A Study to Determine the Immunogenicity and Oral Tolerance to Keyhole Limpet Hemocyanin (KLH): Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for A Study to Determine the Immunogenicity and Oral Tolerance to Keyhole Limpet Hemocyanin (KLH) (NCT01489956), a non-randomized early/phase 1 study. The primary outcome is Participants With a Positive Immune Response to T Cell Stimulation Index (SI) as Measured by 3H-thymidine Incorporation After in Vitro KLH Stimulation of PBMC (Part A).

2. Clinical Question

The broader purpose was to examine oral tolerance to KLH and whether Immucothel alone was strong enough to trigger an immune response. If that response was not observed according to the Part A condition, the plan described testing Immucothel with Montanide. Part B was intended to assess whether oral KLH feeding was associated with suppression or non-activation in T-cell measures, as well as other mechanistic measures. These aims describe questions the study set out to investigate; they do not establish what the results were. Primary endpoint results and the detailed results for the secondary outcomes are not reported in the registry record.

3. Trial Design

The registry classifies this as an early phase 1, non-randomized, unmasked, single-group study. Enrollment was 19. The record lists a start date of 2011-12, primary completion in 2013-04, and status as terminated. It does not report why the study was terminated. The registry does not identify a randomized control group, and the described treatment groups should not be read as equivalent groups created by random allocation.

Part A began with Immucothel alone, described as 100 µg administered subcutaneously on Day 0 and Day 9. The protocol description made the addition of an Immucothel-plus-Montanide group conditional: if an immune response was not observed in at least nine out of the first 10 participants after Immucothel alone, 10 additional healthy subjects would be recruited for the combination. This is a response-guided sequence, not a randomized comparison. The registry safety information lists no participants at risk in the Immucothel-plus-Montanide Part A group, but does not explain the operational history behind that figure.

Part B was also dependent on Part A results. The record describes feeding 50 mg of native KLH on Days 0 through 4 and Days 10 through 14, for a total dose of 500 mg, and then states that participants were to be immunized. The remainder of that arm description is not reported in the registry record, so the full Part B procedure cannot be reconstructed from this information. The study included mechanistic outcomes involving T-cell stimulation, cytokine secretion, and antibodies. These outcomes broaden the scientific questions but do not substitute for the absent primary endpoint results. Allocation procedures beyond the stated non-randomized design, masking, and the reasons for termination are not reported in the registry record.

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: The accrual objective was 10 to 20 evaluable participants in Part A and 10 evaluable participants in Part B. The definition of an evaluable participant (measured/determined by lymphocyte proliferation assay) was provided in the protocol.

Overall Study

Reasons for not completing this period:

Analysis population for the primary outcome, as defined in the registry: Intent-to-treat

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.

Population: Enrolled Sample

6. Primary Endpoint

The registry lists the primary outcome as Participants With a Positive Immune Response to T Cell Stimulation Index (SI) as Measured by 3H-thymidine Incorporation After in Vitro KLH Stimulation of PBMC (Part A), measured over Day 16.

The primary endpoint counted participants with a positive immune response in Part A, using the T-cell stimulation index measured by 3H-thymidine incorporation after in vitro KLH stimulation of peripheral blood mononuclear cells. The endpoint was assessed on Day 16. The stimulation index is defined as incorporation by T cells exposed to KLH divided by incorporation by T cells without stimulation. The registry defines an SI of 3 or higher as a positive immune response, and notes that higher values correspond with lower tolerance to KLH.

A threshold-based endpoint turns a laboratory measurement into a count of participants meeting a prespecified criterion. That can make a biological response easier to describe as a proportion or event count, but it also discards information about how far an individual measurement lies above or below the threshold. Participants with different SI values may therefore be counted the same way if both meet the criterion. The continuous SI results and their distribution are not reported in the registry record.

This endpoint was used to assess immunogenicity in Part A, consistent with the study’s stated aim of determining whether Immucothel triggered an immune response. However, the registry record gives the endpoint definition rather than observed participant counts. It does not report how many participants had a positive response, the total analyzed for this endpoint, or an uncertainty estimate. The endpoint’s intended meaning can be described, but the actual response cannot be inferred from the definition alone.

7. Why No Re-analysis Is Shown

8. Statistical Interpretation

9. Statistical Methods Explained

What does a threshold endpoint represent?

A threshold endpoint classifies each measured response according to whether it meets a defined boundary. In this study, the registry states that an SI of 3 or higher on Day 16 indicates a positive immune response. The resulting outcome is a participant count: participants meeting the rule are counted as events, while those who do not meet it are not counted as events. The registry’s endpoint definition does not report the actual count or the number evaluated.

Thresholding simplifies interpretation, but it changes the information being analyzed. A participant just at the threshold and a participant with a much higher SI both satisfy the positive-response definition. Conversely, measurements on opposite sides of the threshold are classified differently even if they are close. Reporting the distribution of the underlying continuous measurements alongside the threshold count can help readers understand what the classification leaves out. Such continuous results are not reported in the registry record.

