Treatment of Rituximab in Pemphigus Vulgaris Patients

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5th Sep 2017 Health Reference this

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Part 3: Clinical Research 2

INTRODUCTION

Pemphigus is the name of a group of life-threatening blistering diseases of the skin and mucous membranes. The base of treatment for this disease is corticosteroids; however, recently, new drugs, such as rituximab, have been verified for more severe forms of it.

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In the author’s previously unpublished study, the effect of rituximab on variation in the laboratory indices of pemphigus vulgaris patients is addressed. After investigation of the files of pemphigus patients who received rituximab in Razi Hospital, Tehran, Iran from 2008 to 2013, 39 patients were entered into the study. All patients had lab sheets containing CR (creatinine), urea, ALT (alanine aminotransferase), AST (aspartate aminotransferase), Plt (platelet), Hgb (hemoglobin), and WBC (white blood cell) before and after receiving rituximab. The patients received rituximab 4 times at a dosage of 500 mg in 4 successive weeks. The lab results before receiving the first dose of rituximab were compared to the results after receiving treatment. The effect of rituximab on the variation in lab indices with the adjustment effect of age, gender, disease duration, sites of involvement, received adjoins, and the background disease were also investigated.

In the initial analysis, rituximab only had a significant effect on urea reduction. In the CellCept® (mycophenolate mofetil) receiving subgroup, the mixed consumption of rituximab led to a significant reduction in WBC. In the subgroup having background disease, rituximab had a statistically significant impact on platelet reduction. In the subgroup having no background disease, rituximab had a statistically significant effect on urea reduction.

The lab indices were shown to have no significant relationship with age and disease duration. Thus, it can be predicted that disease duration and age would have no effect in the relationship between rituximab and lab indices’ variations.

Although in stratified single-variable analysis for adjusting the effect of other variables (involvement sites and received adjoins) on the relation of rituximab and lab indices, some of these variables showed interacting effects with rituximab on the variations of lab indices. However, due to the low volume of sample and non-normal distribution of most of these variables, it was impossible to do multivariable analysis for investigation of their independent and interactive effects on variations of lab indices in an integrated manner, therefore, we can not make certain comments about their relationships.

Chapter 1

Pemphigus is the name of a group of life-threatening blistering diseases that have characteristic acantholysis leading to formation of intraepithelial blisters in mucus and skin [1]. The acantholysis process is induced via attachments of flowing autoantibodies to adhesion molecules in the cells [2]. Patients with pemphigus have mucosal erosions, blisters, papules, and cutaneous erosions.

The different types of pemphigus are pemphigus vulgaris, pemphigus foliaceus, immunoglobulin A (IgA) pemphigus, and paraneoplastic pemphigus. Different types of pemphigus are differentiated by clinical symptoms, related autoantigens, and histological methods. Pemphigus vulgaris has mucosal and mucocutaneous involvement. The blisters are acantholytic and suprabasal. The autoantibodies responsible for the disease are against desmoglein (DSG) 1 or both desmoglein 3 and 1.

Pemphigus foliaceus only involves the skin. The blisters are acantholytic and subcorneal. The responsible autoantibodies are against desmoglein 1.

IgA pemphigus has the form of grouped erythematous crusts, papules, and vesicle plucks. Blisters can be subcorneal or intraepithelial and acantholytic. The responsible autoantibodies are against desmocollin (DSC) 1 [3].

Paraneoplastic pemphigus involves vast and resistant stomatite along with different cutaneous findings. The responsible autoantibodies are against desmoplakin (DSP) or other desmosomal antigens. Pemphigus vulgaris is the most common type of pemphigus, but is still very rare. The chance of its occurrence is between 0.1 to 0.5 per 100,000 people [4]. Pemphigus often happens among adults and the average age of onset is 40 to 60 years old. It is very rare among children [5,6]. Its prevalence is almost the same in the 2 sexes [7]. Almost all the pemphigus vulgaris patients have mucosal involvement. The mouth is the most common site of involvement and is often the first site of involvement. Other mucosal membranes such as conjunctivae, nose, esophagus, vulva, vagina, cervix and anus are rarely involved [8]. As mucosal blisters are fragile and burst easily, in clinical examination it is difficult to find intact blisters, and instead the examiner tends to find mucosal erosions. Buccal and palatal mucosa are the most common sites of blister involvement in the mouth cavity [9].

Mucosal involvement can be very painful. This pain often increases by chewing and swallowing, which can result in improper alimentation and weight reduction. Most of the patients also have cutaneous involvement appearing in the form of soft blisters in healthy skin or erythematosus. The blisters easily break, resulting in painful erosions. Pemphigus vulgaris rarely causes pruritis. Almost any part of body skin can be involved, but the palmar aspects of the foot and hands are rarely involved. The Nikolsky sign is often observed among these patients (mechanical pressure on the healthy skin results in blistering). Pemphigus is diagnosed based on the clinical, histological, immuno-pathological symptoms and laboratory findings. Even in cases where the clinical symptoms are intensively supporting pemphigus, laboratory investigation is still needed to confirm the diagnosis, as other diseases may have the same symptoms. The first line of treatment of pemphigus is systemic corticosteroids, and addition of adjuvants may also be needed. Patients who do not respond to the first line of treatment might need additional interventions. In such patients, cyclophosphamides, rituximab, intravenous immunoglobulin (IVIG) or plasmapheresis may be helpful.

Initial treatment of pemphigus vulgaris is systemic glucocorticoid, which is often applied in combination with other non-steroidal immunosuppressants such as azathioprine and mycophenolate mofetil. Pemphigus resistant to treatment is a type of pemphigus that does not respond to the aforementioned treatments.

