Study of Docosahexaenoic Acid (DHA) in Triple Negative Breast Cancer Survivors: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Study of Docosahexaenoic Acid (DHA) in Triple Negative Breast Cancer Survivors (NCT01849250), a randomized phase 2 study. The primary outcome is Normal Breast Tissue Expression of Tumor Necrosis Factor Alpha (TNF-alpha) Levels.

2. Clinical Question

In plain terms, the study asked whether the assigned supplement and placebo groups differed in a measured breast-tissue marker after the 12-week treatment period, taking the baseline marker measurement into account. This is a question about a laboratory measure in tissue, not a direct comparison of breast cancer recurrence or survival. The registry's brief summary mentions prevention of recurrence as the broader study aim, but the specified primary endpoint is TNF-alpha expression. Outcomes related to recurrence are not reported as the primary measure in the record.

The registry reports enrollment of 65, but does not provide enough information to describe the enrolled participants' diagnostic mix, baseline characteristics, or the number allocated to each arm for the primary endpoint. It reports that some participants lacked tissue for quantitative RT-PCR and were not evaluable for the primary endpoint. Those missing tissue measurements mean the primary analysis population was not necessarily the same as all enrolled participants.

3. Trial Design

This was a randomized, double-masked, parallel-group phase II study sponsored by the National Cancer Institute. The registry reports enrollment of 65. The study began in 2013-05, reached primary completion on 2016-01-11, and has a completed status. The registry does not report the allocation sequence method, who specifically was masked, or how masking was maintained.

Participants were assigned to docosahexaenoic acid or placebo. The DHA arm took the supplement orally twice a day for 12 weeks; the placebo arm followed the same stated schedule. A parallel-group design assigns participants to one study arm and compares their outcomes across arms, rather than having every participant receive both conditions in sequence. Randomization is intended to support a fair comparison by making assignment independent of measured and unmeasured participant characteristics on average, although it cannot guarantee that groups are identical in a trial of any particular size. This is general information about randomized designs, not a claim about the balance achieved in this study.

The official study title describes patients with a history of breast cancer, premalignant lesions, or benign breast disease, while the brief summary emphasizes breast cancer survivors. The registry lists multiple breast conditions, including benign neoplasm, ductal and lobular carcinoma in situ, invasive carcinoma, Paget disease, and stages of breast cancer. The record does not report the number enrolled within each condition, so the precise composition of the study population cannot be established from the listed information.

The main endpoint was normal breast tissue TNF-alpha expression, measured at baseline and 12 weeks. The analysis description specifies analysis of covariance (ANCOVA), using the post-treatment measurement as the dependent variable and the pretreatment measurement as a covariate. Six participants in the placebo group and one participant in the DHA group were not evaluable for this endpoint because tissue was unavailable for quantitative RT-PCR. The registry does not report the full protocol, sample-size justification, randomization details, or a complete statistical analysis plan. These details should not be inferred from the phase designation or the stated analysis.

4. Participant Flow and Accounting

The registry reports how participants moved through each stage of the study. All counts below are exactly as posted; missing counts are not inferred from other stages.

Before assignment: Of 65 participants enrolled, one was not eligible thus excluded from the study.

Overall Study

Reasons for not completing this period:

Analysis population for the primary outcome, as defined in the registry: 6 participants in the Placebo group and 1 participant in the DHA group were not evaluable due to tissue was not available quantitative RT-PCR.

5. Baseline Characteristics

The table reproduces the baseline characteristics posted in the registry, which describe the participants analysed at baseline and help show whether the randomized groups were comparable.

6. Primary Endpoint

The registry lists the primary outcome as Normal Breast Tissue Expression of Tumor Necrosis Factor Alpha (TNF-alpha) Levels, measured over Baseline to 12 weeks.

The primary endpoint was normal breast tissue expression of tumor necrosis factor alpha (TNF-alpha), reported on a log-transformed relative-expression scale. The registry describes measurements at baseline and 12 weeks, with the principal comparison based on post-treatment expression and adjustment for pretreatment expression. Tissue was obtained by breast core biopsy, and TNF-alpha messenger RNA was measured using quantitative real-time polymerase chain reaction on extracted RNA. Relative expression was determined using the stated delta-delta CT analysis protocol.

This endpoint is a tissue biomarker rather than a direct clinical event such as recurrence. In general, measuring a biological marker can help characterize whether groups differ in a biological process over the study period. That can answer a narrower question than whether a treatment changes a patient's clinical outcome. The registry does not state that TNF-alpha expression is a validated surrogate for recurrence, nor does it report a clinical benefit linked to the marker.

