of 51 to 60 years, and only 3%  were in the age group of <=20 years.

 Table 1: Distribution of patients according to their age                                   Table 2: Gender wise distribution of patients

Age group Number Percent
<=20 3 3
21 to 30 6 5
31 to 40 18 16
41 to 50 26 24
51 to 60 29 27
61 to 70 20 18
71 to 80 8 7
Total 110 100
Gender No. Percent
Female 42 38
Male 68 62
Total 110 100

 

It is shown in the table 2 that 62% (68/110) patients with B-cell lymphoma cases were males and only 38% (42/110) of patients were females.

Fig. 1: Gender wise distribution of patients

Figure 2: Distribution of patients according to type of B cell lymphoma

It is shown in the table 3 that majority of patients 60% (67/110) had diffuse large B-cell lymphoma, 30%(33/110) had follicular lymphoma, 5%(5/110) had marginal zone lymphoma, and 5%(5/110) had  mental cell lymphoma .

Table 4 indicate that 35%(39/110) of patients were bidi smokers, about 18%(20/110) of patients were chewing either mava or gutkha containing tobacco.

 Table 3: Distribution of patients according to type of B cell lymphoma                Table 4: Distribution of patients according to their habits

Abuse No. Percent
None 50 46
Bidi-10/day 16 14
Bidi-20/day                                10  9
Bidi-25/day 13 12
Snuffing-2/day 1 1
Tobacco-8/day 20 18
Total 110 100

 

Type of B cell lymphoma Frequency Percent
Diffuse large B-Cell lymphoma 67 60
FOLLICULAR LYMPHOMA 33 30
MARGINAL ZONE LYMPHOMA 5 5
MENTAL CELL LYMPHOMA 5 5
Total 110 100

 

It is shown in the Table 5 that majority of patients that is 51%(56/110) had high grade lymphoma, and 42%(46/110) had low grade lymphoma were only 7%(8/110) had intermediate lymphoma.

Table 5: Distribution of patients according to grade of lymphoma

GRADE No. Percent
High 56 51
Intermediate 8 7
Low 46 42
Total 110 100

Figure 3: Distribution of patients according to grade of lymphoma

 Table 6: Distribution of patients according to their general condition

General Condition No. Percent
GC FAIR 53 48
METASTASIS 22 20
PERSISTANT 35 32
Total 110 100

It shown that majority of patients that is 48%(53/110) responded to treatment, only 20%(22/110) of patients  developed metastasis and 32%(35/110) of patients had persistant disease.

Figure 4: distribution of patients according to their general condition

 

Figure 5.1: CD20 expression in low grade B-cell lymphoma can be seen by brown colour staining

Figure 5.2: CD20 expression in high grade B-cell lymphoma can be seen by brown colour staining

Figure 5.3: Himatoxyline-eosin staning in low grade B-cell lymphoma

Figure 5.4: Himatoxyline-eosin staning in high grade B-cell lymphoma

 

Table 7: Distribution of patients according stage of lymphoma

 

 

Stage

No. Percent
I 12 11
IA             8 7
IB 1 1
IE 7 6
II 8 7
IIA             1   1
IIB 4 4
IIE 1 1
III 10 9
IIIB              2  2
IIIE 1 1
IV 55 50
Total 110 100

Table 8: Distribution of patients according to treatment given to them

Treatment No. Percent
CHOP 32 29
CHOP+IFRT 3 3
CHOP+R-CHOP 15 14
CHOP+RT 10 9
COP 2 2
COP+IFRT 1 1
COP+RT 1 1
CVP 16 14
CVP-RT+CHOP-RT 1 1
CVP+CHOP 8 7
CVP+CHOP+RT 1 1
CVP+IFRT 3 3
CVP+R-CHOP 2 2
CVP+R-CVP 1 1
CVP+R-CVP+RT 1 1
CVP+RT 6 5
R-CHOP 7 6
Total 110 100

It shown that in the present study 110 patients were enrolled and received different type of treatment like chemotherapy (CHOP, COP, CVP), radiation therapy (RT) and Rituximab(R) either alone or in combination, it was found that most of the patients were received chemotherapy alone or on combination with other therapy.

CHOP Cyclophosphamide, Doxorubicin or Hydroxydauiorubicin, Vincristine, Prednisone
CVP Cyclophosphamide, Vincristine, Prednisone
COP Cyclophosphamide, Oncovin,  Prednisone

 

Table 9 shown that in the present study above mentioned differentiation markers used for the diagnosis of cancer of patients. Among them mostly CD markers were used.

Table 10 shows the distribution of patients according to expression of differentiation markers.  Patients with CD20 Positivity classified as B-cell lymphoma. It was found that CD20 was used in all the patients and it was positive in all of them. When looking at the expression of other markers it was found that LCA was used in 102 patients and it was positive in 94% (96/102) of them. UCHL1 was used in 46 patients and it was positive in only 24% (11/46) of them.