Pemphigus is a chronic disease that needs long-term treatment. A retrospective study was conducted during 1982-1993 on 40 patients [8]. On average, these patients achieved complete remission after 7.7 years; 25% had remission after 2 years; 50% after 5 years; and 75% after 10 years [8]. Most pemphigus vulgaris patients respond to initial treatments [9]. The first step, in the patients who do not respond to initial treatment, is increasing the dosage of systemic corticosteroids (1.5-2 mg/kg of prednisolone per day) or adjuvant drug. The adjuvant drug can also be changed (changing azathioprine to mycophenolate mofetil). In resistant cases, cyclophosphamides, rituximab, IVIG, and plasmapheresis could also be used.

As pemphigus is an auto-immune disease caused by autoantibodies, treatments that reduce B cells are investigated [10-13]. Rituximab is a monoclonal antibody that targets CD20, located on B-lymphocytes, as its antigen. This drug has been shown to have profound effects on pemphigus treatments [13,14]. In a multicenter study conducted on 14 pemphigus vulgaris patients and 7 pemphigus foliaceus patients, both groups were resistant to systemic glucocorticoids and experienced several relapses during glucocorticoid tapering. They were then put on 1 cycle of rituximab with a weekly dosage of 275 mg/m2 for 4 weeks, and this addition proved advantageous [15]. Although, severe infections were reported in the patients under rituximab treatment, its effect on risk of infection is not clear, as other immunosuppressants were also concurrently used. Reactions during injection are among the most common side effects of rituximab. Deep vein thrombosis (DVT), pulmonary embolism, long-term hypogammaglobulinemia, and neutropenia were also common among the patients under rituximab treatment. Regarding the excellent impact of this drug on treatment of resistant pemphigus, and also on other diseases such as idiopathic thrombocytopenic purpura (ITP), vasculitis, lymphocytic leukemia, systemic lupus erythematosus (SLE), we decided to evaluate the effects of this drug on the variation of lab parameters such as white blood cell (WBC), Hemoglobin (Hg), platelet (Plt), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (CR). So far, no study has been conducted on investigation of these variations due to receiving rituximab.

OBJECTIVES AND HYPOTHESES

Major Objective

Investigation of laboratory variations after injection of rituximab in pemphigus vulgaris patients.

Minor objectives of the project:

  • Determination of rituximab impact on laboratory indices
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of age
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of gender
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of other treatment methods
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of disease duration
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of disease involved sites
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of Underlying disease

Application objectives:

  • Enhancement of health level among pemphigus vulgaris patients and paying attention to laboratory effect of patients after rituximab consumption.

Research questions or hypotheses:

  • Rituximab affects the laboratory indices
  • Rituximab affects the laboratory indices with age effect adjustment
  • Rituximab affects the laboratory indices with gender effect adjustment
  • Rituximab affects the laboratory indices with disease duration effect adjustment
  • Rituximab affects the laboratory indices with previous treatment effect adjustment
  • Rituximab affects the laboratory indices with other disease effect adjustment
  • Rituximab affects the laboratory indices with involved sites’ effect adjustment

Chapter 2

Literature Review

In 1997, rituximab was approved by the US Food and Drug Administration (FDA) as a treatment for non-Hodgkin’s lymphoma of B-cell that was resistant to chemotherapy. After that, it was applied for treatment for other diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Wegener’s granulomatosis, idiopathic thrombocytopenic purpura (ITP), and Sjögren’s syndrome. Ten years later, its impact on the treatment of blister diseases such as pemphigus was shown [16].

In a 2006 study by Larrar et al, 2 children with autoimmune hemolytic anemia who were treated with rituximab experienced acute thrombocytopenia and neutropenia [17]. They resolved in several days, which showed that these hematologic effects are directly dependent on the toxicity of rituximab.

In a study by Chairwatanatorn et al in 2003, neutropenia following application of rituximab was tested in 53 patients [18]. All patients except one were under Hodgkin’s lymphoma treatment. Eight cases of grade 4 neutropenia were observed after 1 to 5 months of rituximab treatment (5 patients only received rituximab and 3 patients were also under additional chemotherapy); 3 patients advanced toward sepsis. Neutropenia was not related to other diseases or treatments, and was related with reduction of neutrophil precursors, except for one of the patients whose bone marrow had hypoplasia. All cases of neutropenia occurred among the patients whose polymorphonuclear neutrophils (PMN) were normally or weakly reduced [18].

In a study by Tesfa et al in 2008, neutropenia occurred 4 or more weeks after rituximab treatment in lymphoma patients [19]. However, the mechanism of how rituximab causes neutropenia is still unknown. In a retrospective study of 113 lymphoma patients under rituximab treatment (alone or along with chemotherapy), 8 patients (7%) had neutropenia. The average onset was 88 days after receiving their last dosage of rituximab. The average time interval of neutropenia was 54 days. Four of the 8 patients underwent stem cell transplantation, 3 patients had neutropenia with fever and 2 of them needed granulocyte-colony stimulating factor (G-CSF) treatment. In the patients who had neutropenia, a cessation in maturation was observed in the promyelocytes category (the same as congenital neutropenia or Kostmann disease) [19].

A study by Otrock in 2005 addressed 2 patients who had acute thrombocytopenia after receiving rituximab [20]. One of the patients had hairy cell leukemia and the other one suffered from mantle cell lymphoma. In these patients, thrombocytopenia improved without the need of any treatment after several days. The reason for this is unknown.

A study by Leo et al was conducted in 2004 for investigating the safety of rituximab [21]. In this study, the mixture of fludarabine, rituximab and cyclophosphamide was applied for treatment of follicular lymphoma. Surprisingly, severe thrombocytopenia with World Health Organization (WHO) grades 3 and 4 were observed in the patients, which resulted in the end of trial. Cytological and serological analysis was based on direct toxicity of rituximab.

