Factors associated with Gall Bladder

cancer in Bihar-A case control study

By

Dr. Varsha Singh

(MAE- FETP Scholar 2004-2006)

NATIONAL INSTITUTE OF EPIDEMIOLOGY

(Indian Council of Medical Research)

Mayor VR Ramanathan Road, Chetpet, Chennai, 600031

Factors associated with Gall Bladder

cancer in Bihar-A case control study By

Dr. Varsha Singh

(MAE- FETP Scholar 2004-2006)

Dissertation project.

Submitted in partial fulfillment of the requirements for the degree of

Master of Applied Epidemiology (M.A.E) of

Sree Chitra Tirunal Institute for Medical Sciences and

Technology,

Thiruvananthapuram Kerala-695 011.

This workhas been done as part of the two year Field Epidemidlogy

Training Programme (FETP) conducted at

National Institute of Epidemiology,

(Indian Council of Medical Research),

Mayor V .R. Ramanathan Road, Chennai -600 031.

January 2006

11

CERTIFICATION

This is to certify that this dissertation, entitled "Factors associated with Gall

Bladder cancer in Bihar -A Case control study" submitted by.

Dr Varsha Singh in partial fulfillment of the requirements for the degree of

Master of ApJ?lied Epidemiology is the original work done by her and has

not been submitted earlier in part or whole for any other (Publication or

degree) purpose.

Date Director

lll

Table of contents

List of tables

Acknowledgements

Abstract

l.Introduction

2.Rationale for study

3.0bjectives

4.Review of literature

4.1.Introduction

4.2.Incidence and prevalence

4.3.Age & sex distribution

4.4.Histopathol~gy

4.5.Molecular pathology

4.6.Genetic factors

4.7.Staging

4.8.TNM Classification

4.9.Risk factors

4.10.Prevention and control of Gall bladder cancer

4.11.Conclusion

5.Methodology

5.1.Study' area

5.2.Study population

5.3 Study design

5.4.0perational definitions

5.5.Study team

5.6. Sample size

5.7. Data collection techniques and tools

5.8.Ethical issues

6.Results.

6.1.study population

Page

number

vi

vii

8

9

10

10

11

12

12

13

13

14

14

16

16

17

24

25

26

26

26

26

26

26

27

27

28

29

33

iv

6.2.sociodemographic factors of cases and controls 29

6.3.stratified analysis 31

6.4.Dose response 31

6.5.Multivariate analysis 32

7. Discussion 33

7.1. Incidence of gall bladder cancer in Bihar and associated factors 36

7.2.Causality criteria 36

7.3.Errors and biases 37

8.Conclusion 38

9.Recommendation 38

10.references 39

List of abbreviation 55

Annexure 56

v

List of tables Page Number

Tablel.Univariate analysis for all cases and controls, Mahavir Cancer 45 Sansthan, Patna, Bihar, India

Table 2. Univariate analysis for cases with gall stones and controls, 46 Mahavir Cancer Sansthan, Patna, Bihar, India

Table 3. Univariate analysis for cases without gall stones and controls, 47 Mahavir Cancer Sansthan, Patna, Bihar, India

Table 4.Association of missing breakfast to gall bladder cancer 48

stratified by gender,Mahavir Cancer Sansthan, Patna, Bihar, India

Table 5. Association of missing breakfast to gall bladder cancer 49

stratified by age,Mahavir Cancer Sansthan, Patna, Bihar, India

Table 6. Association of reuse of oil to gall bladder cancer stratified by 50

gender,Mahavir Cancer Sansthan, Patna, Bihar, India

Table7. Associatjon of reuse of oil to gall bladder cancer stratified by 51

age ,Mahavir Cancer Sansthan, Patna, Bihar, India

Table 8. Association of gall bladder cancer to frequency of missing 52

breakfast ,Mahavir Cancer Sansthan, Patna, Bihar, India

Table 9.Association of gall bladder cancer to frequency of reuse of oil 53

Mahavir Cancer Sansthan, Patna, Bihar, India

Table 1 O.Logistic regression analysis of factors associated with gall 54

bladder cancer Mahavir Cancer Sansthan, Patna, Bihar, India

Vl

I i ...

I ACKNOWLEDGEMENT

Several dignitaries and institutions have extended their valuable time, advice and

assistance to me during preparation of this thesis. I extend with gratitude my sincere

thanks to:

Prof. M.D. Gupte, Director National Institute of Epidemiology (NIE), Chennai for his

valuable guidance amidst his very tight schedule.

· Prof. K. Ramachandran, Formerly Professor and Head ofthe Department of Biostatistics,

All India Institute of M_edical Sciences, New Delhi and presently Adviser to DG ICMR

for Field Epide_n;iology Training Programme at NIE,. Chennai for his valuable teaching

comments suggestions and advice.

Dr. Manoj Murerkar,Deputy Director ,NIE and MAE-FETP course-coordinator for his

close guidance and encouragement

Dr R Ramakrishnan, Assistant Director, NIE, my preceptor for his valuable guidance and

encouragement.

Dr. Vidya Ramchandran, Assistant Director, NIE and Dr P Manickam Research Officer

for their advice. and Mr. S. Satish librarian and Uma Manoharan Secretary to the FETP

for their constant support and guidance.

Dr. J.K.Singh, Director, Mahavir Cancer Sansthan and Dr.C.Khandelwal who were my

local preceptors for their constant support and guidance

My special thanks to Dr Yvan Hutin Resident advisor WHO to NIE for his guidance and

boost up for work

My family especially my husband Dr. Sunil Kumar Singh for bearing with me in this

endeavor of hard work with patience.

Last but not the least all the respondents who very graciously spared me their valuable

time and information in addition to extending their cooperation and generous hospitality,

which rendered the entire research, endeavor a very memorable, pleasant and profitable

expenence.

'' ~. 2-o-06 Date

\/~~ Dr. Varsha Singh

Vll

Abstract

Background The annual incidence of gall bladder cancer in northern India (1 0 per 1 00,000) is one of the highest of the world. While studies elsewhere suggest that dietary factors are important, no data are available from Bihar to propose prevention activities. Methods We conducted a case-control study in a tertiary reference cancer treatment center in Patna, Bihar. We recruited incident, histopathologically-diagnosed cases of gall bladder cancer and selected controls among incident, histopathologically-proven malignancies of equivalent severity, including lung, leukemia and lymphoma between May and September 2005. We compared cases and controls with respect to demographic characteristics, education and dietary practices using odds ratios in univariate and multivariate analysis. Results In multivariate analysis the people of age more than 40 years (odds ratio [OR]: 2.82, 95% confidence interval [CI]: 1.44-5.56), female (OR: 7.04, 95% CI: 3.92-12.64), illiterate (OR: 2.67, 95% CI: 1.49-4.80), and missing breakfast (OR: 3.4, 95% CI: 1.74-6.77) were found to be at greater risk of getting gall bladder cancer. Conclusion Skipping breakfast may increase the risk of cancer gall bladder in Bihar. Nutritional interventions should not only focus on the type and quantity of nutrients but also emphasize the importance of eating regularly.

Key words: Gall bladder cancer, dietary factors,risk factor studies.

8

Factors associated with gall bladder cancer in Bihar-A hospital Based

case control study

Introduction

Gall bladder carcinoma was first described in 1777, even after two centuries of its first

description; the tumor remains characterized by an unfavorable prognosis due to silent

progression of the clinical course and limited knowledge of its etiology and poor

scientific capability for epidemiological forecasting. The incidence of gallbladder cancer

varies in different parts of the world. Overall incidence ranges from 0.11100000 to

10.6/100000 in different cancer registries worldwide. 1 The prognosis is poor-only about

a 32 percent fiv€.~year survival rate for lesions confined to the gallbladder mucosa and a

10 percent one-year survival rate for more advanced stages2. The highest mortality rates

have been reported among Chilean Mapuche Indians and Hispanics, among Bolivians,

North American Indians, and Mexican Americans? There is no conclusive evidence that

dietary factors ac! through cholelithiasis only. Increased parity, early age at first child

have also been implicated as one of the risk factors in some of the studies. 3 Vegetables

and fruits have protective effect and there has been some suggestion of inverse

association with fiber intake, vitamin C and vitamin E that seems to ho.ld true for many

other cancers. 3

In India incidence varies in the different registries. The incidence in northern Indian

cities is much higher than the southern Indian cities. In Delhi and Bhopal the incidence is . 7 and 1.6 per 100,000 for male and 10.6 and 4 per 100,000 for females. 1 In Chennai and

Bangalore the incidence is 0.5 and 0.6 per 100,000 for male and 0.8 and 0.7 per 100,000

for females. 1 Its incidence has said to be increasing in the Ganges delta. The average cost

in the diagnosis and treatment of one patient exceeds Rs.l 00, 0001= in Indian currency. In

spite of large number of gall bladder cancer patients in Bihar it has not been reported to

International Research Agencies due to lack of authentic data (no population based

cancer registry, no epidemiologic studies done on cancer gall bladder in Bihar so far). In

Other states Uttar Pradesh, New Delhi and Kerala some studies has been done on risk

factors for cancer gall bladder but no screening, control or prevention activities are being

9

done .. Mortality data on cancer gall bladder in India is not available.

