Journal Club Presentation

Speaker
Dr. Abin K Rajan
Junior Resident

Moderator
Dr. Kathirvel S
Associate Professor

DCM & SPH

PGIMER, CHD
03/13/2024 1
 Title of the article & authors’ affiliation

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 No of citations received by a journal in one year for articles
published in the past 2 years / no of citable documents indexed

Journal Information*
in Web of science of the same journal during the same two years
No of citations received by a journal in one year for articles
published in the past 3 years / no of all documents indexed in
Scopus of the same journal during the same three years

• “frontiers in Nutrition” (2014), sub category of “frontiers” (2007) .

• 10th most cited journal in Nutrition and Dietetics category

• Composed of 12 different specialties including, Nutritional epidemiology,

Nutri-genomics, Clinical nutrition, Nutrition methodology and so on

• Total publications – 4,666

• Impact factor – 6.5, Cite score- 4.3, Total citations- 20,000+

• Current article has 1,104 views, 288 downloads and 5 citations

03/13/2024 https://www.frontiersin.org/about/impact 3
Abstract
• Large number of studies and reviews on association of cruciferous vegetable (CV) /
Isothiocynate (ITC) intake & cancer risk

• First of a kind study to identify the association of CV/ITC intake and ovarian cancer (OC)
survival

• Association between pre diagnosis CV/ITC intake with OC survival among 853 Chinese
patients between 2015-2020

• Kaplan-Meier technique plot survival curve and to estimate crude survival probabilities

• Cox-proportional hazard regression model for the association between variables

• Higher intake of CV/ITC was associated with better survival

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Introduction and Background

• Worldwide, 3 lakh new diagnosed cases of OC and 2 lakh new deaths

associated with OC were reported in 2020

• Overall 5 year survival rate for invasive epithelial OC is around 49

percentage*

• Previous research suggested intake of CV might increase OC survival

• CVs belonging to Cruciferae & Brassicaceae families, consist of

Broccoli, Cauliflower, Kohlrabi, Bokchoi, rape among others.

03/13/2024 *https://www.cancer.org/cancer/ovarian-cancer/detectio 5
n-diagnosis-staging/survival-rates.html
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Cont.
.
• Glucosinolates-GLS (Glucoraphanin) are a class of compounds ubiquitously contained in
CVs, when metabolized yields ITC (Sulforaphene), indole and nitrile derivatives which causes
cancer prevention & suppression

• The ITC yield depends on the cooking practice and the gut microbiota composition

• Different CVs contain different GLS and ITCs , some are volatile and lost while cooking

• CVS are associated with not only cancer prevention but also delay the progression of other
chronic diseases such as Diabetes & Cardiovascular diseases

• No of studies has been done to elucidate the protective effect of CVs on cancer initiation
and progression

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Cont…

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 Does dietary habit has any role
in the risk and mortality
associated with ovarian cancer
???

03/13/2024 https://gco.iarc.fr/2018 9
Methods
Study setting and duration

• Ovarian cancer follow up study (OOPS) to explore relationship between

pre/post-diagnostic, environmental exposure factors and OC survival

• Follow up and medical treatment was conducted at “Shengjing

Hospital of China Medical University, Shenyang, China”

• Total study duration was from January 2015- December 2020

Study population

• 18-79 years old females with histologically confirmed OC diagnosis

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 Recruitment of study participants  Flow of participants through study
853 eligible participants
enrolled as of
December 2020

796 agreed to
participate

744 (93%) returned • 24 excluded

completed self
for leaving
administered
more than 11
questionnaire
food items
empty in FFQ
• 17 excluded
703 participants for
considered for final implausible
study analysis calorie intake

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Data collection
• Relevant data of each participant was collected by means of Questionnaires,
Anthropometric measurements, Clinical information and biorepository.

• Questionnaires were self administered and filled out by trained interviewers during face
to face to interview

• A total of 10 relevant domains were included in the food frequency questionnaires

• Anthropometric evaluation were done as per the SOP

• Clinical information was extracted from the hospital database

• Biological specimen (blood, urine, feces) were collected at base line survey following SOP

and preserved at -80°C.

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Dietary Assessment
• Dietary intake was assessed at recruitment with a 111-item food frequency questionnaire
(FFQ), which has been verified to have reasonable reliability and validity.

• The reproducibility coefficients (spearman correlation coefficients and intra-class

correlation coefficients) were above 0.5 for most food groups, and correlation
coefficients (spearman correlation coefficients) were between 0.3 and 0.7 for most food
groups between the FFQ and weighed diet records.