Chapter 3

Investigation Method

39 Therapy resistant pemphigus patients in Razi Hospital in Tehran, who had received rituximab from 2008 to 2012 were considered for inclusion in this retrospective cohort study. Data was collected before and after rituximab treatment. The variables included WBC, Hg, Plt, AST, ALT, Urea, and Cr and age, gender, involved sites, previous therapies, underlying disease, and disease duration. Test sheets associated to before and after rituximab application, containing WBC, Hg, Plt, AST, ALT, Urea and Cr were compared.

Type of Study

This study is a retrospective cohort study conducted on the pemphigus patients resistant to therapies who had received rituximab in 2008-2012.

Studied Population

Therapy-resistant pemphigus patients who were treated with rituximab in Razi Hospital, Tehran, Iran in 2008-2012.

Inclusion Criteria

Pemphigus patients who did not respond to the initial therapies (therapy-resistant pemphigus), and then were treated with rituximab.

Exclusion Criteria

  1. Patients with no required tests before application of rituximab in their file
  2. Patients with no follow-up after receiving rituximab
  3. Patients whose first follow-up, after the last dosage of rituximab, is greater than 1 month.

Sampling Method

According to the available files, files of all the patients who had received rituximab from 2008 to 2012 were considered for inclusion.

Data Collection

The data collection tool included a checklist divided into 2 parts: one for the data before and one for the data collection after rituximab treatment. The variables included WBC, Hg, Plt, AST, ALT, Urea, and Cr and age, gender, involved sites, previous therapies, underlying disease, and disease duration.

Project Implementation

After studying the files of therapy-resistant pemphigus patients, the patients who had required data in their files were entered into the research. Rituximab treatment was defined as receiving 4 doses of 500 mg for 4 weeks, along with normal saline. Test sheets associated to before and after rituximab application, containing WBC, Hg, Plt, AST, ALT, Urea and Cr were compared. (The maximum time interval between the second test sheet and the last dosage of rituximab could be 1 month.)

Data Analysis

Finally, the finalized cases that had the inclusion criteria, were analyzed in Stata statistical software (StataCorp, Texas, USA) in terms of variations in WBC, Hg, Plt, AST, ALT, Urea and Cr after application of rituximab as the major variable and investigation of minor variables.

Problems and Limitations

As the base of this research was on filed files of hospital, inadequacy of data either before or after rituximab application excluded a bunch of samples from the study in a way that among 105 available files, only 39 files had the required data.

Variables

Major variables: quantitative measurement of white blood cells (WBC), Hemoglobin (Hgb), platelets (Plt), aspartate aminotransferase (AST), ALT, urea, and creatinine (Cr) before and after application of rituximab

Minor variables:

  1. Gender
  2. Age
  3. Involved sites
  4. Previous therapies
  5. Underlying disease
  6. Disease duration

The data of variables were collected according to the positive findings in the patients’ files (Table 1).

 

 

Table 1 – Patient variables.

Title

Variable Type

Quantitative

Qualitative

Scientific Practical Definition

Measurement Method

Scale

Independent

Dependent

Continuous

Discrete

Nominal

Ranking

WBC

Number of WBC per μ(mu)L of blood

File reading

Cell/mCl

Hgb

Amount of hemoglobin

File reading

Gr/dl

Plt

Number of plackets in patient blood

File reading

Cell/mcl

AST

*

Amount of AST

File reading

IU/L

ALT

Amount of ALT

File reading

IU/L

Urea

Microgram of urea per deciliter of blood

File reading

Mg/dl

Cr

*

Keratin amount

File reading

Mg/dl

age

*

Years from birth

File reading

year

gender

*

According to patient phenotype

File reading

Male/female

Underlying disease

*

Existence of systemic disease

File reading

Having/not having

Previous therapies

*

Received adjoin before rituximab

File reading

Azathioprine, IVIG

Cyclophosphamide,

CellCept®, methotrexate

Involved sites

*

Involved sites before starting rituximab

File reading

Upper body, lower body, face. Genitalia, sculp, mucus

Disease Duration

Months passed from onset to receiving rituximab

File reading

Month

WBC – white blood cell, Hgb – hemoglobin, Plt – platelet, AST – aspartate aminotransferase, ALT – alanine aminotransferase, CR – creatinine, IVIG – intravenous immunoglobulin

 

Chapter 4

Results

Among 105 therapy-resistant pemphigus patients who received rituximab treatment in Razi Hospital, Tehran, Iran from 2008 to 2012, only 39 patients managed to enter the study. The others were excluded due to inadequate data. Also in the included patient group, the maximum time interval between the last dosage of rituximab and follow-up was 1 month. The data of the remaining 39 patients were analyzed by Stata statistical software (StataCorp, Texas, USA) and the following results were obtained:

  • The age of the patients ranged from 16 to 67 with a mean of 36.46 years.
  • Their disease duration from the beginning of the disease until receiving rituximab ranged from 5 to 84 months with a mean of 39.30 months.
  • Of the patients, 25 (64%) were men and 14 (36%) were women. It does not seem that the sex difference is related to therapy-resistant pemphigus, it is rather associated with the data collection method and exclusion of patients with incomplete files.

Investigation of the involved sites showed that 25 patients (64%) had mucosal involvement, 20 patients (51.3%) had upper body involvement, 18 patients (46.2%) had lower body involvement, 19 people (48.7%) had genitalia involvement, 23 people had facial involvement, 36 people (92%) had body involvement, and in 22 patients (56.4%) the scalp was involved. The lab result variations of the mentioned patients were investigated in terms of the involved sites.