Incidence of cancer gall bladder in Bihar is high. In Bihar Hospital based cancer registry

data indicates it is the third most common malignancy among the females after carcinoma

cervix and carcinoma breast. Relative frequency of this cancer among all female cancers

in a cancer hospital is 9.6% in Bihar. 1 An increase in incidence is observed for cancer gall

bladder as compared to other common cancers. May be due to improved imaging

techniques we are able to diagnose more cases. In spite of large number of cancer gall

bladder patients in Bihar it has not been reported to International Research Agencies

because of inefficient evidence (no population based cancer registry, no epidemiologic

studies done on cancer gall bladder in Bihar so far). There is a need for studies on factors

associated with gall bladder cancer in Bihar.

3. Objective

1. To measure strength of association between selected risk factors and gall bladder

cancer

2. To gener;;tte hypothesis on basis ofthe available information.

3. To suggest further prospective studies concerning risk factors for carcinoma gall

bladder in Bihar.

10

Factors associated with Gall bladder cancer in Bihar-A hospital Based

case control study

1. Introduction

Gall bladder carcinoma was first described in 1777, even after two centuries of its first

description; the tumor remains characterized by an unfavorable prognosis due to silent

progression of the clinical course and limited knowledge of its etiology and poor

scientific capability for epidemiological forecasting. The incidence of gallbladder cancer

varies in different parts of the world. Overall incidence ranges from 0.1/100000 to

10.6/100000 in different cancer registries worldwide. 1 The prognosis is poor-only about

a 32 percent five-year survival rate for lesions confined to the gallbladder mucosa and a

10 percent one-year survival rate for more advanced stages2 . The highest mortality rates

have been reported among Chilean Mapuche Indians and Hispanics, among Bolivians,

North American Indians, and Mexican Americans.2 There is no conclusive evidence that

dietary factors act through cholelithiasis only. Increased parity, early age at first child

have also been implicated as one of the risk factors in some of the studies. 3 Vegetables

and fruits have protective effect and there has been some suggestion of inverse

association with fiber intake, vitamin C and vitamin E that seems to hold true for many

other cancers.3

In India incidence varies in the different registries. The incidence in northern Indian

cities is much higher than the southern Indian cities. In Delhi and Bhopal the incidence is

7 and 1.6 per 100,000 for male and 10.6 and 4 per 100,000 for females. 1 In Chennai and

Bangalore the incidence is 0.5 and 0.6 per 100,000 for male and 0.8 and 0.7 per 100,000

for females. 1 Its incidence has said to be increasing in the Ganges delta. The average cost

in the diagnosis and treatment of one patient exceeds Rs.1 00, 0001= in Indian currency. In

spite of large number of gall bladder cancer patients in Bihar it has not been reported to

International Research Agencies due to lack of authentic data (no population based

cancer registry, no epidemiologic studies done on cancer gall bladder in Bihar so far). In

Other states Uttar Pradesh, New Delhi and Kerala some studies has been done on risk

factors for cancer gall bladder but no screening, control or prevention activities are being

9

done .. Mortality data on cancer gall bladder in India is not available.

2. Rationale (or Study

Incidence of cancer gall bladder in Bihar is high. In Bihar Hospital based cancer registry

data indicates it is the third most common malignancy among the females after carcinoma

cervix and carcinoma breast. Relative frequency of this cancer among all female cancers

in a cancer hospital is 9.6% in Bihar. 1 An increase in incidence is observed for cancer gall

bladder as compared to other common cancers. May be due to improved imaging

techniques we are able to diagnose more cases. In spite of large number of cancer gall

bladder patients in Bihar it has not been reported to International Research Agencies

because of inefficient evidence (no population based cancer registry, no epidemiologic

studies done on cancer gall bladder in Bihar so far). There is a need for studies on factors

associated with gall bladder cancer in Bihar. '

3. Objective ·

1. To measure strength of association between selected risk factors and gall bladder

cancer

2. To generate hypothesis on basis of the available information.

3. To suggest further prospective studies concerning risk factors for carcinoma gall

bladder in Bihar.

10

4.3. Age & sex distribution

Gradiser and Kelly reported average age of gall bladder cancer to be 70 years ranging

from 52-86 years.Perpetuo et al (1978) reported peak incidence in 7th and 8th decade of

life. Guo et al (!981) found peak incidence in ih and 8th decade of life.Maringhini et al

(1987) in an epidemiological study in Rochester, found median age of cholelithiasis to be

63 years among men and 57 years among women while median age for cancer gall

bladder was 79.6 years and 72.3 years among women. In India Shukla et al reported mean

age of the patient to be 50 years (range-40-60 years) Khandelwal et al reported it to be

33-70 years.

Carcinoma of the gallbladder is predominantly a disease of elderly females. Of 2998

patients from 51 series reported over last 20 years all over the world there were 2292

females and 706 males. A female to male ratio of 3.2: 1. Shukla et al (1985) in their series

of 315 patients found a female to male ratio of 2.5: l.Khandelwal et al in their series of

313 patients found a male to female ratio of (1 :2~9).In Males the highest incidence rates . are reported from La Paz,Bolivia(7.5 /100,000). And Quito, Equador(4.1/100,000).The

highest female to male ratio is reported from porto Alegre ,Brazil(4.69),Israel(3.6) and

among Hispanics in Central Califomia(3.0).The female predominance is less marked in

Nagasaki,Japan,UK and among blacks in US.

4.4. Histopathology

Over 90 percent of gallbladder carcinomas are adenocarcinoma?425 On gross

examination, approximately 10 to ) 7 percent of the gallbladder carcinomas cannot be

identified with certainty,24 and their macroscopic findings are similar to those of chronic

cholecystitis. Gallstones are found in almost all cases of gallbladder cancer (78 percent to

85 percent).82226 Most carcinomas (60percent) originate in the fundus ofthe gallbladder,

30 percent in the'body, and 10 percent in the neck.22 The prevalence of gallbladder cancer

associated with diffuse calcification ofthe gallbladder (so-called porcelain gallbladder) is

12 to 21 percent.26 Most gallbladder cancers are well-to-moderately differentiated

adeno-carcinomas. Some of these are papillary lesions that grow predominantly into the

lumen of the gallbladder.

13

4.5. Molecular pathology

Dysplasia and carcinoma in situ precede most gallbladder carcinomas. However, there is

indirect evidence that progression could occur from precursor lesions to infiltrating

carcinoma. Dysplasia and carcinoma in situ are found in the mucosa adjacent to most

carcinomas of the gallbladder, sometimes separated by histologically normal

. h 1" 26 ep1t e mm.

' Patients with dysplasia and carcinoma in situ are 15 and 5 years younger, respectively,

than those with invasive carcinoma. If multiple sections of gallbladders removed for

cholelithiasis are examined, dysplasia and carcinoma in situ are detected in 13.5 percent

and 3.5 percent of the cases, respectively. There is consensus that if dysplasia and

carcinoma in situ are found, multiple additional sections of the gallbladder should be

examined to rule out invasive cancer.

4.6. Genetic factors

Studies on genetic changes involved in gallbladder carcinogenesis are limited 35 . Most of

these studies have focused at-.ras, 36•. TP53, and. p16111k4/CDKN2 gene abnormalities and

deletions ("loss dfheterozygosis").37

The reported prevalence rates of ras gene mutations in series of gallbladder carcinomas

are quite variable. While ras mutations were not detected in some small series, two other

groups reported greater of K-ras mutations in 39 to 59 percent of patients. All the

mutations occurred at codon 12 of the K-ras gene. However, this site has been the most

intensively analyzed ras gene region. A greater frequency (50 to 83 percent) of K-ras

gene mutations has been reported in gallbladder carcinomas from patients having

anomalous junction of the pancreatico-biliary duct suggesting that reflux of pancreatic

juice might contribute to the carcinogenic process.

TP53 gene abnormalities are frequent in gallbladder cancers. Although the frequency of

p53 immunostaining in gallbladder carcinoma varies widely (ranging from 35 to 92

percent), two thirds of the studies show a frequency greater than 50 percent. Most of the

TP53 mutation studies on gallbladder carcinoma have confirmed the immuno-

14

percent), two thirds of the studies show a frequency greater than 50 percent. Most of the

TP53 mutation · studies on gallbladder carcinoma have confirmed the immuno­

histochemical findings. Analyses of exon 5 to 8 of TP53 have demonstrated point

mutations in 31 to 70 percent of gallbladder carcinomas, and no particular "hot spot" has

been identified.

In addition, deletions at the TP53 locus (17p13) have been frequently (58 to 92 percent)

reported in gallbladder carcinomas, indicating that the inactivation of the TP53 plays an

important role in the pathogenesis of this neoplasm. Yokoyama et al compared the TP 53

mutations in gallbladder carcinomas from two high-prevalence areas, Japan and Chile. No

differences in the frequencies of TP53 mutations between both groups were detected.

However, mutations from Japanese cases comprised transversions in 31 percent of cases . with 46 percent of all mutations taking place at the A:T pair. The exact sequence of

molecular changes that lead to neoplastic transformation in the gallbladder epithelium

remains uncertain. More detailed understanding of the earliest molecular abnormalities

may eventually provide methods for risk assessment and early detection of gallbladder

carcinoma. The excess accumulation of p53 protein in gallbladder dysplasia (0 to 32

percent) and carcinoma in situ lesions ( 45 to 86 percent) suggests that TP 53 abnormality

is an early event. The presence of deletions at the TP53 locus in histologically normal

epithelium near gallbladder carcinoma, and of TP53 muta-tions in precursor lesions,

(Miquel et al., in preparation), indicate an early and important role of TP53 inactivation.