Using the baseline value in an ANCOVA comparison can account statistically for initial differences in the measured marker and focus the comparison on post-treatment values conditional on baseline. This is a general explanation of the method. It does not establish what the adjusted estimate was in this trial: the group means, standard deviations, adjusted difference, confidence interval, and p-value for TNF-alpha are not reported in the registry record. Because tissue was unavailable for some participants, the endpoint also does not represent an observed measurement for everyone enrolled.

7. Why No Re-analysis Is Shown

8. Statistical Interpretation

9. Statistical Methods Explained

The primary analysis description is an analysis of covariance, commonly abbreviated ANCOVA. It uses the post-treatment TNF-alpha measurement as the outcome and includes the pretreatment TNF-alpha measurement as a covariate. Treatment group is the comparison of interest. This description comes from the registry record; details such as the exact model specification, diagnostics, and handling of missing observations are not reported there.

What does baseline adjustment do?

In general, baseline adjustment compares groups at follow-up while accounting for their measured starting values. This can improve precision when baseline and follow-up measurements are related, and it can account for an observed imbalance in baseline values. ANCOVA is not the same as calculating a change score for each participant and comparing those changes, even though both approaches use baseline and follow-up information. The registry specifies ANCOVA, so it would be inaccurate to describe the primary analysis as a simple comparison of mean changes.

The adjusted contrast is interpretable only alongside its estimate and uncertainty. A confidence interval gives a range of values compatible with the data under the model and its assumptions; narrower intervals generally indicate greater precision. A p-value addresses compatibility with a specified null hypothesis under the analysis assumptions, but does not quantify the size or clinical importance of a difference. These are general statistical explanations. The registry record does not report the primary endpoint's adjusted estimate, confidence interval, or p-value, so none can be interpreted for this trial.

Why use a log-transformed expression measure?

The primary endpoint is stated as log-transformed relative expression. In general, transforming a measure can make its distribution or relationship with other model terms more suitable for a statistical analysis, depending on the data. On a log scale, differences do not have the same direct interpretation as differences on the original scale. An interpretation on the original scale may require an appropriate transformation of an estimate and its interval. The registry does not report a numerical estimate or specify additional details about the scale used for reporting results, so no back-transformed interpretation is possible.

The tissue assay was quantitative real-time PCR on RNA extracted from breast core biopsies, with relative expression determined using a delta-delta CT protocol. This identifies the stated measurement approach, but the record does not report assay precision, laboratory quality-control results, sample processing details, or how measurements below assay limits were handled. Those aspects should not be assumed.

How should the randomized comparison be understood?

In general, random allocation helps make treatment groups comparable in expectation, reducing systematic confounding by participant characteristics. It does not guarantee balance in a particular trial, especially when the study population is limited. Masking can reduce the influence of expectations on participant behavior, care, and outcome assessment, depending on who is masked and how outcomes are measured. Here the registry calls the study randomized and double-masked, but does not state which parties were masked or provide the allocation procedure.

The parallel design means the assigned arms were intended to be compared over the same treatment period. The record specifies DHA or placebo taken orally twice a day for 12 weeks. The posted registry description does not establish adherence, treatment discontinuation, crossover, or exposure beyond the reported schedule. RBC fatty-acid level is listed among secondary outcomes as a surrogate of compliance, but numerical results for it are not reported.

What does missing tissue mean for analysis?

Six participants in the placebo group and one participant in the DHA group were not evaluable for the primary endpoint because tissue was unavailable for quantitative RT-PCR. In general, missing outcome data can reduce precision and can introduce bias if the likelihood of having an observed measurement is related to the outcome or differs in important ways between groups. The impact depends on why the data are missing and how the analysis handles them. The registry does not report the missing-data method, the full analysis population, or any sensitivity analyses. Therefore, the effect of these unavailable samples on the primary comparison cannot be quantified.

The registry lists seven outcome measures and four analyses. Multiple outcomes or analyses can raise multiplicity concerns: when many hypotheses are tested, the chance of at least one apparently notable result can increase. In general, prespecification and an appropriate adjustment or interpretation plan can address this issue. The record does not report a multiplicity strategy, nor does it provide enough details to determine which outcomes were treated as confirmatory or exploratory.

Sample-size calculations in general are planning tools that depend on assumptions about the effect to detect, variability, significance level, allocation, and statistical method. The record describes planning illustrations based on an observed effect and states assumptions of a two-sided significance level of 0.05, equal allocation, and a normal approximation. Such illustrations are not observed power and do not replace the trial's original design justification. The trial's own sample-size justification is not reported in the registry record. No numerical illustration is interpreted because the corresponding calculation values are not reported in the registry record for this account.