The patients, before application of rituximab, were simultaneously under treatment with prednisolone and other adjoins. To summarize the unsuccessful treatments, 5 patients had cyclophosphamide, 18 of them received CellCept® (mycophenolate mofetil), 7 people (17.9%) had intravenous immunoglobulin (IVIG), 5 patients were treated with methotrexate, and 22 patients had azathioprine. All these patients did not respond to corticosteroid and had active disease.

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In terms of variation in lab test results after receiving rituximab, the patients were investigated in terms of the previous adjuvants as well. Among 9 patients, 12 of them (30.8%) had systemic underlying diseases such as hypertension (HTN), diabetes mellitus (DM), Ischemic Heart Disease (IHD) and many more.

The major variables were WBC, Hgb, Plt, AST, ALT, Urea and Cr before and after application of rituximab.

Before Receiving Rituximab:

  • The WBC range was 4,000-14,800 with average of 10,092.
  • The Hgb range was 9.1-16.8 with average of 13.8.
  • The Plt range was 100,000-683,000 with an average of 243,384.
  • The AST range was 6-64 with average of 24.56.
  • The ALT range was 10-143 with average of 43.92.
  • The Urea range was 12-145 with average of 37.25.
  • The Cr range was 0.5-1.2 with average of 0.87.

After Receiving Rituximab:

  • The WBC range was 5,400-19,000 with average of 9,964.
  • The Hgb range was 7.4-16.7 with average of 13.42.
  • The Plt range was 110,000-440,000 with average of 232,512.
  • The AST range was 10-121 with average of 25.43.
  • The ALT range was 12-144 with average of 48.46.
  • The Urea range was 15-54 with average of 29.12.
  • The Cr range was 0.6-1.2 with average of 0.85.
  • The WBC had no statistically significant variations.
  • The Hgb had no statistically significant variations.
  • The Plt had no statistically significant variations.
  • The AST had no statistically significant variations.
  • The ALT had no statistically significant variations.
  • The Urea had statistically significant variations.
  • The Cr had no statistically significant variations.

After receiving rituximab and adjusting for the effect of gender:

  • The WBC had no statistically significant variations.
  • The Hgb had no statistically significant variations.
  • The Plt had no statistically significant variations.
  • The AST had no statistically significant variations.
  • The ALT had no statistically significant variations.
  • The Cr had no statistically significant variations.
  • In the case of Urea, we concluded that it depends on gender, as in men the variation was significant while in women the variations were not statistically significant.

When investigating the results with adjustment of the involved sites, the following results were obtained:

  • In patients with lower body involvement, rituximab had no significant effect on WBC, Plt, AST, ALT, Urea and Cr, but it had significant impact on Hgb reduction.
  • In patients whose lower body was not involved, Urea significantly increased after receiving rituximab.
  • In patients whose lower body was involved, rituximab caused a significant reduction in Cr, Urea, and Hgb.
  • In patients whose upper body was not involved, rituximab had no significant effect on the variables.
  • In the patients with or without facial involvement, rituximab had no significant impact on any of the variables.
  • In patients whose genitalia region was involved, rituximab has no significant impact on any of the major variables.
  • In patients with no genitalia involvement, rituximab resulted in significant reduction of urea.
  • In patients with body involvement, rituximab resulted in significant reduction of urea.
  • In patients with scalp involvement, rituximab resulted in significant reduction of urea.

The adjustment of previous therapies was also addressed. As all the patients received prednisolone, the effect of adjoins (azathioprine, CellCept®, cyclophosphamide, IVIG and methotrexate) was addressed:

  • In patients who had received cyclophosphamide, rituximab has no statistically significant impact on the major variables.
  • In patients who had not received cyclophosphamide, rituximab led to statistically significant reduction of urea.
  • In patients who had received CellCept (mycophenolate mofetil), rituximab has statistically significant impact on reduction of urea and WBC.
  • In patients who did not use IVIG adjoin, rituximab had a significant impact on reduction of urea.
  • In patients who did not use methotrexate adjoin, rituximab had significant impact on reduction of urea.
  • In patients who used azathioprine adjoin, rituximab had significant impact on reduction of urea.

The adjustment impact of systemic underlying diseases (such as HTN, DM, IHD) was also addressed.

  • In patients with systemic underlying disease, rituximab had significant impact on platelet reduction.
  • In patients with no systemic underlying disease, rituximab had significant impact on urea reduction.

There was no statistically significant relationship between the lab test result variations and disease duration and age (Table 1 through Table 8).

 

TABLES

Table 1 – Age distribution in the studied patients

Min

Max

Standard Deviation

Average

Age

16

67

13.48

36.48

 

Table 2 – Disease duration distribution in the studies patients

Min

Max

Standard Deviation

Average

Disease duration

5

84

20.28

29.30

 

Table 3 – Absolute and relative frequency distribution of patients based on their gender

Number

%

Men

25

64.1

Women

14

35.9

Total

39

100

 

Table 4 – Absolute and relative frequency of involved sites at the time of rituximab injection.

Frequency

%

Upper body

20

51.3

Lower body

18

46.2

Face

23

59

Genitalia

19

48.7

Body

36

92.3

Mucus

25

64.1

Scalp

22

56.4

 

Table 5 – Absolute and relative frequency of received adjoins before application of rituximab

Frequency

%

cyclophosphamide

5

12.8

CellCept®

18

46.2

IVIG

7

17.9

methotrexate

5

12.8

azathioprine

22

56.4

IVIG – intravenous immunoglobulin

 

Table 6 – Absolute and relative frequency of the patients based on having or not having underlying disease.