CDKN2 region (9p21) have been reported in half of gallbladder cancers. However, there

are no comprehensive studies at present on the CDKN2 gene status and the mechanism of

inactivation in gallbladder carcinoma, including deletion, mutation, methylation,

homozygous deletions, and protein expression analysis.

Other early genetic changes include loss of heterozygosity at 9p21 (CDKN2 gene) and

18q21 (DCC gene) regions. Data regardingK-ras gene mutations in precursor lesions are

controversial. While some studies fail to demonstrate K-ras mutation in precursor

gallbladder lesions others report a relatively high prevalence (22 to 44 percent) in

precursor lesions accompanying invasive tumors. 39, 40

15

4. 7. Staging

Stage I cancer is limited to the mucosa; Stage II to the muscular layer; and Stage III to the

peri-muscular layer. Stage IV shows metastases in the lymph nodes; and Stage V has

hepatic or other sJistant metastases. There is a correlation between level of tumor invasion

in the gallbladder wall and the presence of lymph node metastases. 28

Patients with localized stage disease have much better survival rates (Stages I to III, five­

year survival rate of 41.9 percent) than those with regional (Stage IV, 3.8 percent) or

distant (Stage V, 0.7 percent) metastasis. Similar results have been obtained using the

TNM staging system of the International Union against Cancer (UICC) and American

Joint Committee on Cancer (AJCC), which has proven to be a good system for

comparison of surgical results and prediction of patient outcome.

4.8. TNM classification:

Stage I is a tumor limited to the mucosa or muscular layers; Stage II tumors invade the

peri-muscular tissue; Stage III tumors invade serosa, liver less than two centimeters, or

have regional (hepato-duodenalligament) lymph node metastasis; and Stage IV shows

liver invasion greater than two centimeters (Stage IVA), or metastasis to nonregional

lymph nodes and/or distant organs (Stage IVB).

The gallbladder's very thin wall and the discontinuous muscular layer are believed to

· facilitate tumor invasion and contribute to the advanced local and regional disease usually

present at the time of diagnosis.

Tsukada et al. reported that the five-year survival rate in patients with TNM Stage I

tumors was 91 percent; 85 percent in patients with Stage II tumors; 40 percent in patients

with Stage III tumors; and 19 percent in patients with Stage IV tumors. In the same study,

16

in patients with TNM Stage III and Stage IV tumors the five-year survival rate was 52

..,., ........ ,-.r after curative resection. This was significantly better than the five percent five-~-~-~-~~

year survival rate after a non curative resection?5

4.9. Risk factors

Gall stones

The association between cholelithiasis and gallbladder cancer has been known since 1861

and is supported by autopsy studies, screening surveys, and hospital-based case-control

studies Cholelithiasis is more frequent in gallbladder cancer than in extrahepatic bile

ducts cancer. In a recent case-control study performed in Australia, Canada, Holland, and

Poland, a history of gallbladder symptoms requiring medical attention was identified as

one of the major risk factors for gallbladder cancer.

The theoretical basis for this phenomenon is that the inflammation, chronic trauma, and

infection in approximately one third of gallstone patients promotes epithelial dysplasia

and adenocarcinoma formation. For this reason, it has been suggested that larger stones

have a greater impact on the risk of developing gallbladder cancer, possibly reflecting

greater duration ,and intensity of epithelial irritation. Diehl reported that in subjects with

gallstones larger than three centimeters, the risk of gallbladder cancer is 1 0 times greater

than in subjects with gallstones smaller than one centimeter.60 Warren et al. reported a

larger mean stone diameter among 19 subjects with gallbladder cancer (20.3 mm) when

compared with 883 subjects undergoing surgery for gallstones (11.9 mm). In contrast,

Moerman et al found no association between.stone size and gallbladder cancer.

Cholesterol gallstones represent approximately 80 to 90 percent of all gallstone cases in

the Western world and are considered to be a promoting factor. There is little available

information as to whether pigment or cholesterol stones have a different activity as

promoters of gallbladder cancer development.

17

Reproductive factors

Together with thyroid cancer, cancer of the gallbladder is the only non-sex hormone­

related cancer displaying a female preponderance, with incidence being 3-4 times more

common among women. A case control study of 64 newly diagnosed cases of

gallbladder cancer and 1 01 cases of cholelithiasis carried out at department of

gastroenterology, Regional cancer centre, Trivendrum suggests that mean age of

menarche was 13.4+/-1.2 years among female patients with cancer while it was 14.0+/-

1.4 years for gallstone patients. Higher age at menarche (> 13 years, OR 2.48, 95%

confidence interval (CI) 1.16-5.3), higher number of childbirths (>3 births, OR 3.92; 95%

CI 1.4-10.3), hig)ler number of pregnancies (>3 pregnancies, OR 6.66, 95% CI 1.8-23.4),

and higher age at last childbirth (>25 years, OR 2.97, 95% CI 1.04-8.5) were found to

have significantly higher risk of developing gallbladder cancer. Early menarche, late

menopause, multiple pregnancies and childbirth increases the risk of gallbladder cancer.

Dietary Risk factors

Several factors however have been suggested, although none has stood the test of time.

Consumption of Staple diet

In India there is striking peculiarity of North- South variation in occurrence of GBC

.India making one suspect that variation in dietary habits could be an important factor

related to the etiology of GBC. Malhotra (1967), reported the variation of dietary habit in

Northern and Southern parts of India. The pattern of diet and eating in North Indian are

masticatory in n~ture in contrast to sloppy diets of South Indians. A potential association

of masticatory diet with GBC might be that the mucus concentration in the saliva rises

with masticatory meals as compared to sloppy diets and since mucus is precipitated by

strong acid in the stomach, and in this state might be less permeable to stimuli which are

responsible for Cholecystokinin release, which are thus prevented from reaching the

receptors. The cholecystokinin is responsible for gallbladder contraction. Changes in the

gallbladder contraction as well as in the flow rate of bile are greatly affected by these

dietetic variations. Bile remains for longer duration in the gallbladder and biliary system,

when the gallbladder contraction decreases. The derivatives of cholic acid are the most

potent of the chemical carcinogens. The specific mechanism of how they act as

carcinogens is unclear, but it seems to be an exciting task to find out whether or not the

18

I 1 " stasis of bile in the gallbladder and the biliary system, caused by the dietary habits, could

induce frequently the development of Gallbladder cancer in North Indians compared with

South Indians.

Consumption of vegetables and fruits

Findings in various studies on the consumption of vegetables indicate an inverse

association with Gall bladder cancer risk. Lower risk of GBC was also reported with

frequent intake (ranging from daily to three times or more per week) of fruits, odds ratio

(OR)= 0.30 (0.19-0.49); boiled vegetables, OR= 0.37 (0.19-0.75) and salad, OR= 0.45

(0.21-1.00). Further indications of a protective role of vegetables and fruits were found in

an integrated series of case control studies conducted in Northern Italy, in which

researchers found a weak inverse association with high consumption of fruits and

vegetables for OBC?0-23 Low intake of fresh fruits was associated with gallbladder

cancer with OR of 6.40 (1.40-30.30) in a case- control study in Chilean population.45

Consumption of vegetables in higher amount has been found to be associated with a

reduced risk of many epithelial cancers of the alimentary tract. A protective effect of

vegetables and fruits on gallbladder cancer was also reported by Pandey et al (2002).23

There are many possible reasons given as to how a diet with a high consumption of / '

vegetables and fruits prevents the Occurrence of cancer. A large number of potentially

anti-carcinogenic agents i.e. carotenoids, vitamin ·c, vitamin E, selenium, folic acid,

dietary fiber, indoles, phenols, flavinoids, protease inhibitors, allium compounds and

plant sterols are found in these food sources. These. have both complementary and

overlapping mechanism of action which include the induction of detoxification of

enzymes, inhibition of nitrosamine formation, provision of substrate for formation of

antineoplastic agents, dilution and binding of carcinogens in the digestive tract, alteration

of hormone metabolism, stimulation of immune system, regulation of gene expression in

cell proliferation ,apoptosis and scavenging of oxidative agents. Further, plant- based

diets have a lower calorie density and increased nutrient density which are important

factors in preventing obesity epidemic?0-24

Consumption of flesh foods and egg

A case control study of cancer of the biliary tract in Japan reported decreased risk with

greater consumption of fresh or salted fish with odds ratio in the range of 0.1 to 0.4

19

(P<0.05) and eggs with OR= 0.21 (0.10- 0.45). Another study in Japan reported a positive

association between the mortality rate of biliary tract cancer and expenditure for foods

like pork. Tavani et al (2000) demonstrated a positive relationship with consumption of

red meat and several neoplasms; but no statistically significant association was found '

with red meat intake and risk of GBC. Strom et al (1995) suggested non- vegetarian diet

as a risk factor but they emphasized more on the cooking habit; baking/ roasting meat vs.

frying was a risk factor for GBC. In a case- control study, red meat consumption was

found to be associated with increased risk of gallbladder cancer.

Consumption of Sugar

Sugars have been stated to be one of the risk factors for biliary tract cancer. For

gallbladder cancer the association with the intake of sugar was significant (P< 0.05) and

there was a two fold increase in risk in the upper quartile of intake, OR= 2.38 (1.03-5.46).

Specific dietary constituents like monosaccharide and disaccharides are potential risk

factors for gallbladder cancer. Sugar may influence the bile composition through

lipoprotein metabolism.22 Sugar increases caloric intake without providing any of the

nutrients that reduce cancer risk. By promoting obesity and elevating insulin levels, high

sugar intake may increase cancer risk. However, a case- control study conducted by Seera

et al (2002) found a positive association between low intake of sugar as soft drinks with

gallbladder cancer, OR=3.60 (1.30-10.10).