10. Multiplicity, Missing Data and Analysis Populations

The registry lists seven outcome measures and four analyses. The outcomes cover the primary TNF-alpha expression measure and secondary measures involving crown-like structures, other breast-tissue biomarkers, and RBC fatty-acid level as a compliance surrogate. Considering multiple outcomes can create multiplicity: in general, testing many hypotheses without an appropriate plan can increase the chance of a false-positive finding somewhere among them. The registry record does not report whether a multiplicity adjustment was used, how outcomes were prioritized beyond identifying the primary endpoint, or which analyses were considered confirmatory.

For the primary endpoint, the registry specifically identifies six participants in the placebo group and one participant in the DHA group as not evaluable because tissue was unavailable for quantitative RT-PCR. These measurements therefore could not contribute as observed assay results. The record does not report the method used to handle missing values, whether the analysis was restricted to evaluable participants, whether any imputation was conducted, or whether sensitivity analyses assessed the impact of missingness.

In general, analyses restricted to participants with observed outcomes can be biased if availability of an outcome is related to the outcome itself or to factors associated with it. Imputation and other missing-data approaches also depend on assumptions, which should be described and examined. The reason for missing tissue is reported, but that alone does not establish whether the missingness was unrelated to the endpoint or whether the analysis remains unbiased.

The record does not report a complete definition of the analysis population, such as whether all randomized participants were included in the primary analysis, or whether additional participants were excluded for other reasons. It therefore is not possible to determine the analysis denominator for the primary endpoint from the available information. The registry's enrollment figure should not be treated as the number with an observed primary endpoint.

11. Safety Results

The registry reports serious adverse events as affected participants divided by participants at risk.

Other (non-serious) adverse events

Participants with at least one non-serious event at or above the registry's 0% reporting threshold.

Most frequent non-serious adverse event terms

The registry reports zero serious adverse events among 32 participants at risk in the placebo group and zero among 32 at risk in the DHA group. It reports other adverse events among 17 participants in the placebo group and 15 in the DHA group, with 32 at risk in each group. These are participant counts as categorized in the registry, not descriptions of the event types, severity distribution beyond the serious category, or timing.

The adverse-event information should be read as descriptive. Zero reported serious events in each group does not prove that serious harms are impossible or that the risks are identical. Likewise, the counts for other adverse events do not by themselves establish a meaningful difference between groups. No statistical comparison or uncertainty interval for these counts is reported in the registry record.

The registry does not list serious adverse-event terms, and death counts are not reported in the registry record. The event definitions, collection procedures, exposure-adjusted rates, and detailed follow-up information are also not reported. The at-risk counts refer to the participants shown for each group in the adverse-event record and should not be assumed to describe the denominator for every outcome or analysis.

12. What the Result Does, and Does Not, Mean

The record establishes that a randomized, double-masked, parallel-group phase II trial compared DHA with placebo over 12 weeks, with normal breast tissue TNF-alpha expression as the primary endpoint. The primary analysis was described as ANCOVA, adjusting post-treatment expression for the pretreatment value. These registry entries define the intended comparison and the type of measurement used.

What the record does not establish is the primary endpoint result. It does not report an adjusted difference, arm-specific TNF-alpha values, a confidence interval, or a p-value. It is therefore not possible to say from this record whether expression was higher or lower in either group, how large any difference was, or how precisely it was estimated. The description of an analysis method is not a substitute for its result.

Even if a biomarker result were available, a difference in tissue expression would be evidence about that measured marker, not automatically evidence about recurrence or another clinical outcome. The registry's broader study summary refers to prevention of recurrence, but the listed primary endpoint is a tissue biomarker. The record does not report that this biomarker is a validated surrogate for recurrence, nor does it provide a recurrence comparison as the primary result.

For the same reason, secondary outcome titles should not be mistaken for findings. The record lists crown-like structures, COX-2, IL-beta, aromatase, and RBC fatty-acid level, but their numerical results are not reported. The adverse-event counts describe reported event categories but do not establish comparative safety, particularly without event details and uncertainty estimates.

The appropriate interpretation is bounded by what the record reports: the design, treatment schedule, endpoint measurement approach, endpoint-specific tissue unavailability, and selected adverse-event counts. Claims about the size, direction, clinical importance, or statistical significance of the primary treatment contrast are not supported by the registry information available for this analysis.