Frequency

Part 3: Clinical Research 2

INTRODUCTION

Pemphigus is the name of a group of life-threatening blistering diseases of the skin and mucous membranes. The base of treatment for this disease is corticosteroids; however, recently, new drugs, such as rituximab, have been verified for more severe forms of it.

In the author’s previously unpublished study, the effect of rituximab on variation in the laboratory indices of pemphigus vulgaris patients is addressed. After investigation of the files of pemphigus patients who received rituximab in Razi Hospital, Tehran, Iran from 2008 to 2013, 39 patients were entered into the study. All patients had lab sheets containing CR (creatinine), urea, ALT (alanine aminotransferase), AST (aspartate aminotransferase), Plt (platelet), Hgb (hemoglobin), and WBC (white blood cell) before and after receiving rituximab. The patients received rituximab 4 times at a dosage of 500 mg in 4 successive weeks. The lab results before receiving the first dose of rituximab were compared to the results after receiving treatment. The effect of rituximab on the variation in lab indices with the adjustment effect of age, gender, disease duration, sites of involvement, received adjoins, and the background disease were also investigated.

In the initial analysis, rituximab only had a significant effect on urea reduction. In the CellCept® (mycophenolate mofetil) receiving subgroup, the mixed consumption of rituximab led to a significant reduction in WBC. In the subgroup having background disease, rituximab had a statistically significant impact on platelet reduction. In the subgroup having no background disease, rituximab had a statistically significant effect on urea reduction.

The lab indices were shown to have no significant relationship with age and disease duration. Thus, it can be predicted that disease duration and age would have no effect in the relationship between rituximab and lab indices’ variations.

Although in stratified single-variable analysis for adjusting the effect of other variables (involvement sites and received adjoins) on the relation of rituximab and lab indices, some of these variables showed interacting effects with rituximab on the variations of lab indices. However, due to the low volume of sample and non-normal distribution of most of these variables, it was impossible to do multivariable analysis for investigation of their independent and interactive effects on variations of lab indices in an integrated manner, therefore, we can not make certain comments about their relationships.

Chapter 1

Pemphigus is the name of a group of life-threatening blistering diseases that have characteristic acantholysis leading to formation of intraepithelial blisters in mucus and skin [1]. The acantholysis process is induced via attachments of flowing autoantibodies to adhesion molecules in the cells [2]. Patients with pemphigus have mucosal erosions, blisters, papules, and cutaneous erosions.

The different types of pemphigus are pemphigus vulgaris, pemphigus foliaceus, immunoglobulin A (IgA) pemphigus, and paraneoplastic pemphigus. Different types of pemphigus are differentiated by clinical symptoms, related autoantigens, and histological methods. Pemphigus vulgaris has mucosal and mucocutaneous involvement. The blisters are acantholytic and suprabasal. The autoantibodies responsible for the disease are against desmoglein (DSG) 1 or both desmoglein 3 and 1.

Pemphigus foliaceus only involves the skin. The blisters are acantholytic and subcorneal. The responsible autoantibodies are against desmoglein 1.

IgA pemphigus has the form of grouped erythematous crusts, papules, and vesicle plucks. Blisters can be subcorneal or intraepithelial and acantholytic. The responsible autoantibodies are against desmocollin (DSC) 1 [3].

Paraneoplastic pemphigus involves vast and resistant stomatite along with different cutaneous findings. The responsible autoantibodies are against desmoplakin (DSP) or other desmosomal antigens. Pemphigus vulgaris is the most common type of pemphigus, but is still very rare. The chance of its occurrence is between 0.1 to 0.5 per 100,000 people [4]. Pemphigus often happens among adults and the average age of onset is 40 to 60 years old. It is very rare among children [5,6]. Its prevalence is almost the same in the 2 sexes [7]. Almost all the pemphigus vulgaris patients have mucosal involvement. The mouth is the most common site of involvement and is often the first site of involvement. Other mucosal membranes such as conjunctivae, nose, esophagus, vulva, vagina, cervix and anus are rarely involved [8]. As mucosal blisters are fragile and burst easily, in clinical examination it is difficult to find intact blisters, and instead the examiner tends to find mucosal erosions. Buccal and palatal mucosa are the most common sites of blister involvement in the mouth cavity [9].

Mucosal involvement can be very painful. This pain often increases by chewing and swallowing, which can result in improper alimentation and weight reduction. Most of the patients also have cutaneous involvement appearing in the form of soft blisters in healthy skin or erythematosus. The blisters easily break, resulting in painful erosions. Pemphigus vulgaris rarely causes pruritis. Almost any part of body skin can be involved, but the palmar aspects of the foot and hands are rarely involved. The Nikolsky sign is often observed among these patients (mechanical pressure on the healthy skin results in blistering). Pemphigus is diagnosed based on the clinical, histological, immuno-pathological symptoms and laboratory findings. Even in cases where the clinical symptoms are intensively supporting pemphigus, laboratory investigation is still needed to confirm the diagnosis, as other diseases may have the same symptoms. The first line of treatment of pemphigus is systemic corticosteroids, and addition of adjuvants may also be needed. Patients who do not respond to the first line of treatment might need additional interventions. In such patients, cyclophosphamides, rituximab, intravenous immunoglobulin (IVIG) or plasmapheresis may be helpful.

Initial treatment of pemphigus vulgaris is systemic glucocorticoid, which is often applied in combination with other non-steroidal immunosuppressants such as azathioprine and mycophenolate mofetil. Pemphigus resistant to treatment is a type of pemphigus that does not respond to the aforementioned treatments.