Table 9: Different markers use in Study for the diagnosis of cancer

Sr. no. Marker Diagnosis
1 CD 20 Lymphoma(B-cell) marker
2 LCA Leucocyte marker
3 UCHL1 B & T cell marker
4 CD30 Hodgkin’s Lymphoma activated B & T cell marker
5 CD15 Hodgkin’s Lymphoma B-cell marker
6 CD10 Lymphoid cell marker
7 CD2 Lymphoma(T-cell)
8 CD3 Lymphoma(T-cell)
9 CD5 Lymphoma(T-cell)
10 CD99 (MIC-2) Erwings sarcoma
11 CD23 Lymphoma B-cell marker
12 CD43 Lymphoma
13 CK Carcinoma
14 EMA Carcinoma
15 BCL2 Lymphoma B-cell marker
16 VIM Sarcoma
17 CYCLIND1  Mental cell Lymphoma
18 AE1 Carcinoma
19 S100 Melanoma

 

Table 10: Distribution of patients according to expression of different markers

Sr.No Marker Positive Negative Not used
No % No % No %
1 CD 20 110 100 0 0 0 0
2 LCA 96 87 6 6 8 7
3 UCHL1 11 10 35 32 64 58
4 CD30 0 0 18 16 92 84
5 CD15 5 4 0 0 105 96
6 CD10 3 3 22 20 85 77
7 CD2 2 2 22 20 86 78
8 CD3 1 1 25 23 84 76
9 CD5 4 4 12 11 94 85
10 CD99 0 0 10 9 100 91
11 CD23 1 1 16 14 93 85
12 CD43 2 2 7 6 101 92
13 CK 1 1 52 47 57 52
14 EMA 2 2 37 34 71 64
15 BCL2 22 20 11 10 77 70
16 VIM 2 2 27 25 81 73
17 CYCLIND1 1 1 21 18 88 81
18 AE1 1 1 40 36 69 63
19 S100 0 0 5 4 105 96

 

Table 11:  Distribution of patients who received treatment without Rituximab according to their general condition. (N=84)

Treatment without Rituximab GC Total
GC fair Metastasis Persistant
CHOP 10 8 14 32
CHOP+IFRT 0 1 2 3
CHOP+RT 6 2 2 10
COP 0 0 2 2
COP+IFRT 0 0 1 1
COP+RT 0 0 1 1
CVP 5 3 8 16
CVP-RT+CHOP-RT 0 1 0 1
CVP+CHOP 4 4 0 8
CVP+CHOP+RT 1 0 0 1
CVP+IFRT 1 1 1 3
CVP+RT 2 2 2 6
Total 29 (35%) 22 (26%) 33 (39%) 84 (100)

 

Table 11 indicate that out of total 110 patients 84 patients were received chemotherapy and/or radiotherapy without Rituximab out of them 35%(29/84) of patients had responded to  treatment, 26%(22/84) of them developed metastasis, and 39%(33/84) of patients had persistant disease.

Table 12:  Distribution of patients who received treatment with Rituximab according to    their general condition. (N=26)

Treatment with Rituximab GC Total
GC fair Metastasis Persistant  
CHOP+R-CHOP 13 0 2 15
CVP+R-CHOP 2 0 0 2
CVP+R-CVP 1 0 0 1
CVP+R-CVP+RT 1 0 0 1
R-CHOP 7 0 0 7
Total 24 (92%) 0 (0%) 2 (8%) 26 (100%)

 

Table 12 shown that out of total 110 patients 26 patients were received chemotherapy and/or radiotherapy with Rituximab, out of them 92% (24/26) of patients responded to treatment, none of them had develop metastasis, and 8%(2/26) of patients persistant disease.

Table 13 indicate that out of total 110 patients 84 patients were received chemotherapy and/or radiotherapy without Rituximab out of them 35%(29/84) of patients had responded to  treatment, 26%(22/84) of them developed metastasis, and 39%(33/84) of patients had persistant disease.

Table 13: Impect of treatment on survival of patients. (Cross tabulation of patients disease status vs treatment without rituximab or with rituximab)

Disease Status Total
Responded well Metastasis Persistant disease
RITUXIMAB Not Given No. 29 22 33 84
Percentage 35 26 39 100
Given No. 24 0 2 26
Percentage 92 0 8 100
Total No. 53 22 35 110
Percentage 48 20 32 100
χ2=26.79, d.f.=2, P<0.001, Statistically highly significant.

 

Out of total 110 patients 26 patients were received chemotherapy and/or radiotherapy with Rituximab, out of them 92% (24/26) of patients responded to treatment, none of them had develop metastasis, and only 8% (2/26) of patients persistant disease.