13. Why This Trial Matters Statistically

This trial illustrates the distinction between a clinically motivated research aim and the endpoint used to evaluate it. The study summary frames the work around recurrence prevention, while the primary endpoint is normal breast tissue TNF-alpha expression measured from baseline to 12 weeks. Statistical interpretation must stay close to the measured outcome: a marker comparison and a recurrence comparison answer different questions.

It also highlights why the analysis model matters. The registry describes an ANCOVA that compares post-treatment values while accounting for pretreatment expression. That is more specific than saying the study compared simple changes, and it shows why the exact endpoint and model should be read together. At the same time, a method description without its estimate and uncertainty does not reveal what the comparison found.

The endpoint-specific tissue unavailability is another important statistical feature. The record identifies participants who could not be evaluated because assay tissue was unavailable, but does not explain how this affected the analysis population or whether missing-data sensitivity analyses were performed. This illustrates how conclusions depend not only on model choice but also on which participants have analyzable outcomes.

Finally, the registry contains a broader set of outcome measures, while its primary numerical results are not reported in the record examined. Multiple outcomes require a clear analysis and interpretation plan, and descriptive event counts need their denominators and context. These considerations are relevant beyond this study: readers need the endpoint estimate, its uncertainty, the analysis population, and information about missing observations to judge how much the data establish.

14. Limitations

• The posted summary statistics are the only data these results rest on; patient-level data, full protocols and statistical analysis plans are not available.
• Recalculated results are simple, unadjusted calculations from posted summaries. They do not replace the sponsor's prespecified analysis.

The central limitation for statistical interpretation is that numerical results for the primary TNF-alpha endpoint are not reported in the registry record. There is no adjusted contrast, group-level summary, confidence interval, or p-value with which to assess direction, magnitude, or precision. The same absence applies to the listed secondary outcomes in the information.

The record reports endpoint-specific missing tissue: six participants in the placebo group and one in the DHA group were not evaluable for quantitative RT-PCR. It does not report the full analysis denominator, the method for handling missing outcomes, or sensitivity analyses. The reason tissue was unavailable is stated, but the relationship between sample availability and the endpoint is not reported. This prevents assessment of potential missing-data bias.

Although the study is identified as randomized and double-masked, the sequence-generation process, allocation concealment, and the parties included in masking are not reported in the registry record. The record also does not provide a full protocol or complete statistical analysis plan, so the prespecification of analyses and the handling of multiple outcomes cannot be assessed. The trial's own sample-size justification is not reported.

The listed conditions span benign, premalignant, and cancer diagnoses, but the number of participants in each subgroup and detailed baseline characteristics are not reported. Therefore, the exact population composition and the applicability of any result to a particular subgroup cannot be determined from this record. The record also does not supply detailed assay-quality information or numerical results for the RBC fatty-acid compliance surrogate.

Adverse-event information is limited to participant counts in serious and other categories. Event terms, timing, collection methods, and death counts are not reported in the registry record. These omissions limit interpretation of both efficacy and safety; they do not show that an unreported result was favorable or unfavorable.

15. Related Tutorials

16. Related Statistical Calculators

17. Sources

• ClinicalTrials.gov: Study of Docosahexaenoic Acid (DHA) in Triple Negative Breast Cancer Survivors — NCT01849250.
• Registry record last updated 2021-12-28; status last verified 2021-12.
• Results first posted 2021-12-28.
• Data retrieved from ClinicalTrials.gov on 2026-11-09.
• Group sizes, means, event counts and sponsor-reported p-values are reproduced as posted. Effect estimates, confidence intervals and p-values not attributed to the sponsor are independently calculated from the posted summary statistics.

18. Record Summary

NCT01849250 is a completed randomized, double-masked, parallel-group phase II study sponsored by the National Cancer Institute. Enrollment was 65. The study began in 2013-05 and reached primary completion on 2016-01-11.

Participants received oral DHA or placebo twice a day for 12 weeks. The primary endpoint was normal breast tissue TNF-alpha expression at baseline and 12 weeks, measured by quantitative real-time PCR and analyzed using ANCOVA as described in the registry. Six placebo participants and one DHA participant were not evaluable for the primary endpoint because tissue was unavailable for quantitative RT-PCR.

Numerical primary and secondary outcome results are not reported in the registry record. Adverse-event information reports zero serious events among 32 at risk in each group, and other adverse events among 17 placebo and 15 DHA participants. The registry does not report the trial's sample-size justification, full statistical analysis plan, or detailed missing-data methods.