Consumption of Tea and Coffee

Researchers have proposed that tea might protect against cancer because of its

antioxidant content. One of the studies has reported an inverse association with the

amount of tea drunk throughout life. Coffee has been suggested to be carcinogenic due to

presence of mutagens. Kratzer et al (1997reported that regular coffee was consumed by

74.5% of the subjects and trend was seen towards slightly higher prevalence of '

gallbladder stone.s in coffee drinkers. 53 But a study in Japan, reported a low risk of GBC

with frequent intake of coffee, OR= 0.25 (0.09-0.68).62

Role of Dietary constituents

Energy intake

A case-control study in Poland, found gallbladder cancer risk was positively associated

with total calorie intake, with an odds ratio of 4.13 reported for the upper versus the

20

lowest quartile (P<O.Ol).lO Another study in Poland also found, total energy intake was

related to gallbladder cancer with an OR of2.00 (1.10-3.70) for the highest vs. the lowest

quartile.9

Body Mass Index

Higher body mass may play a role- either direct or indirect- in gallbladder cancer. In a

case- control study in Poland, researchers noted an elevated risk of GBC for females with

higher values of,BMI, the OR for females in the highest vs the lowest quartile was 2.10

(1.20,.3.80). A prospective cohort study in the USA, found that gallbladder mortality rates

were associated with obesity in women.57 Other studies also revealed that obesity was

positively associated with a higher incidence of GBC. Several reports have demonstrated

a strong relationship between obesity, gallstone and gallbladder cancer. 58

Carbohydrate

In a case-control study of GBC in Poland, a weak positive effect of carbohydrate

consumption was observed, although the trends in risk were not significant (OR= 1.18,

P= 0. 77). 11 • The intake of total carbohydrates was positively related to the disease of the

biliary tract (P< 0.01). For the upper quartile of intake, total carbohydrates showed an

almost fourfold Increase in risk. A positive association was observed between total

/ carbohydrate intake and GBC. The risk ratio of 11.30 { 4.60-28.00) reported for the

highest vs the lowest quartile (P < 0.0001)Y

Fiber

Low intake of dietary fiber has been suggested as a possible cause of a variety of

disorders prevalent among western societies including cholesterol gallstones. In a case­

control study in Poland, a weak inverse association with high fiber consumption, OR=

0.31 (0.09-1.1 0) P = 0.08 was observed. Higher intake of dietary fiber appeared to be

protective for GBC. Another case control study also showed that low fiber intake was

positively associated with GBC risk.9

Fat

Gallbladder can<;er rates are higher in Japan than in the USA, even though dietary fat

intake is higher in the USA. The major risk factors for gallbladder cancer include a

history of gallstones and obesity. To the extent that dietary fat contributes to either

gallstone formation or obesity, it may indirectly increase the risk of gallbladder cancer. A

21

J -. case-control study observed no apparent trend with total fat intake and gallbladder cancer

OR= 0.86 (0.23-3.22) P=0.91. Another case control study in Japan reported increased

risk for GBC with greater preference for "Oily foods", with an odds ratio of 3.29 (1.68-

6.43).Still another case control study in Chilean population, found a weakly direct

association for high consumption of fried foods and gallbladder cancer. A positive

association of high fat intake with GBC risk was found in a case- control Study in

Karachi.61

Cholesterol

A population based case-control study in Poland, found a positive association of GBC

with cholesterol;an adjusted OR of 1.54 (0.51-4.65).9

Protein

In a case- control study in Poland, a weaker direct association was observed for protein

consumption with an adjusted OR of 3.88 (0.92-16.30). On the other hand a study in

Japan reported that intake of animal proteins was low risk factor for gallbladder cancer.

Vitamins

Vitamin A

A case- control study in Karachi showed that risk of GBC was positively associated with

low intake of vitamin A.

Vitamin B

Epidemiological studies that have investigated association of cancer risk at specific sites . and diets high in vitamin B are limited in number. In a case- control study a significant

reduction in risk of GBC associated with increased intake of vitamin B6 (pyridoxine) was - II

observed by Zatonski et al (1997).

Vitamin C

Vitamin C is a potential cancer inhibiting agent. It prevents the formation of carcinogen

from precursor compounds.Epidemiological evidences show a protective effect of diets

high in vegetables and fruits containing vitamin C for cancer of the gallbladder. A case­

control study in Poland, found an inverse association between GBC and vitamin C, which

was statistically significant for the three highest consumption quartile, OR = 0.29 (0.1 0-

0.86). Another epidemiological case- control study found inverse associations of vitamin

C with GBC. Ascorbic acid deficient guinea pigs frequently develop gallstones and

22

ascorbic acid status may also affect the risk of gallbladder disease in human. 64

Vitamin E

Researchers in a study found an inverse association of vitamin E with GBC. 29 In a case­

control study in Poland, possible protective association with vitamin E intake was found,

OR for the highest vs. the lowest quartile of intake was 0.30 (0.09-0.96).

Obesity and risk factors

The nutrient composition of the diet is important in determining the amount of energy

that is consumed and ultimately stored by the body. In addition daily eating patterns can

also influence the amount of food eaten, Missing breakfast, snacking and eating to

compensate for s,tress all tend to promote over eating and therefore obesity.

Missing breakfast/meals

A case control study done in Karachi found a significant association between missing

meals and cancer gall bladder among women.61 A survey was conducted in south East

Asian countries to see the breakfast patterns. It was found that more than 1 0% of people

miss their breakfast. Fasting is associated with stone and sludge formation in the gall

/ bladder which are known to be associated with the etiopathogenesis of cancer gall

bladder. During fasting Bile remains for longer duration in the gallbladder and biliary

system, when the gallbladder contraction decreases. The derivatives of cholic acid are the

most potent of the chemical carcinogens. The specific mechanism of how they act as

carcinogens is unclear, but it seems to be an exciting task to find out whether or not the

stasis of bile in the gallbladder and the biliary system, caused by the dietary habits, could

induce frequently the development of Gallbladder cancer.

23

I 4.10. Prevention and control of cancer gall bladder

Cholecystectomy is the most frequent of all intra-abdominal operations. It is estimated

that in the US more than 550,000 cholecystectomies are performed yearly within an

estimated population of 20,000,000 at-risk patients with asymptomatic gallstones. The US

cholecystectomy rate was 47.6 per 1000 inhabitants in 1993, whereas the rate was only

25.1 per 1000 inhabitants in 1992 in Chile, in spite of a three to four times higher

prevalence of cholelithiasis.45The incidence of gallbladder cancer has diminished

considerably, in an inverse correlation to the increase in cholecystectomies in developed

countries.". A descriptive survey published in 1995 showed that gallbladder cancer

mortality rates were over four times higher in towns with a high proportion of Mapuche

Indians (35 per .100,000 inhabitants in the region of the Araucania, Southern Chile),

compared with towns with a predominantly Hispanic population (8 per 100,000

inhabitants in the municipality of La Florida, Santiago). 46 Three studies have analyzed the

potential benefits of prophylactic cholecystectomy for low-risk populations. Based on

decision analysis models, it was concluded that the benefits of prophylactic

cholecystectomy were irrelevant when compared with expectant management of gallstone

disease.44The role of prophylactic cholecystectomy in high-risk populations, including

North American Indians, Mexican _Americans, Chileans, and Bolivians, has yet to be

reported.2 A recent cost-effectiveness analysis of screening and treatment among Chilean

women under 40 years old with asymptomatic cholelithiasis showed that prophylactic

laparoscopic cholecystectomy can significantly benefit the population at a very low '

incremental cost (Puschel et al., personal communication, Santiago, 2000).However these

interventions at mass scale need policy recommendations and political will.

24

4.11. Conclusion

1. The process of gallbladder carcinogenesis is usually related to a history of

cholelithiasis, which is frequently present for at least 20 years before the tumor

appears.

2. Because only a small fraction of patients with cholesterol gallstones develop

gallbladder cancer, it is important to identify the factors( dietary or other) that

induce progression from cholelithiasis to gallbladder cancer or cause cancer gall

bladder independent of cholelithiasis. This may allow the identification and early

treatment of gallstones of susceptible individuals within high-risk populations.

3. The limitations of epidemiologic gall-bladder cancer studies have included the

small size of populations studied and problems in quantifying exposure to putative

exogenous risk factors, particularly potential carcinogenic xenobiotics. '

Multicenter case-control studies in high-risk populations in which accurate

biomarkers of exposure to various risk factors are used and the genetic factors are

assessed will be of great value in answering several of the questions raised in this

rev1ew.

4. Primary prevention of gallbladder cancer is not expected in the near future.

However, secondary prevention, primarily oriented to treatment of symptomatic

gallstones, must be emphasized in endemic areas where cholelithiasis is highly

prevalent. Prophylactic laparoscopic cholecystectomy might be cost effective in

developed countries but it is not advisable for a country like India with this huge

population.. It is apparent that interventional programs are urgently needed to

decrease the number of gallbladders at risk for gallbladder cancer development in

high-risk areas.