In rituximab group 92%(24/26) of patients responded to treatment while only 35% (29/84) of patients responded to the treatment in subgroup of patients who did not received rituximab. The difference was statistically significant (χ 2=26.79, d.f. =2, P<0.001). It indicate that when Rituximab was used in combination of chemotherapy and radiation therapy is the best choice of treatment in comparison with only chemotherapy and radiation therapy either alone or combined for B-cell lymphoma patients.

DISCUSSION

The B-cell lymphomas are types of lymphoma affecting B cells. B cell lymphomas include both Hodgkin’s lymphomas and most non-Hodgkins lymphomas. They are often divided into indolent (slow-growing) lymphomas and aggressive lymphomas. Indolent lymphomas respond rapidly to treatment and are kept under control (in remission) with long-term survival of many years, but are not cured. Aggressive lymphomas usually require intensive treatments, but have good prospects for a permanent cure.

Monoclonal antibodies target one particular protein found on the surface of cells. Rituximab targets a protein called CD20. All B cells have the CD20 protein on the outside of the cell. The most common types of non Hodgkin’s lymphoma develop when some of the B cells become cancerous. The cancerous B cells also carry the CD20 protein. The antibody sticks to all the B cells it finds. The cells of the immune system then pick out these B cells and kill them.B cells develop from cells in the bone marrow called stem cells. B cell stem cells do not have the CD20 protein. So they are not killed by rituximab, and normal healthy B cells can grow to replace the ones that have been killed. Normal B cell levels in the blood are restored within a few months of having the treatment.

Rituximab is part of standard treatment for high grade diffuse B cell lymphoma diagnosed at stage 2, 3 or 4. Rituximab is also part of standard treatment for low grade follicular NHL that is resistant to chemotherapy or has relapsed at least twice after successful treatment with chemotherapy. And it is now used in certain situations to treat follicular lymphoma.

In this study 110 patients diagnosed as B-cell Lymphoma were enrolled. The median age of patients is 51 years. Present study represent that males (62%) were more affected then females (38%). According to WHO classification of lymphoma there is about 13 subtypes of B-cell lymphoma but in present study only four type of B-cell lymphoma was found among them (60%) was Diffuse large B-cell lymphoma, followed by (30%) was follicular lymphoma. Marginal cell lymphoma and mental cell lymphoma each share (5%) of total patients.

The finding of present study in relation to habits is that 46% patients had no habits like smoking bidi, snuffing, and any type of tobacco consumption. Whereas 35% of patients had habits of smoking bidi, out of them (14%) consume around 10 bidi per day, (9%) consume around 20 bidi per day, (12%) consume around 25 bidi per day. Out of total 110 patients, (18%) had habit of tobacco consumption in different forms like mava, gutkha.

In relation of clinic-pathological parameters majority of patients (51%) had high grade lymphoma, followed by (42%) with low grade lymphoma. and only (7%) with intermediate grade of lymphoma. In relation to stage majority of patients (50%) had stage IV B-cell lymphoma, 25% had subgroups of stage I B-cell Lymphoma, 13% had subgroups of stage II B-cell Lymphoma and only 12% had subgroups of stage III B-cell Lymphoma.

Out of 110 patients 48% were responded to treatment. Metastasis was found in 20% of patients and 32% of patients had no improvement in their general condition.

These patients were mainly treated with chemotherapy and/or radiation therapy. Rituximab therapy was given to the patients either alone or in combination with Chemotherapy and/or radiotherapy.

Regarding CD20 expression, CD20 positive cases were diagnosed as B-cell lymphoma.  When looking at the expression of other markers it was found that LCA was used in 102 patients and it was positive in 94% (96/102) of them. UCHL1 was used in 46 patients and it was positive in only 24% (11/46) of them. BCL2 was used in 33 patients and it was positive in 67% (22/33).

It was found that 92% of patients who received chemotherapy and/or radiotherapy with Rituximab were responded to treatment while only 35% of patients who received  chemotherapy and/or radiotherapy without Rituximab were responded to treatment and this difference was found statistically significant. None of the patients treated with rituximab developed metastatic disease. It indicate that Rituximab is more effective in treatment of B-cell lymphoma when used in combination of other therapy like chemotherapy, radiation therapy.

 

CONCLUSION

In the present study it was found that detection of CD20 protein is most important in diagnosing B-cell lymphoma and identifying candidates who will benefit from rituximab therapy. Rituximab a monoclonal antibody has antitumor activity against CD20 protien positive B-cell Lymphoma patients. Current research is assessing Rituximab effect when it is given with certain chemotherapy drugs. If is given with CHOP, CVP, COP and RT, it is increases the effectiveness of that therapy and may improve survival.

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