How should a count be interpreted?

A count of participants with an event is descriptive unless it is accompanied by an analysis that defines the population, denominator, and uncertainty. A count alone does not establish how common the event was among all participants enrolled or among those eligible for assessment. For this endpoint, the intent-to-treat population is named, but the number analyzed and handling of participants without a Day 16 measurement are not reported in the registry record.

In general, a confidence interval expresses uncertainty around an estimate using the observed data and a stated statistical method. It is not a guarantee that the true value falls within the interval, nor does it resolve limitations in the design or missing data. No confidence interval for the primary response is reported in the registry record. A p-value, in general, describes how compatible data are with a specified statistical model and null hypothesis; it is not the probability that the null hypothesis is true. No primary endpoint p-value or null hypothesis test is reported in the registry record.

Why does allocation matter?

Random allocation is a method for making treatment assignment independent of participant characteristics on average. This study is explicitly described as non-randomized, so that protection against systematic differences between groups is not established. The Part A plan also made the combination group conditional on the response observed in the Immucothel-alone participants. That creates a sequence in which group membership and treatment are linked to prior study findings. It is therefore not appropriate to interpret the groups as if they were randomized arms in a controlled trial.

The study is classified as single-group, while its intervention descriptions refer to different parts and treatment options. Those labels do not turn the study into a randomized parallel-group trial. A comparison between Part A and Part B would also need to account for the different study procedures and the dependency of Part B on Part A. The registry record does not provide a comparative analysis plan or reported comparative results.

What do the planning assumptions mean?

The record describes a planning illustration based on the observed effect, with a two-sided significance level of 0.05, equal allocation between two arms, a normal approximation, and an effect defined as a stated fraction of the observed effect. These are assumptions for a planning calculation, not evidence that the trial itself used that design or those assumptions. No numeric planning estimates are reported in the registry record. Such planning values are not observed power and do not describe the certainty of the study’s results.

The assumptions also do not match the study’s non-randomized, response-guided structure in a straightforward way. In general, sample-size calculations depend on the outcome, target effect, variability or event frequency, allocation, and chosen error rates. If planning inputs do not reflect the actual design and analysis, the resulting illustration should not be treated as a study-specific justification. The trial’s own sample-size justification is not reported in the registry record. The record also does not report a planned interim analysis or stopping boundary.

What can mechanistic outcomes add?

The secondary outcome descriptions include T-cell cytokine secretion, T-cell stimulation measured by CFSE staining, suppression or non-activation after oral feeding, assessments of archived serum samples, and comparison of KLH-specific antibody levels between parts. These measures relate to different biological processes and may provide complementary descriptions. They are not interchangeable with the primary endpoint, which is a thresholded T-cell stimulation index based on 3H-thymidine incorporation.

Each outcome can have its own measurement timing, analysis population, and missingness. Those details and the outcome results are not reported in the registry record. Without them, it is not possible to determine whether the mechanistic measures support, differ from, or qualify the primary endpoint. Their listing should be read as a description of planned assessment domains, not as evidence that a particular biological effect was observed.

10. Exact Estimates for Each Arm

No two-arm comparison is shown for this trial, but each arm's result can still be summarized with exact interval estimates computed from the posted counts. The Clopper-Pearson interval is exact and conservative; the Wilson interval is a common closer-to-nominal alternative; the Bayesian credible interval uses a uniform Beta(1, 1) prior. For small groups the three can differ noticeably.

R code and output

arm    <- c("Immucothel Alone (Part A)")
n      <- c(10)
events <- c(9)
for (i in seq_along(n)) {
  ci <- binom.test(events[i], n[i])$conf.int          # Clopper-Pearson exact interval
  cr <- qbeta(c(0.025, 0.975), 1 + events[i], 1 + n[i] - events[i])   # Bayesian, uniform Beta(1,1) prior
  wi <- (function(x, n) { p <- x / n; z <- qnorm(0.975); d <- 1 + z^2 / n
    c((p + z^2 / (2 * n) - z * sqrt(p * (1 - p) / n + z^2 / (4 * n^2))) / d, (p + z^2 / (2 * n) + z * sqrt(p * (1 - p) / n + z^2 / (4 * n^2))) / d) })(events[i], n[i])
  cat(sprintf("ARM|%s|%d|%d|%.1f|%.1f|%.1f|%.1f|%.1f|%.1f|%.1f\n", arm[i], n[i], events[i], 100 * events[i] / n[i],
              100 * ci[1], 100 * ci[2], 100 * wi[1], 100 * wi[2], 100 * cr[1], 100 * cr[2]))
}

ARM  Immucothel Alone (Part A)  10  9  90.0  55.5  99.7  59.6  98.2  58.7  97.7

11. Multiplicity, Missing Data and Analysis Populations

The registry lists a primary endpoint and multiple secondary outcome domains, including cytokine profiles, CFSE-based stimulation measures, serum assessments, and antibody comparisons. Testing many outcomes can increase the chance of at least one apparently notable result if each is assessed separately. Statistical methods for addressing multiplicity may include prioritizing a primary hypothesis, adjusting significance thresholds, or treating secondary findings as exploratory. Which, if any, approach was used is not reported in the registry record. The number of analyses reported is 0, but that registry field does not establish whether analyses were conducted outside the record.