Pemphigus is a chronic disease that needs long-term treatment. A retrospective study was conducted during 1982-1993 on 40 patients [8]. On average, these patients achieved complete remission after 7.7 years; 25% had remission after 2 years; 50% after 5 years; and 75% after 10 years [8]. Most pemphigus vulgaris patients respond to initial treatments [9]. The first step, in the patients who do not respond to initial treatment, is increasing the dosage of systemic corticosteroids (1.5-2 mg/kg of prednisolone per day) or adjuvant drug. The adjuvant drug can also be changed (changing azathioprine to mycophenolate mofetil). In resistant cases, cyclophosphamides, rituximab, IVIG, and plasmapheresis could also be used.

As pemphigus is an auto-immune disease caused by autoantibodies, treatments that reduce B cells are investigated [10-13]. Rituximab is a monoclonal antibody that targets CD20, located on B-lymphocytes, as its antigen. This drug has been shown to have profound effects on pemphigus treatments [13,14]. In a multicenter study conducted on 14 pemphigus vulgaris patients and 7 pemphigus foliaceus patients, both groups were resistant to systemic glucocorticoids and experienced several relapses during glucocorticoid tapering. They were then put on 1 cycle of rituximab with a weekly dosage of 275 mg/m2 for 4 weeks, and this addition proved advantageous [15]. Although, severe infections were reported in the patients under rituximab treatment, its effect on risk of infection is not clear, as other immunosuppressants were also concurrently used. Reactions during injection are among the most common side effects of rituximab. Deep vein thrombosis (DVT), pulmonary embolism, long-term hypogammaglobulinemia, and neutropenia were also common among the patients under rituximab treatment. Regarding the excellent impact of this drug on treatment of resistant pemphigus, and also on other diseases such as idiopathic thrombocytopenic purpura (ITP), vasculitis, lymphocytic leukemia, systemic lupus erythematosus (SLE), we decided to evaluate the effects of this drug on the variation of lab parameters such as white blood cell (WBC), Hemoglobin (Hg), platelet (Plt), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (CR). So far, no study has been conducted on investigation of these variations due to receiving rituximab.

OBJECTIVES AND HYPOTHESES

Major Objective

Investigation of laboratory variations after injection of rituximab in pemphigus vulgaris patients.

Minor objectives of the project:

  • Determination of rituximab impact on laboratory indices
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of age
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of gender
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of other treatment methods
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of disease duration
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of disease involved sites
  • Determination of rituximab impact on laboratory indices by adjusting for the effect of Underlying disease

Application objectives:

  • Enhancement of health level among pemphigus vulgaris patients and paying attention to laboratory effect of patients after rituximab consumption.

Research questions or hypotheses:

  • Rituximab affects the laboratory indices
  • Rituximab affects the laboratory indices with age effect adjustment
  • Rituximab affects the laboratory indices with gender effect adjustment
  • Rituximab affects the laboratory indices with disease duration effect adjustment
  • Rituximab affects the laboratory indices with previous treatment effect adjustment
  • Rituximab affects the laboratory indices with other disease effect adjustment
  • Rituximab affects the laboratory indices with involved sites’ effect adjustment

Chapter 2

Literature Review

In 1997, rituximab was approved by the US Food and Drug Administration (FDA) as a treatment for non-Hodgkin’s lymphoma of B-cell that was resistant to chemotherapy. After that, it was applied for treatment for other diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Wegener’s granulomatosis, idiopathic thrombocytopenic purpura (ITP), and Sjögren’s syndrome. Ten years later, its impact on the treatment of blister diseases such as pemphigus was shown [16].

In a 2006 study by Larrar et al, 2 children with autoimmune hemolytic anemia who were treated with rituximab experienced acute thrombocytopenia and neutropenia [17]. They resolved in several days, which showed that these hematologic effects are directly dependent on the toxicity of rituximab.

In a study by Chairwatanatorn et al in 2003, neutropenia following application of rituximab was tested in 53 patients [18]. All patients except one were under Hodgkin’s lymphoma treatment. Eight cases of grade 4 neutropenia were observed after 1 to 5 months of rituximab treatment (5 patients only received rituximab and 3 patients were also under additional chemotherapy); 3 patients advanced toward sepsis. Neutropenia was not related to other diseases or treatments, and was related with reduction of neutrophil precursors, except for one of the patients whose bone marrow had hypoplasia. All cases of neutropenia occurred among the patients whose polymorphonuclear neutrophils (PMN) were normally or weakly reduced [18].

In a study by Tesfa et al in 2008, neutropenia occurred 4 or more weeks after rituximab treatment in lymphoma patients [19]. However, the mechanism of how rituximab causes neutropenia is still unknown. In a retrospective study of 113 lymphoma patients under rituximab treatment (alone or along with chemotherapy), 8 patients (7%) had neutropenia. The average onset was 88 days after receiving their last dosage of rituximab. The average time interval of neutropenia was 54 days. Four of the 8 patients underwent stem cell transplantation, 3 patients had neutropenia with fever and 2 of them needed granulocyte-colony stimulating factor (G-CSF) treatment. In the patients who had neutropenia, a cessation in maturation was observed in the promyelocytes category (the same as congenital neutropenia or Kostmann disease) [19].

A study by Otrock in 2005 addressed 2 patients who had acute thrombocytopenia after receiving rituximab [20]. One of the patients had hairy cell leukemia and the other one suffered from mantle cell lymphoma. In these patients, thrombocytopenia improved without the need of any treatment after several days. The reason for this is unknown.

A study by Leo et al was conducted in 2004 for investigating the safety of rituximab [21]. In this study, the mixture of fludarabine, rituximab and cyclophosphamide was applied for treatment of follicular lymphoma. Surprisingly, severe thrombocytopenia with World Health Organization (WHO) grades 3 and 4 were observed in the patients, which resulted in the end of trial. Cytological and serological analysis was based on direct toxicity of rituximab.