25

I i

1 I

5. Methodology

5.1. Study area-We selected Mahavir Cancer Sansthan, Patna, Bihar, India for the study.

5.2. Study population - Annually 10,000 patients come to Mahavir cancer Sansthan

from whole of Bihar. We defined the study population as patients coming to Mahavir

cancer Sansthan, Patna, Bihar, India for diagnosis, treatment or palliation. Specific

inclusion criteria were (1) having histological/cytological (including bone marrow

examination) diagnosis of malignancy done (2) residing in Bihar for>lO years (3) cases

who are willing to participate in the study.

5.3. Study design- We conducted a hospital based case control study by review of

. records and interview of cases and controls using structured questionnaire directly

administered in person by trained health worker.

5.4. Operational definitions

Cases-We defined cases as all histopathologically/cytologically confirmed incident cases

of cancer gall bladder coming to Mahavir cancer Sansthan between May 2005 to

September 2005.'·

Controls-We defined controls as all histopathologically or cytologically confirmed

incident cases of carcinoma lung and/or hematopoietic and lymphoreticular malignancy

coming to Mahavir cancer Sansthan (from May 2005 to September 2005) for

diagnosis/treatment/palliation.

5.5. Study team-We constituted a six member study team

1. Principal investigator

2. Pathologist

3. Radiologist

4. Three medical social workers

26

I I

1 Training of the principal investigator done at NIE, Chennai. Pathologist and radiologist

were trained at Mahavir Cancer Sansthan. Health workers /medical social workers were

also trained at Mahavir Cancer Sansthan in the data collection procedures.

5.6.Sample size- We calculated the sample size (using statcalc of epi-2000) assuming

that 10% of people have dietary malpractices (missing meals, reuse of cooking oil) in the

control group and with a 95% confidence level, 90% power and case: control ratio of 1, a

total of 146 patients were required. (Odds Ratio=3). Adding 10% refusal or dropout a

total of 160 case patients were recruited. Equal no. of controls was selected from the

same hospital suffering from carcinoma lung and/ or hematopoietic and lymphoreticular

malignancies. Cases -160 (carcinoma gall bladder). Controls- 160 (carcinoma lung

and/or acute leukemia).

5.7. Data collection technique and tools '

We reviewed the records to collect information regarding age, sex and place of residence.

Trained health workers administered structured questionnaire to cases and controls in

person to collect information on dietary malpractices (habit of missing meal, reuse of

cooking oil), demographic characteristics, marital status, smoking habit, previous

infection of gall bladder, past medical and surgical history and parity in case of females.

/ . The question on dietary factors addressed frequency of meals, time of breakfast, Reuse of

oil, missing breakfast, for a period often to twenty years in subjects' lives before onset of

symptoms of current illness. We assessed the exposure dietary malpractices both for

intensity and duration. For assessment of intensity we categorized the exposures into 5

levels. Duration of exposure was kept more than ten years in subjects' lives. We defined

missing breakfast as 'Intake of less than 100 calories till 12 o'clock in the noon'. We

defined Reuse of oil as 'the oil left after deep frying being reused for cooking'after being

kept at room temperature'. We defined non vegetarian 'as person taking non vegetarian

food at least twice a week'. We defined frequency of fresh vegetables as 'at least twice a

week or more intake'.

The data collected related to the time period prior to onset of any symptoms was defined

as '1 0 to 20 years before any symptoms of current illness were perceived by the cases and

controls'.

27

Quality control measures-The principle investigator validated a tenth of interviews

through observation of field procedure of data collection for quality assurance and

consistency.

Data analysis and statistical tests

Univariate analysis-we calculated univariate relative risk for the potential exposure

factors, 95% confidence intervals, chi square tests and p values to test the statistical

significance. We analyzed the data on Epi Info 2005 version 3.3.2.

Stratified analysis -We did stratified analysis of some of the exposure factors to check

confounding and interaction.

To check causality we studied the linear trend for mcrease m intensity of dietary

malpractices in relation to the study disease ..

Multivariate analysis-We did multivariate analysis for six of the exposure factors.

/" 5.8. Ethical Issues

Human subject protection

Ethics committee at NIE reviewed & cleared the protocol of this study.

We obtained written consent from the study subjects in the local language Hindi.

Institute Ethics Committee reviewed and cleared protocol of my study.

28

6. Results

6.1. Study population

Cases

There were 167 incident cases of cancer gall bladder registered from May 2005 to

September 2005, at Mahavir Cancer Sansthan, Patna. Fourteen (8.3%) cases did not

undergo microscopic confirmation of the disease, six (3.5%) cases refused to participate

in the study. We interviewed and analyzed 147 cases, who met all the inclusion criteria.

Controls

Similarly, there were 171 incident cases of control group registered during the same

period at Mahavir Cancer Sansthan. 15(8. 7%) of controls did not undergo microscopic

confirmation of the disease and eight ( 4.6%) of them refused to participate in the study.

We interviewed and analyzed 148 controls who met all the inclusion criteria. Thirty one

controls had carcinoma lung, 71had lymphoma and 46 controls had leukemia.

6.2. Sociodemographic and dietary exposure factors among cases and controls

/ (Tablel, 2 and 3)

1. Age distribution- Age of the cases ranged from 16 to 80 years (mean: 51.7,

SD: 12.5). Age of the controls ranged from 15 to 100 years (mean: 43.9, SD: 17.8).

Among cases mean age for males was 55.6 years. Mean age for females was 50.5 years.

Mean age among controls was 43.4 years for males and 45 years for females. Proportion

of people aged 40 years or more was more among cases (82%) than controls (54%).

2. Sex distribution- Proportion of females was higher among cases (76%) than controls

(27% ). In our study female to male ratio is 3.17:1 for cases and 0.3 7:1 for controls.

3. Parity (among females) - Multiparty (parity2. 3) was not significantly different among

cases (82%) and,controls (73%).

29

4. Residence in gangetic basin -53 (36%) of 147 cases resided in the districts forming the

gangetic basin. These districts are Patna, Vaishali, Muzaffarpur, Bcgusarai, Darbhanga

and Gaya.64 (43%) of 148 controls resided in these districts.

5. Illiteracy -Illiteracy was higher among cases than control and was found to be

associated with the study disease (OR: 2.79, CI 1.69-4.62).

6. Tobacco use- Tobacco addiction among cases (22%) and controls (27%) were similar.

7. Religion-129 (87%) of 147 cases and 131(88%) of 148 controls belonged to Hindu

religion.

8. Gall bladder stones-This emerged as single most important risk factor for gall bladder

cancer. 86/147(59%) of cases had gall bladder stones on ultrasound examination of the

gall bladder. None ofthe controls had gall bladder stones.

9. Non vegetarian diet- We categorized frequency of non vegetarian diet into 4 levels,

namely, occasional, once a week, twice a week and daily .It was not associated with the

study disease as compared to the controls at all levels. If we consider consuming non

vegetarian food at least twice weekly as an exposure factor, the association was not

significant (OR-0.74, CI-0.46-1.15).

10. Consumption of fresh vegetables-Similarly, we categorized frequency of intake of

~ fresh vegetables. Consumption of fresh vegetable at least twice a week was not associated

with the study disease. (OR-0.9, CI-0.6-1.3).

11. Missing breakfast-It was strongly associated with carcinoma gall bladder. Missing

breakfast at least twice a week had a strong association (OR: 4.19, CI: 2.59-6.29) with

Gall bladder cases. Missing breakfast was more commonly seen among those cases who

did not have gall bladder stones. (OR-6.8, CI: 3.32-14.14)

12. Frequent Reuse of cooking oil-It was positively associated with cancer gall bladder

(OR: 1.95, CI: 1.14-3.35). Similar to missing breakfast this exposure factor was also seen

more commonly among those cases that did not have gall bladder stones. (OR:4.22

CI:2.14-8.38)

30

6.3. Stratified analysis for the exposure factors (Tables 2, 3, 4, 5, 6, 7)

Confounding & Effect modification

Table -2 and 3 shows the association between various factors and gall bladder cancer

stratified by presence or absence of gall bladder stones in the cases. The odds ratio across

the strata was different for reuse of oil suggesting effect modification by presence of

stones.

Table-4 describes the association between missing breakfast and gall bladder cancer

stratified by gender. The crude odds ratio and Mantel Haenszel adjusted odds ratio were

not similar suggesting confounding.

Table-5 describes the association between missing breakfast and gall bladder cancer

stratified by age. The crude odds ratio and Mantel Henszel adjusted odds ratio were '

similar in the case of age. The odds ratio across the strata were different but the

confidence intervals were overlapping indicating the absence of effect medication.

Table-6 describes the association between reuse of oil and cancer gall bladder stratified

by gender. The crude and adjusted odds ratios are similar to the stratum specific odds ratio

/ in the case of reusing the cooking oil, again indicating the absence of confounding or

effect modification by gender.

Table-7 describes the association between reuse of oil and cancer gall bladder stratified

by age .The crude, adjusted and stratum specific odds ratio are similar, suggesting no

confounding or effect modification by age. Overall there was some confounding by

gender in case of missing breakfast, for other stratifications there was no difference in

the strength of association of potential risk factor with the study disease after adjustment

for age and sex.

6.4. Dose response (Table 8,9)

We have categorized the dietary exposure variables missing breakfast and reuse of oil

according to intensity-never, occasionally, once a week, twice a week, daily. Strength of

association of exposure factor missing breakfast & reuse of oil with the study disease

appeared to increase with the increase in the intensity of exposure. A linear trend is seen.

31

We observe a significant dose response relationship for both the dietary exposure factors

missing breakfast and reuse of oil.

