Title: The distribution of reproductive risk factors disclosed the heterogeneity of receptor-
defined breast cancer subtypes among Tanzanian women
Version: 1 Date: 09 Jun 2021
Reviewer's report:
This hospital-based, cross-sectional, case-only study provides a detailed summary of molecular
subtypes and other breast cancer characteristics among 263 breast cancer cases identified from a
single center in Tanzania, including an assessment of associations between various reproductive
factors and molecular subtypes. While the study provides valuable information about breast
cancer subtypes in an understudied population, there are multiple concerns with respect to the
study design, analysis, interpretation, and presentation of results. Please see below for specific
comments.
General:
• A major concern is the lack of consideration of selection bias, particularly survivor bias, as it
appears that prevalent breast cancer cases were included and survival is known to differ across
molecular subtypes.
• Please be cautious of the use of the words “risk”, “incidence”, and “protection” throughout
the paper. Since this is a case-only study (and a cross-sectional study) without a breast cancer-
free control group, associations cannot be interpreted as risk of developing a certain breast
cancer subtype. The case-only study design is only useful for assessing potential heterogeneity
of associations across subtypes; it cannot provide risk estimates of subtype-specific exposure-
disease associations (PMID: 26619806). For example, it may be more appropriate to state:
“Among breast cancer cases, those with the exposure (vs. those without the exposure) are more
likely to be (or have higher odds of being) subtype Y than subtype Z” or “…the prevalence of
subtype Y is higher than subtype Z among those with the exposure…”.
• Please proofread and check for grammatical errors throughout.
Abstract:
• A space is missing between many of the sentences in the abstract.
• Please spell out IDC-NST.
• In the Results section, the authors stated “Women aged #40 years showed 2-fold (p=0.004)
while post-menopausal status 1.3-fold (p=0.001) increased risk of luminal-A subtypes compared
to their counterparts”. By “counterparts”, I assume the authors meant comparing women aged
#40 to <40 years and post- to pre-menopausal. However, it is unclear what the comparison group
was for the outcome of interest. Were comparisons made for luminal-A vs. all other breast cancer
subtypes? Since a control group (women without breast cancer) was not specified in Methods,
it should be made clear that this is a case-only study. Please also see comment above to revise
interpretation/wording throughout the abstract.
Introduction:
• It would be helpful to provide a bit more background summarizing current literature on specific
reproductive factors associated with breast cancer molecular subtypes, with citation of more
recent studies (for example: PMID 30185547, PMID 26684063).
Methods:
• Study design and setting (line 105): Please specify that this is a case-only study.
• Study population:
- When stating the inclusion criteria, please specify the time period of case diagnosis. Also, was
the recruitment period between September 2019 and June 2020?
- Were cases incident or prevalent cases, i.e., were they recruited within a relatively short time
of their diagnosis? This is important to consider especially given possibly higher breast cancer
mortality rates in this population.
- Were both in situ and invasive breast cancer cases included?
- Why were cases with a score of 2+ for HER-2 expression excluded?
- What was the response rate? It is unclear if all cases who were alive at the time of recruitment
were included in the study.
• Data collection:
- It would be helpful to provide more details on the definitions of the main variables of interest.
For example, how were parity and menopausal status defined? And was it menopausal status at
the time of breast cancer diagnosis?
• Sampling procedure:
- Consider changing the title of this subsection to “Characterization of molecular subtypes”
or something similar. “Sampling procedure” is vague and can refer to sampling of study
participants.
- Please specify the percentage of cells stained for the receptors to be considered “positive”.
• Statistical analysis:
- Instead of performing separate analyses comparing luminal-A vs. non-luminal-A, luminal-
B vs. non-luminal-B etc., it may be more appropriate and efficient to conduct polytomous
logistic regression analysis to assess heterogeneity across the 4 molecular subtypes (see PMID:
26619806), using a single comparison group (usually the most common subtype). A p-value for
heterogeneity can be calculated using the Wald test. If pairwise comparisons are also desired,
the issue of multiple comparisons should be considered.
- What was the criterion used to evaluate confounding (e.g., change in OR)? It appears that only
age, BMI, and family history were included in final models. Were other potential confounders,
such as alcohol and smoking, considered?
- Please describe how the “combined effect” of parity and breastfeeding was evaluated.
- It should say “multivariable” (not “multivariate”) logistic regression throughout.
Results:
• Please see above comment re: interpretation of results
• Table 2: In addition to percentages, please also provide the numbers of cases.
• Table 3:
- For each risk factor, please specify the reference category.
- For age at menarche, there are three categories but only one OR estimate was presented
- Please clearly specify in footnotes which variables were adjusted for in the analysis.
- Were associations for breastfeeding duration evaluated?
• Pages 12 and 13: please check the text carefully. Many statements do not match the OR
estimates presented: for example: “…breast-feeding had 1.2-fold protection against luminal-A
subtype (OR: 1.98, 95%CI: 1.09-3.61, p=0.025).”’
• Suggest including Table 4 as a supplementary table as it is not included as part of results.
Instead, consider including supplementary tables 1 and 2 as main tables.
Discussion:
• First paragraph: As a case-only study with no information on denominators, these findings
cannot be interpreted as breast cancer incidence being higher in certain subgroups.
• Could differences in the prevalence of molecular subtypes compared to other African studies
(Table 4) reflect differences in how the cases were recruited with respect to time of diagnosis?
It seems that the present study only included those who were alive at the time of recruitment
and it is unclear if a long time has passed since diagnosis. If so, this may partially explain why
triple-negative cases (more aggressive) are less common in the present study.
• Again, the interpretation of results and conclusions need to be revised substantially, to not over-
interpret certain factors as “increases risk” or “protective against” of specific subtypes given the
lack of a control group.