Chapter 3

Investigation Method

39 Therapy resistant pemphigus patients in Razi Hospital in Tehran, who had received rituximab from 2008 to 2012 were considered for inclusion in this retrospective cohort study. Data was collected before and after rituximab treatment. The variables included WBC, Hg, Plt, AST, ALT, Urea, and Cr and age, gender, involved sites, previous therapies, underlying disease, and disease duration. Test sheets associated to before and after rituximab application, containing WBC, Hg, Plt, AST, ALT, Urea and Cr were compared.

Type of Study

This study is a retrospective cohort study conducted on the pemphigus patients resistant to therapies who had received rituximab in 2008-2012.

Studied Population

Therapy-resistant pemphigus patients who were treated with rituximab in Razi Hospital, Tehran, Iran in 2008-2012.

Inclusion Criteria

Pemphigus patients who did not respond to the initial therapies (therapy-resistant pemphigus), and then were treated with rituximab.

Exclusion Criteria

  1. Patients with no required tests before application of rituximab in their file
  2. Patients with no follow-up after receiving rituximab
  3. Patients whose first follow-up, after the last dosage of rituximab, is greater than 1 month.

Sampling Method

According to the available files, files of all the patients who had received rituximab from 2008 to 2012 were considered for inclusion.

Data Collection

The data collection tool included a checklist divided into 2 parts: one for the data before and one for the data collection after rituximab treatment. The variables included WBC, Hg, Plt, AST, ALT, Urea, and Cr and age, gender, involved sites, previous therapies, underlying disease, and disease duration.

Project Implementation

After studying the files of therapy-resistant pemphigus patients, the patients who had required data in their files were entered into the research. Rituximab treatment was defined as receiving 4 doses of 500 mg for 4 weeks, along with normal saline. Test sheets associated to before and after rituximab application, containing WBC, Hg, Plt, AST, ALT, Urea and Cr were compared. (The maximum time interval between the second test sheet and the last dosage of rituximab could be 1 month.)

Data Analysis

Finally, the finalized cases that had the inclusion criteria, were analyzed in Stata statistical software (StataCorp, Texas, USA) in terms of variations in WBC, Hg, Plt, AST, ALT, Urea and Cr after application of rituximab as the major variable and investigation of minor variables.

Problems and Limitations

As the base of this research was on filed files of hospital, inadequacy of data either before or after rituximab application excluded a bunch of samples from the study in a way that among 105 available files, only 39 files had the required data.

Variables

Major variables: quantitative measurement of white blood cells (WBC), Hemoglobin (Hgb), platelets (Plt), aspartate aminotransferase (AST), ALT, urea, and creatinine (Cr) before and after application of rituximab

Minor variables:

  1. Gender
  2. Age
  3. Involved sites
  4. Previous therapies
  5. Underlying disease
  6. Disease duration

The data of variables were collected according to the positive findings in the patients’ files (Table 1).

 

 

Table 1 – Patient variables.

Title

Variable Type

Quantitative

Qualitative

Scientific Practical Definition

Measurement Method

Scale

Independent

Dependent

Continuous

Discrete

Nominal

Ranking

WBC

Number of WBC per μ(mu)L of blood

File reading

Cell/mCl

Hgb

Amount of hemoglobin

File reading

Gr/dl

Plt

Number of plackets in patient blood

File reading

Cell/mcl

AST

*

Amount of AST

File reading

IU/L

ALT

Amount of ALT

File reading

IU/L

Urea

Microgram of urea per deciliter of blood

File reading

Mg/dl

Cr

*

Keratin amount

File reading

Mg/dl

age

*

Years from birth

File reading

year

gender

*

According to patient phenotype

File reading

Male/female

Underlying disease

*

Existence of systemic disease

File reading

Having/not having

Previous therapies

*

Received adjoin before rituximab

File reading

Azathioprine, IVIG

Cyclophosphamide,

CellCept®, methotrexate

Involved sites

*

Involved sites before starting rituximab

File reading

Upper body, lower body, face. Genitalia, sculp, mucus

Disease Duration

Months passed from onset to receiving rituximab

File reading

Month

WBC – white blood cell, Hgb – hemoglobin, Plt – platelet, AST – aspartate aminotransferase, ALT – alanine aminotransferase, CR – creatinine, IVIG – intravenous immunoglobulin

 

Chapter 4

Results

Among 105 therapy-resistant pemphigus patients who received rituximab treatment in Razi Hospital, Tehran, Iran from 2008 to 2012, only 39 patients managed to enter the study. The others were excluded due to inadequate data. Also in the included patient group, the maximum time interval between the last dosage of rituximab and follow-up was 1 month. The data of the remaining 39 patients were analyzed by Stata statistical software (StataCorp, Texas, USA) and the following results were obtained:

  • The age of the patients ranged from 16 to 67 with a mean of 36.46 years.
  • Their disease duration from the beginning of the disease until receiving rituximab ranged from 5 to 84 months with a mean of 39.30 months.
  • Of the patients, 25 (64%) were men and 14 (36%) were women. It does not seem that the sex difference is related to therapy-resistant pemphigus, it is rather associated with the data collection method and exclusion of patients with incomplete files.

Investigation of the involved sites showed that 25 patients (64%) had mucosal involvement, 20 patients (51.3%) had upper body involvement, 18 patients (46.2%) had lower body involvement, 19 people (48.7%) had genitalia involvement, 23 people had facial involvement, 36 people (92%) had body involvement, and in 22 patients (56.4%) the scalp was involved. The lab result variations of the mentioned patients were investigated in terms of the involved sites.