6.5. Multivariate analysis (table 10)

Stepwise logistic.regression analysis by forward selection

We carried out a multivariate logistic regression analysis entering all the exposure

variables for which we had done a univariate analysis. Out of ten factors, four factors,

namely, age, gender, literacy status and missing breakfast were identified as independent

risk factors associated with carcinoma gall bladder. The model explained about 30%-40%

of the variability (R2 =0.4). Reusing cooking oil did not emerge as a significant factor in

this regression model.

32

r ... 7. Discussion

This study is an exploratory study because data concerning the distribution of potentially

important characteristics among cases and controls are unavailable and knowledge of the

natural history of the disease is insufficient to justify study of a specific hypothesis. Here

an attempt is made to gather data concerning possible differences on a variety of factors

among the cases as compared to controls in the hope that etiologic clues worthy of further

study will emerge.

7.1. Incidence of gall bladder cancer in Bihar and associated factors

Relative frequency of ~all bladder cancer in Bihar is 9.6% among all female malignant

cases in a cancer hospital. There is no population based cancer registry in Bihar. There is

population based cancer registry in adjacent district capital Kolkata that shows high

incidence of gall bladder cancer in Kolkata (6.4%) among all female cancer cases. 1

This study give_s a view of profile of gall bladder cancer in Bihar, which was not

documented so far. The demographic characteristics of the cases are similar to cases in

other studies done in India. In a case control study done in Uttar Pradesh Shukla et al

reported mean age of the cancer gall bladder patient to be 50 years (range:40-60 years).

Khandelwal et al reported it to be 52 years (range: 33-70 years).In this study age of the

/cases ranged from 16 to 80 years (mean: 51.7 years). Studies done in Europe reported

higher mean age of gall bladder cancer patients than that of studies done in India.

Carcinoma of the gallbladder is predominantly a disease of elderly females. Of 2998

.. patients from 51 series reported by IARC over last 20 years there were 2292 females and

706 males with a female to male ratio of 3.2:1.10-11 Shukla et al (1985) in their series of

315 patients found a female to male ratio of 2.5: l.Khandelwal et al in their series of 313 '

patients found a ·male to female ratio of 2, 7: 1. In this study female to male ratio was

3.17:1 in cases while it was 0.37:1 in case of controls. Being female was strongly

. associated with cancer gall bladder.

Case control studies done in other parts of the world and India suggest strong association

with increase in parity among women with cancer gall bladder. However this association

was not seen in this study, both cases and cont~ols were not significantly different for

parity.

33

According to studies conducted by TMH and liPS Mumbai there are areas in Bihar

comprising of Gangetic basin endemic for cancer gall bladder? These areas are Patna,

Vaishali, Muzaffarpur, Begusarai, Darbhanga and Gaya. This study did not show any

preponderance of cases in the districts of Gangetic basin. There was no difference in the

number of cancer gall bladder patients and cases of other malignant conditions coming

from these districts. These suggested that referral patterns for case patients and control

subjects were same for these districts. Same proportions of patients were coming from

these areas for different malignant conditions.

Literacy was independently associated with the disease in the multivariate analysis,

suggesting that various social factors directly associated with literacy and not considered

in the study could be the indirect cause of this association.

Other Studies have found association of cancer gall bladder with tobacco addiction.

Studies done on other cancers indicate that many of the cancers are related to tobacco

addiction including cancer lung and Leukemia (the controls in this study) so this study

failed to identify an association with cancer gall bladder and tobacco habit.

Religion did not emerge as a risk factor, both Hindus and Muslims possessing equal risk

/ of cancer gall bladder in this study.

Findings in various studies on the consumption of vegetables indicate an inverse

association with,Gall bladder cancer risk. Lower risk of GBC was also reported with

frequent intake (ranging from daily to three times or more per week) of fruits, odds ratio

(OR: 0.30, CI: 0.19-0.49); boiled vegetables, (OR: 0.37,19-0.75) and salad,(OR:0.45

CI0.21-1.00).3 Further indications of a protective role of vegetables and fruits were found

in an integrated series of case control Studies conducted in Northern Italy, in which

researchers found a weak inverse association with high consumption of fruits and

vegetables for GBC. Low intake of fresh fruits was associated with gallbladder cancer

with OR of 6.40 (CI: 1.40-30.30) in a case- control study in Chilean population?0-21 The

controls in these studies were not patients of other cancers, so an association could be

shown with respect to fresh fruits & vegetables consumption However in this study

controls were chosen from many sub-groups of cancer, fruits and vegetables in general

have a protective effect from cancer, so among the cancer patients there was no

34

association suggesting both groups were similar in fruits and vegetable consumption.

Consumption of fresh vegetables at least twice weekly was not associated positively or

negatively with the study disease. The same result was observed even when we analyzed

for daily consumption (not tabulated).

Risk factors

Gall stones-These are the most common risk factors for gall bladder cancer, based on

autopsy surveys and hospital based case control studies. A relation has been established

by Nervi et al (WHO, 1989) between gall stones and subsequent carcinogenesis. In a

cohort study of gall bladder patients who did not have a cholecystectomy ,Maringhini et

al (1987) estimated the cumulative incidence of gall bladder cancer after 20 years to be

about 1% with a 3 fold elevation in the relative risk. The risk of gall bladder cancer is

related to gall st~:me size also. Subjects with stones over 3 em in diameter have shown a

risk that is ten times that seen with stones less than 1 em. (Diehl 1983).60

Skipping breakfast An elevated risk of gall bladder cancer is seen with skipping

breakfast in this study. A study done in Karachi has shown similar results. Prolonged

fasting affects the size of bile acid pool and promotes biliary sludge formation. Stasis of

bile in the gall bladder for longer hours causes irritation of gall bladder mucosa by

/precipitation of bile salts. 61

Reuse of cooking oil -repeated heating and keeping the oil at room temperature before

using it again has shown strong association with cancer gall bladder. Oil containing

polyunsaturated fatty acids like vegetable oils (cases and controls predominantly use

vegetable oil for'-deep frying) produce HNE ( 4-hydroxy trans-2 nonenal) when heated at

frying temperature. Heating butter, ghee, olive oil or coconut oil produces HNE or other

lipid peroxidation products in a lesser amount. HNE is a toxic compound that is easily

absorbed from the diet. The toxicity arises because the compound is highly reactive with

proteins, nucleic acids, DNA , RNA and other biomolecules. HNE was found in the bile

of gall bladder cancer patients in more concentration than the bile of healthy

subjects65 .this suggests that lipid peroxidation product may be carcinogenic in gall

bladder cancer. Defective storage, especially in hot and humid climate promotes

35

contamination with carcinogenic ·mycotoxin such as Aflatoxin B produced by Aspergillus

Flavus61 •

7.2.Causality criterion in the study-Ideally the experimental approach provides a direct

method for establishing or refuting whether any association between two factors is

causal or not, in the absence of experimentation, several lines of reasoning have been

advocated for causality ,this study does provide factors essential to fulfill the causality

criteria. Missing breakfast has emerged as an independent risk factor satisfying some of

the causal criteria.

1. Temporal association-A causal association reqmres that factors thought to be

causative must precede those events that are regarded as their effects .This requirement is

basic to all explications of the causal concept. Occasionally a definite temporal sequence

is difficult to establish for a chronic disease. In this study we assessed the dietary

exposure factors for the cases and controls in the time frame which was before the onset

of any symptoms of cancer gall bladder or control group malignancies. However, the '

design of the present study limits this causal criterion.

2. Strength of association- Odds ratio of about four for the association between the

exposure and the disease both in the univariate and multivariate analyses suggest strong

/' strength of association.

3. Not due to chance- The association was statistically significant both in the univariate

, and multivariate analyses indicating evidence against the role of chance in the observed

association.

4. Biological plausibility-It is difficult to prove but there are evidences that stasis of bile

in the gall bladder for longer hours causes epithelial damage and dysplasia8.Prolonged

fasting affects the size of bile acid pool and promotes gall stone formation. Defective

storage of oil, ·~specially in hot and humid climate promotes contamination with

carcinogenic mycotoxin such as Aflatoxin B produced by Aspergillus Flavus. Improper

processing and repeated heating of food produces HNE , which is toxic for the living

cells. Reuse of oil may expose the subjects repeatedly to these toxic substances. Missing

breakfast tends to decrease the overall micronutrient absorption for the whole day.

Missing breakfast tends to increase binge eating in the next meal, promoting obesity

36

.Obesity has complex relations with physical activity and diabetes.These metabolic

disorders are related with chronic gallbladder disease and cancer gall bladder also.

5. Consistency-We do not have reports from other case control studies considering the

same exposure factors suggesting similar results. However within the study the results are

consistent with respect to different age groups.

6. Dose response-The dose response relationship is one of the main features of this study,

as the intensity•. of exposure was categorized over a considerable range. We have

categorized the dietary exposure variables missing breakfast and reuse of oil according to

intensity-never, occasionally, once a week, twice a week, daily. Strength of association of

exposure factor missing breakfast & reuse of oil with the study disease appeared to

increase with the increase in the intensity of exposure. A linear trend was significant.

However, reusing of cooking oil was not significantly associated in the multivariate

analysis. But in Univariate analysis when cases were stratified on the basis of presence of

gall bladder stones, reuse of oil emerged as a significant factor in the cases without gall

stones.

7. Specificity-It is difficult to assess specificity in this study.To summarize, missing

breakfast and reuse of oil is associated with cancer gall bladder satisfying at least three

/' causality conditions, namely, strength of association, association not due to chance and

dose response in our study.