• Participants reported their usual frequency of intake for standard serving sizes of food
items in the 12 months before OC diagnosis

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Cont…
• CV intake was calculated by summing the intake of Chinese
cabbage, pakchoi, kohlrabi, rape, broccoli, cauliflower, and
Never
2-3 times per month raphanus sativus
One time per week • Intake of each CV in grams/day was calculated through
2-3 times per week
multiplying consumption frequencies per day by fitted portion
4-6 times per week
1-2 times per day sizes (g/time).
>2 times per day • Spearman correlation coefficients and intra-class correlation
coefficients of reproducibility for CV were 0.61 and 0.53,
respectively
• The Chinese Food Composition Tables were used as the
nutrient database to calculate the daily ITC intake

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Follow-up
• Both active and passive follow up strategies

• Passive follow up by data extraction from Liaoning Providence Center for Disease Control
and Prevention and Shengjing Hospital database, every 6 months

• In the active mode, all the enrolled and surviving OC patients were invited for face to face
interview every 6 months

• The status of lifestyle factors was re-estimated using the same measurements as those in
the baseline survey

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Outcome of interest

• Death due to any cause

• Survival time was defined as the interval between histologic diagnosis and the

date of death due to any cause or the date of the last follow-up (March 31, 2021)

for patients who were still alive.

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Data analysis
• Distribution of categorical variables between the study groups were examined by chi-
squared test

• For continuous variables One-way ANOVA and Kruskal-wallis test used for normal and non
normal distributions respectively.

• The Kaplan–Meier technique was used to plot crude survival curves and estimate the
crude overall survival probabilities.

• Cox proportional hazards regression models were used to assess the associations of pre-
diagnosis CVs and ITC intake with overall survival

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 • SURVIVAL TABLE
• SURVIVAL CURVE
• LOG RANK TEST
• HAZARD RATIO
&
PROPORTIONAL HAZARD ASSUMPTION
• COX REGRESSION

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 SURVIVAL TABLE

Eg: 5 year survival after radiotherapy in 20 parathyroid cancer patients

rX= nX-1/2wx qX= dX/rX pX= 1-qX PX= pX*pX-1

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• SURVIVAL CURVE

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 Log Rank test: Non parametric significance test for comparing survival curves
H0: No difference in the rate of event between the two groups

pd= (d1+d2)/(n1+n2) e1= pd*n1 e2= pd*n2

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 Hazard ratio: ratio of risk of event (death) between 2 groups
Proportional Hazards Assumption: Hazard ratio is same between both groups at all times

Risk of event (death) = observed deaths/ total population at risk

Population at risk is directly proportional to number of expected deaths

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Cox proportional hazards regression model

• For a person surviving up to a time point t, the probability of an endpoint (death)

instantaneously at time t is h(t), which is an unknown function of time.

• The probability is called HAZARD and h(t) is the hazard function

• h0(t) ; when all predictor variables are zero

• h(t) ; when predictor variables are non zero

• h(t)/h0(t) ; hazard ratio, depends only on value of predictor variables, not on time

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Cont…

• The proportional hazards assumption was tested through inclusion of an interaction term
between each activity variable and log survival time, and no violations were found (all p >
0.05).

• CV and ITC intake was categorized by tertile distribution, wherein the lowest tertile
served as the reference group

• The linear trend tests were performed by assigning the median value of consumption for
each tertile of CVs and ITCs and treating it as a continuous variable in the respective
regression models

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Cont…
• Methods for checking Proportional Hazard Assumption

1. Graphical method
• Comparing –ln (-ln) curves of both groups: Curves should be parallel to each other
and the vertical distance should be constant at all times
• Comparing observed vs expected curves of groups: both observed and expected
curves should be close to each other

2. Goodness of fit method: Using residual of predictor variables

3. Time dependent variable method: By adding cross product term

• Multiply the time independent variable with time (cross product term)

• Run the regression statistic

• If p<0.05, then Proportional hazard assumption is not valid 26

Cont…

• Stratified analyses were used to assess potential effect modification by menopausal

status (no compared with yes), residual lesions (no compared with yes), histological type
(serous compared with non-serous), and BMI (<25 compared with 25 kg/m2).

• Potential interactions of CV and ITC intake with these stratifying variables were assessed
by the addition of cross-product terms to the multivariable Cox models.

• All analyses were performed in SAS version 9.4 (SAS Institute, Cary, NC, USA). Two-sided
P < 0.05 were considered statistically significant.