The patients, before application of rituximab, were simultaneously under treatment with prednisolone and other adjoins. To summarize the unsuccessful treatments, 5 patients had cyclophosphamide, 18 of them received CellCept® (mycophenolate mofetil), 7 people (17.9%) had intravenous immunoglobulin (IVIG), 5 patients were treated with methotrexate, and 22 patients had azathioprine. All these patients did not respond to corticosteroid and had active disease.

In terms of variation in lab test results after receiving rituximab, the patients were investigated in terms of the previous adjuvants as well. Among 9 patients, 12 of them (30.8%) had systemic underlying diseases such as hypertension (HTN), diabetes mellitus (DM), Ischemic Heart Disease (IHD) and many more.

The major variables were WBC, Hgb, Plt, AST, ALT, Urea and Cr before and after application of rituximab.

Before Receiving Rituximab:

  • The WBC range was 4,000-14,800 with average of 10,092.
  • The Hgb range was 9.1-16.8 with average of 13.8.
  • The Plt range was 100,000-683,000 with an average of 243,384.
  • The AST range was 6-64 with average of 24.56.
  • The ALT range was 10-143 with average of 43.92.
  • The Urea range was 12-145 with average of 37.25.
  • The Cr range was 0.5-1.2 with average of 0.87.

After Receiving Rituximab:

  • The WBC range was 5,400-19,000 with average of 9,964.
  • The Hgb range was 7.4-16.7 with average of 13.42.
  • The Plt range was 110,000-440,000 with average of 232,512.
  • The AST range was 10-121 with average of 25.43.
  • The ALT range was 12-144 with average of 48.46.
  • The Urea range was 15-54 with average of 29.12.
  • The Cr range was 0.6-1.2 with average of 0.85.
  • The WBC had no statistically significant variations.
  • The Hgb had no statistically significant variations.
  • The Plt had no statistically significant variations.
  • The AST had no statistically significant variations.
  • The ALT had no statistically significant variations.
  • The Urea had statistically significant variations.
  • The Cr had no statistically significant variations.

After receiving rituximab and adjusting for the effect of gender:

  • The WBC had no statistically significant variations.
  • The Hgb had no statistically significant variations.
  • The Plt had no statistically significant variations.
  • The AST had no statistically significant variations.
  • The ALT had no statistically significant variations.
  • The Cr had no statistically significant variations.
  • In the case of Urea, we concluded that it depends on gender, as in men the variation was significant while in women the variations were not statistically significant.

When investigating the results with adjustment of the involved sites, the following results were obtained:

  • In patients with lower body involvement, rituximab had no significant effect on WBC, Plt, AST, ALT, Urea and Cr, but it had significant impact on Hgb reduction.
  • In patients whose lower body was not involved, Urea significantly increased after receiving rituximab.
  • In patients whose lower body was involved, rituximab caused a significant reduction in Cr, Urea, and Hgb.
  • In patients whose upper body was not involved, rituximab had no significant effect on the variables.
  • In the patients with or without facial involvement, rituximab had no significant impact on any of the variables.
  • In patients whose genitalia region was involved, rituximab has no significant impact on any of the major variables.
  • In patients with no genitalia involvement, rituximab resulted in significant reduction of urea.
  • In patients with body involvement, rituximab resulted in significant reduction of urea.
  • In patients with scalp involvement, rituximab resulted in significant reduction of urea.

The adjustment of previous therapies was also addressed. As all the patients received prednisolone, the effect of adjoins (azathioprine, CellCept®, cyclophosphamide, IVIG and methotrexate) was addressed:

  • In patients who had received cyclophosphamide, rituximab has no statistically significant impact on the major variables.
  • In patients who had not received cyclophosphamide, rituximab led to statistically significant reduction of urea.
  • In patients who had received CellCept (mycophenolate mofetil), rituximab has statistically significant impact on reduction of urea and WBC.
  • In patients who did not use IVIG adjoin, rituximab had a significant impact on reduction of urea.
  • In patients who did not use methotrexate adjoin, rituximab had significant impact on reduction of urea.
  • In patients who used azathioprine adjoin, rituximab had significant impact on reduction of urea.

The adjustment impact of systemic underlying diseases (such as HTN, DM, IHD) was also addressed.

  • In patients with systemic underlying disease, rituximab had significant impact on platelet reduction.
  • In patients with no systemic underlying disease, rituximab had significant impact on urea reduction.

There was no statistically significant relationship between the lab test result variations and disease duration and age (Table 1 through Table 8).

 

TABLES

Table 1 – Age distribution in the studied patients

Min

Max

Standard Deviation

Average

Age

16

67

13.48

36.48

 

Table 2 – Disease duration distribution in the studies patients

Min

Max

Standard Deviation

Average

Disease duration

5

84

20.28

29.30

 

Table 3 – Absolute and relative frequency distribution of patients based on their gender

Number

%

Men

25

64.1

Women

14

35.9

Total

39

100

 

Table 4 – Absolute and relative frequency of involved sites at the time of rituximab injection.

Frequency

%

Upper body

20

51.3

Lower body

18

46.2

Face

23

59

Genitalia

19

48.7

Body

36

92.3

Mucus

25

64.1

Scalp

22

56.4

 

Table 5 – Absolute and relative frequency of received adjoins before application of rituximab

Frequency

%

cyclophosphamide

5

12.8

CellCept®

18

46.2

IVIG

7

17.9

methotrexate

5

12.8

azathioprine

22

56.4

IVIG – intravenous immunoglobulin

 

Table 6 – Absolute and relative frequency of the patients based on having or not having underlying disease.

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