•7.3. Errors and biases -We have tried to minimize errors and biases in this study. We

have used standardized questionnaires administered by interviewers who were blinded to

the case control status of the subjects. Any error in the recording of information is

therefore non-differential, underestimating the risk. We have recruited cases and controls

both equal severity malignant cases which itself minimizes diagnostic bias. To minimize

referral bias both cases and controls care selected from the same hospital in the same time

period. Because any particular hospital has its own referral practice. Be it lower income

individuals or upper income individuals. The results of a case control study can be biased

due to overrepresentation of a particular exposure factor. Selection of incident cases has

reduced the bias due to length of stay, and survival. Chance variation is appropriately

addressed as we are getting an odds ratio of higher than three. Dietary exposure factors

are assessed for a ten to twenty year reference period before the onset of any symptoms

37

showing a time relationship .The association of missing breakfast and reuse of oil is

strong with a dose response both for the exposure factor missing breakfast and reuse of

oil. Consistency within the study is assessed by the strength of association of the

exposure factors being same with other similar studies. We do not have evidence for this.

There will be factors which . are intermediate in the association between dietary

malpractices and cancer gall bladder, it is very unlikely that the association if causal is a

direct one in biochemical terms, and dietary malpractices may produce some

metabolic( obesity, diabetes) change that in tum increases the risk of cancer gall bladder.

9. Conclusion-Case control method of investigation is often the research strategy of

choice, however particularly when initiating an exploratory study of disease etiology or

investigating a rare disease. This study is kind of fishing expedition where multiple

hypotheses are proposed for investigation. The purpose of this study is to learn enough

about the possible causes of cancer gall bladder so that specific hypotheses may be

suggested and be sufficiently supported to justify a detailed investigation by apex cancer

research organizations. The strength of association of dietary malpractices with the study

disease indicates that dietary malpractices and irregularities may bring about metabolic

/ and cellular changes precursor of chronic gall bladder disease and consequently gall

bladder cancer.

8. Limitation-The issue of confounding is complex. Most potential confounding factors

have been adequately controlled by multivariate method ,but there remain the probability

of confounding by other factors not included in the study such as-BMI, serum

cholesterol, nutrional status, genetic factors . There could be bias in the absence of a true

difference, if due to any reason cases over report dietary malpractices than controls.

9. Recommendation-Prospective population based studies can be done in the areas

where cancer gall bladder is prevalent that may come up with strong evidences

supporting causality criteria for risk factors of gall bladder cancer.

38

'

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35 Tanno S, Obara T, Fujii T, et al. Proliferative potential and K-ras mutation in epithelial hyperplasia of the gallbladder in patients with anomalous pancreaticobiliary ductal union. Cancer 1998;83:267-275.

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42 Iwase T, Nakazawa S, Yamao K, et al. Ras gene point mutations in gallbladder lesions associated with anomalous connection of pancreatobiliary ducts. Hepatogastroenterology 1997;44: 1457-1462.

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47 Blanken A. Hospital discharges and length of stay: Short stay hospitals, United States, 1972. Vital Health Stat 1976;10:1-66.

48 Ahlberg J, Ewerth S, Hellers G, Holmstrom B. Decreasing frequency of cholecystectomies in the countries of Stockholm and Uppsala, Sweden. Acta Chir Scand 1978; Suppl482:21-23.

49. Herber D. Vegetables, fruits and phytoestrogens in the prevention of disease. J Post grad Med 2004;50: 145-9.

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54. Potter JD. Food, Nutrition and the prevention of cancer: A globalperspective. Washington DC: American Institute of Cancer Research;1997. p. 198-201

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56. Nervi F, Duarte I, Gomez G, Rodreguez G, Delpino G, Ferrerio O,et al. Frequency of the gallbladder carcinoma in Chile. Int J Cancer 1988;41 :657.

57. Hartz A, Rupley D, Rifum A. The association of girth measurementswith disease in 32, 856 women. Am J Epidemio11989;119:71-80

58. Maclure KM, Hayes KC, Colditz GA, Stampfer MJ, Speizer FE,Willett WC. Weight, diet and the risk of symptomatic gallstonesin middle- aged women. New Engl J Med 1989;321 :563-9.

59. Heaton KW. The Epidemiology of gallstones and suggested aetiology.Clin Gastroenterol1973;2:67-83.

60 Diehl AK: Gallstone size and the risk of gallbladder cancer_,_JAMA. 1983 Nov 4; 250(17):2323-6.

61. Rizvi TJ, Zuberi SJ. Risk factors for gallbladder cancer in Karachi.J Ayub Med Coli Abbottabad 2003; 15: 16-8.

62. Tominaga S, Kato I. Changing patterns of cancer and diet in Japan.Recent Progress in Reaserch on Nutrition and Cancer Wiley-Liss, Inc 1990;1-10.

63. Wattenberg LM. Chemoprevention and cancer. Cancer Res1985;45:1-8.

64. Simon JA, Hudes ES. Serum ascorbic acid and gallbladder diseaseprevalence among US adults: The third National Health andNutrition Examination Survey (NHANES III). Arch Intern Med2000; 160:931-6.

43

65 Pandey M, Shukla VK, Singh S, Roy SK, Rao BR. Biliary lipid peroxidation products in gallbladder cancer: increased peroxidation or biliary stasis? EurJCancer Prev. 2000 Dec;9(6):417-22

44

\ Table-1 Univariate analysis for all cases and controls,Mahavir cancer sansthan,Patna,Bihar,India,2005

Sociodemographic characteristics & risk factors

Age > 40years

Female sex

parity2:3

Residence in the gangetic plains

Illiteracy

Tobacco users

Hindu religion

Gall bladder stone

Frequent non veg> 2 days a week Frequent Fresh vegetables>2

days a week Missing breakfast twice a week or more Frequently reusing of oil

Cases N=147

125

111

91

53

100

33

129

86

21

71

64

54

82

76

82

36

68

22

87

59

18

48

43

37

Control N=148

89

40

29

64

64

40

131

0

33

65

23

34

54

27

73

43

43

27

88

22

44

15

23

Odds Ratio 95% C.I.

3.96 2.25-6.99

8.32 4.78-14.56

1.47 0.6-4.04

0.74 0.45-1.21

2.79 1.69-4.62

0.78 0.44-1.37

0.93 0.43-1.99

0.74 0.46-1.15

0.9 0.62-1.3

4.19 2.59-6.29

1.95 1.14-3.35

45

&

\

Table-2 Univariate analysis of cases with gall stone and all controls, Mahavir cancer sansthan,Patna,Bihar,India,2005

Cases with gall Controls stones n=148 n=86

Sociodemographic Characteristics and risk factors # Total (%) # Total (%) Odds ratio 95% confidence interval

Age>40 years 74 86 86 89 148 54 4.1 1.9-8.7

Female sex 71 86 82 40 148 27 12.8 6.2-26.3

Residence in gangetic basin 31 86 36 64 148 73 0.7 0.4-1.3

Illiteracy 61 86 71 64 148 43 3.2 1.7-7.9

Tobacco users 15 86 17 40 148 43 0.5 0.3-1.2

Hindu religion 74 86 86 131 148 88 0.8 0.4-1.6

Frequent non veg>2 days a week. 16 86 19 33 148 22 0.8 0.4-1.6

Frequent fresh vegetable >2 days a week 40 86 46 65 148 44 1.1 0.6-1.9

Missing breakfast twice a week or more 30 86 35 23 148 15 2.9 1.5-5.7

Frequently reusing oil 20 86 23 34 148 23 1 0.5-2

46

\

Table-3 Univariate analysis of cases without gall stones and controls, Mahavir cancer sansthan,Patna,Bihar,India,2005

Cases without gall Controls stone n=61 n=148

Sociodemographic Chaqcteristics and risk factors # Total _(%) # Total (%) Odds ratio 95% confidence interval

Age>40 years 51 61 83 89 148 54 3.4 1.5-7.7

Female sex 40 61 65 40 148 27 5.1 2.6-10.3

Residence in gangetic basin 22 61 36 64 148 73 0.7 0.4-1.4

Illiteracy 39 61 64 64 148 43 2.3 1.2-4.5

--Tobacco users 18 61 29 40 148 43 1.1 0.5-2.3

Hindu religion 55 61 90 131 148 88 1.2 0.4-3.6

Frequent non veg >2 days a week 11 61 18 33 148 22 0.7 0.3-1.7

Frequent fresh vegetables >2 days a week 31 61 51 65 148 44 1.3 0.7-2.5

Missing breakfast twice a week or more 34 61 56 23 148 15 6.8 3.3-14

Frequently reusing of oil 34 61 56 34 148 23 4.2 2.1-8

47

I ~ 'i r

i ~

Table- 4.Association between missing breakfast and gall bladder cancer stratified by gender Mahavir Cancer Sansthan, Patna, Bihar, India, 2005

Cases Controls Odds 95%CI

-n=147 n=148 Ratio

Stratum A1(all) # % # %

Exposed 64 44 23 16 4.19 2.34-7.56

Unexposed 83 56 125 84

Stratum Bl(males)

Exposed 13 36 15 14 3.5 1.35-9.16

Unexposed 23 64 93 86

Stratum B2(females)