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RESULTS

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Discussion
• Subgroup Analysis showed that Chinese cabbage and Kohlrabi has statistically significant
association (protective effect) with survival

• Similar studies conducted in Australia by Dolecek et al, Nagle et al showed same results as of
the present study

• One study by Playdon et al revealed no association between CV intake and mortality, but
included advanced OC patients, advanced age and lower set intake threshold for categories

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Cont…
• Glucosinolates (Glucoraphanin) are a class of compounds ubiquitously contained in CVs,
when metabolized yields ITC (Sulforaphene), indole and nitrile derivatives which causes
cancer prevention & suppression
Gut microbiome

GSH
ITC-GSH
Enterocytes

Blood

ITC-NAC Liver & kidneys

Excreted via feces and urine ITC-GSH
ITC-CYS Mercapturic acid
pathway
Free ITC
Cancer prevention and
+
suppression Tissues Blood
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protein
36
 Pro- carcinogen PHASE 1 reaction carcinogen

- CYP
enzymes
-
SFN
Nrf2
+ +
Keap Nrf2 NQ01 Γ-GCS
Keap 1
1

H01

PHASE 2 Enzymes
03/13/2024 37
 -
SFN

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 Metabolic Fate of Dietary Glucosinolates and Their
Metabolites: A Role for the Microbiome

• Inter individual variation in ITC conversion rates between 11-40 %

• Crop growing condition, cooking practices, individual characteristics

• Myrosinase activity is absent in mammalian cells

• Cooking inactivates the myrosinse enzyme

• Gut microbiome has myrosinase like activity

• Many microbes in human GIT are unculterallable and in vitro mono culture lacks microbe
– microbe interaction, microbe-complex food interaction & microbe-host interaction
Bouranis JA, Beaver LM and Ho E (2021) Metabolic Fate of Dietary Glucosinolates and Their
03/13/2024 Metabolites: A Role for the Microbiome. Front. Nutr. 8:748433. doi: 10.3389/fnut.2021.748433 39
03/13/2024 40
Cont…

• Studies also revealed two distinctive times of CVs metabolism, first right after intake of
food and 2nd during entero-hepatic circulation

• In another study, the participants were fed cooked broccoli and evaluated 24 hour urine
ITC levels. Fecal samples of participants with highest and lowest ITC concentrations were
cultured and incubated with GRP for 1-2 months and found no significant difference in
the microbiome composition between two groups

• Genes and their respective enzymes responsible for GLS conversion is yet to be identified

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Cont…

• Some microbiome has been identified who preferably converts GRP to Nitriles, but these
studies has been done in vitro, lacking simulation of original conditions in gut.

• Eg: microbes with enzymes capable of converting GLS to desulfoglucosinolates, which

only metabolized to Nitriles not ITC

• The GLS can also be reduced to their redox partners (eg: Glucoiberverin) by gut enzymes
or microbiomes and their metabolism is also not well understood

• Microbiome composition of gut is heavily influenced by the diet intake with high or low
levels of CVs.
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Cont…
• Such different composition of micrbiomes in the gut in turn can have effect on the overall
health of the host itself.

• (eg: high intake of CVs decreases the concentration of sulfate reducing bacteriae, which is
associated with IBD. Retrospectively sulfate reducing bacteria convert GLS to desulfo-GLS,
yielding less ITC)

• It was also found that components other than GLS in the CVs were also implicated in the
activity of the gut microbiome, which in turn associated with ITC production

• Chronic exposure with CVs increased the myrosinase like activity of the micrbiomes and
the bioavailability of ITCs

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Cont…
• To address these uncertainties, a system biology based approach can be utilized

• High throughput sequencing helps in rapid and deep sequencing along with advanced
bioinformatics for data analysis has wider scope

• 16S sequencing along with metagenoimic sequencing helps in identifying the composition
and the function of microbiome

• Mulit-omics methods (metatranscriptomics, metaproteomics, and metabolomics) is based

on data driven approach which incorporates machine learning techniques and data
reduction techniques

• These approaches will be the key to better understand the interplay between GLS
metabolism, the gut microbiome and cancer prevention
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Strengths and Limitations

• First of a kind study to evaluate survival associated with CV/ITCs

• Well designed prospective cohort study with high baseline survey participation and
follow up retention rates

• Validated FFQ administered by face to face interview

• Statistical techniques to account for potential confounders

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• Could not assess the intake of CVs directly (information bias)

• Limitations in generalizability of findings to other geographical regions due to different

dietary habits

• Did not account for progression free survival

• Taken all cause mortality instead of disease specific mortality

• Influence of unmeasured and unaccounted confounders.

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Critical evaluation

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Summary

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STROBE Checklist

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