Exposed 51 46- 8 20 3.40 1.35-8.84

Unexposed 60 54 32 80

Woolfe, test for effect modification- x2- 0.0572, p-0.8

Mantel Haenszel odds ratio- 3.45

48

Table-5.Association between missing breakfast and gall bladder cancer stratified by age (<40 years and> 40 years), Mahavir Cancer Sansthan, Patna, Bihar, India, 2005

cases controls Odds 95%CI n=147 n=148 - Ratio -

Stratum A # % # % (all ages)

Exposed 64 44 23 16 4.19 2.34-7.56

Unexposed 83 56 125 84

Stratum Bl (<40years)

Exposed 14 64 8 14 11.16 3.12-41.95

Unexposed 8 36 51 86

Stratum B2 (>40 Years)

Exposed 50 40 15 17 3.29 1.62-6.73

Unexposed 75 60 74 83

Mantel Haenszel estimate of odds ratio-3.8 Woolfe,s test for heterogeneity of odds ratio-x2=0.06 p=O.S

Table-6 Association between reuse of oil and gall bladder cancer stratified Patna, Bihar, India, 2005

cases n=147

Stratum A(all)

Exposed 54

Unexposed 93

Stratum B1(males)

Exposed 13

Unexposed 23

Stratum B2(females)

Exposed 41

Unexposed 70

MH adjusted odds ratio=1.86 P=0.04

Test for interaction x2= o.oo1 p=0.9

37

63

36

64

37

63

controls Odds 95%CI n=148 Ratio

34 23 1.95 1.14-3.35

114 77

24 22 1.98 0.81-4.83

84 78

10 25 1.76 0.73-4.3

30 75

by gender , Mahavir Cancer Sansthan,

'in

Table-7 Association of reuse of oil and gall bladder cancer according to age, Mahavir Cancer Sansthan, Patna, Bihar, India,

cases n=l47

Stratum A( all) ' #

Exposed

Unexposed

Stratum 1 ( <40 years)

Exposed

Unexposed

Stratum B2(>40years)

Exposed

Unexposed

MH adjusted OR=2.27 P=0.004

Test for interaction x2=0.16 p=0.6

54

93

12

10

42

83

%

37

63

55

45

38

62

2005 controls Odds 95%CI n=l47 Ratio

# %

34 23 1.95 1.14-3.35

114 77

16 27 3.22 1.04-10.12

43 73

18 20 2 1.01-3.97

71 80

51

Table-8 Association of gall bladder cancer to frequency of missing

Breakfast habits, Mahavir Cancer Sansthan, Patna, Bihar, India, 2005

Cases Controls Relative Exposure

Stratum-1 Never missed breakfast Stratum-2 Occasionally missed breakfast Stratum-3

n=147 #

29

4

Miss once a week 50 Stratum-4 Miss breakfast twice a week or more

25

Stratum-5 Almost 39 daily

x2 for linear trend-41.152

p<0.005

n=148 odds ratio % # %

19.8 52 35.13 1 (reference)

2.7 47 31.75 .15

34.0 26 17.56 3.52

17 12 8.1 3.74

26.5 11 7.43 6.19

"'~

L _ .. _.. u,1!1!4

F"

Table-9 Association of gall bladder cancer to frequency of Reuse of oil Mahavir Cancer Sansthan, Patna, Bihar, India, 2005

Frequency of

Reuse of cooking oil

Occasionally

Frequently

Daily

x2 for linear trend-7 .835

p value-0.005

-

#

74

34

20

Cases Control

n=147 n=148

% # %

57.8 85 71.43

26.56 28 23.53

15.62 6 5.04

Odds ratio

l(reference)

1.72

4.97

53

r !

. -

Table-10 Logistic regression analysis of factors associated with gall bladder cancer, Mahavir cancer sansthan, Patna, Bihar, 2005

Exposure factors

Socio-Demographic factors

Age More than 40years

Female sex

Residence in the Gangetic plains

Illiteracy

Tobacco users

Muslim religion

Dietary exposure factors .

Frequent consumption ofNon veg (> 2 days a week) Frequent consumption of fresh vegetables(>2 days a week)

. -

Missing breakfast twice a week or more

Frequently reusing oil

Odds Ratio

2.82

7.04

0.94

2.67

1.02

0.98

1.40

0.94

3.43

1.60

95% C.I.

-

1.44-5.56

3.92-12.64

0.53-1.69

1.49-4.80

0.53-1.97

0.41-2.33

0.78-2.55

0.24-3.78

1.74-6.77

0.84-3.06

54

List of Abbreviations

BMI

AJCC

UICC

GBC

TNM

CDKN2

Kras

TP53

Body Mass Index

American Joint Committee on Cancer

International Union against Cancer

Gall Bladder Cancer

Tumor Node Metastasis

Cyclin Dependent Kinase Inhibitor

Kirsten rat sarcoma viral oncogene

Tumor Protein 53

55

Patient information sheet

Name ofthe principal investigator: Dr. Varsha Singh Name of the organization: Mahavir cancer Sansthan, Patna, Bihar Collaborating Organizations: Regional Cancer Centre, Patna and National Institute OfEpidemiology, Chetmai.

Pranam,

I --------------------- a health worker, and my colleagues are part of the team from the department of p:r;eventive oncology Mahavir cancer sansthan, working under Dr. Varsha Singh -in-charge preventive oncology department are doing a survey to know the risk factors associated with cancer gall bladder in Bihar.

The incidence of cancer gall bladder is on increase in recent years. To determine risk factors of this cancer we need to ask questions to those who are suffering from cancer gall bladder and those who are suffering from equally severe different type of cancer. From ---- to ---- we will be asking some questions to you and using some information from your medical records. Answering these questions will take only about 30 minutes of your time. Taking part in this survey is totally voluntary. You can choose not to participate. You can choose not to answer a specific question or can stop answering the questions at any time without giving any reason. There is no benefit from this to you at present but your participation may benefit the community in future if we determine some of the risk factors.

The information collected by us will be totally confidential. We may ask questions about your certain dietary practices and life style. The question, which we ask, does not mean that they are certainly a risk factor for you to get the disease, neither it means that they are protective. You or your guardian may answer these questions. We will not write your name on this only a code/ID number will be used.

If you want to know more about this survey you may contact 1. Dr. Varsha Singh M.O.-In charge Preventive Oncology Department, Mahavir Cancer Sansthan, ph. No.- 9334319975,2275824 2. Miss Lincy Joseph, Cancer Registry Programme Assistant, Mahavir Cancer Sansthan

Thank you

Dated: Place: Patna, Bihar

56

Informed Consent Form

Serial Number: Dated:

Patient's identification number

Name of investigator:

I, ----------------------------------------------------------------------Son of/ daughter of/wife of -----------------------------------------------------------------------and resident of village ----------------------------------------of Panchayat ---------------- have read the foregoing informations/it has been read to me on ----------.

The informations are about the survey to determine the factors associated with carcinoma gall bladder in Bihar and certain dietary and other factors, which may be associated with this disease.

I have been told the purpose of the survey.

I have been told that taking part in this survey is totally voluntary. I consent voluntarily to participate as a subject in this survey and understand that I have the right to withdraw from the study at anytime without it any way affecting my further medical care at the Sans than.

Dated: (signature or thumb impression)

57

IDNUMBER Status

Checklist to confirm patients status

D 1. Cases 2. Controls

!.Registration at Mahavir Cancer Sansthan from May 2005 to September 2005 2. Diagnosis (site) 3 .Method of microscopic diagnosis 4.date of report

Who is answering the questions (put 1 if patient or 2 if attendant) l.where do you live ?

2. actual age( in years)

village area block

district state

3.gender(l. for male 2. for female) 4.Education

1.Graduate 2. > 1 0 years of formal education 3. 7-1 0 years of school 4. 5-7 years of school 5.can read and write 6.illiterate

5 .occupation !.government employee 2.private 3 .self-employed 4.agriculture 5.unemployed 6.retired 7 .others/specify

6.Marital Status(put 1 if married else 2)

For females only

7.Number of children 8.number of pregnancies

9 .Past Medical History

1. History of TB ( 1. for yes/2.no) 2. History of Asthma ( 1. for yes/2.no)

actual number actual number

58

' 3 .History of chronic liver disease ( 1. for yes/2.no) 4. History of hypertension ( 1. for yes/2.no) 5 .History of diabetes( 1. for yes/2.no) 6.History of typhoid ( 1. for yes/2.no) 7.Any other significant complaints (specify)

1 O .. History of past surgeries !.cholecystectomy 2.hysterectomy 3 .nephrolithotomy 4.lobectomy (lung) 5.any other (specify) 6.none

ll..p.resence of gall stone/stones in the gall bladder l.yes (q12-14) 2.no

12.no; of stone- l.single 2.multiple

13.size of stone I .in mm

14.position o;f the stone l.fundus

.Dietary factors

15.Are you a vegetarian-

16 .if non -vegetarian How often do you take non-veg meals

I. yes 2.no

2.body 3.neck 4.any other

!.occasionally

2.once a week 3.Twice a week

59

l 17. How many times do you eat in a day 18. At what time do you take you dinner 19 .at what time do you take your breakfast 20.do you ever miss any of your meals if yes

21. which oil do you use for cooking 22. What do you do with the oil that is left after cooking-

23 .how often do you reuse the leftover oil For cooking

24. Which dal do you take regularly

4.Daily

1. Specify !.specify. !.specify

then specify How often

1.occasionally 2.once a week 3.twice a week 4.Frequently 5.Daily

!.specify

1. Throw 2. Reuse

1. Occasionally 2. Once a week 3. Twice a week 4. Frequently 1. Chana 2. Moong 3. Arhar 4. Masoor

60