Missing-data handling is also not reported in the registry record. The listed intent-to-treat population indicates the named population for the primary outcome, but does not by itself specify which participants were included in an endpoint calculation or how unavailable measurements were handled. The record does not report endpoint-specific denominators, reasons for missing measurements, imputation methods, or sensitivity analyses. These omissions matter because an event count depends on both the number meeting the response rule and the number with usable assessment data.

The study’s non-randomized and conditional structure adds another interpretive concern. The planned route to the combination group depended on Part A findings, while Part B depended on Part A results. The registry does not report an analysis plan explaining how such dependencies, outcome selection, or multiple mechanistic measures were addressed. It also does not report the reason for termination or whether all planned assessments were completed. These details are necessary to judge how planned analyses compare with the analyses ultimately conducted.

12. 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 5% reporting threshold.

Most frequent non-serious adverse event terms

The registry safety information reports zero serious adverse events among 13 participants at risk in the Immucothel Alone Part A group and zero among six participants at risk in the Part B group. The Immucothel plus Montanide Part A group is listed with zero participants at risk and zero serious adverse events. A zero count in a group with no participants at risk does not provide safety experience for that group.

These counts are descriptive. They do not establish that an event is impossible or that the intervention is safe, and the record does not provide a comparative analysis. The number at risk is the relevant reported safety denominator for reading each group’s count; it should not automatically be used as the denominator for the primary endpoint or assumed to represent everyone enrolled.

The record does not list serious adverse event terms, and deaths are not reported in the registry record. It reports four other adverse events in the Immucothel Alone Part A group and four in Part B. The terms, severity, relatedness, timing, and participant-level distribution of those other events are not reported in the registry record. These summary counts therefore do not support conclusions about the nature or cause of the events.

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

The registry record identifies a biological question: whether Immucothel alone produced a T-cell response to KLH and whether oral KLH feeding was associated with suppression or non-activation in Part B assessments. It defines a positive primary response using an SI threshold measured on Day 16. These features make the study question and endpoint understandable, but they do not reveal the observed answer.

The posted information does not report primary endpoint counts, SI values, response estimates, or uncertainty intervals. It also does not report the results of the listed secondary mechanistic outcomes. Consequently, the record cannot establish whether the primary response criterion was met by any participant, how frequently it was met, or how results varied across participants. It cannot support a conclusion that oral tolerance was induced or that a particular intervention did or did not trigger an immune response.

Nor can the study be read as a causal comparison of Immucothel alone against Immucothel with Montanide or against Part B. The design was non-randomized, and the combination option depended on findings in Part A. Part B was also dependent on Part A results. Without random allocation, comparable groups cannot be assumed; without reported results and baseline characteristics, the magnitude and direction of any between-group contrast cannot be assessed.

Enrollment of 19 describes the recorded study enrollment, not necessarily the number with a complete primary endpoint or the number included in every analysis. The safety denominators reported for the groups differ from the overall enrollment, and endpoint-specific denominators are not reported. The terminated status is a registry fact, but the reason for termination is not reported in the registry record.

The appropriate interpretation is therefore limited to the design, endpoint definition, and available safety counts. Those details can clarify what the investigators intended to measure and what the registry records for safety, but they cannot replace the missing efficacy and mechanistic results. No inference about clinical benefit or harm follows from the endpoint definition alone.

14. Why This Trial Matters Statistically

This study is statistically interesting because its primary outcome translates a laboratory measurement into a binary participant-level response. That choice makes the intended outcome readily interpretable as a count of participants reaching a defined SI threshold. It also highlights the trade-off in dichotomization: the response classification is simple, while variation in the actual SI values is obscured. The record does not report the continuous measurements that would help characterize that variation.

The study’s response-guided sequence is another important design feature. The combination treatment was planned conditionally on the immune response observed among initial Immucothel-alone participants. Such a design can be useful for staged investigation, but it differs statistically from a randomized comparison planned in advance with concurrent groups. The resulting treatment groups do not offer a like-for-like causal contrast merely because they are listed separately.

The study also connects an immunogenicity endpoint with planned mechanistic outcomes involving T-cell stimulation, cytokines, and antibodies. Multiple biological measures can provide distinct perspectives, but they raise questions about outcome hierarchy, multiplicity, measurement timing, and missing data. The registry record lists the outcome domains without reporting results or the methods used to interpret them jointly.

Finally, the record illustrates the gap between a well-described endpoint and an interpretable estimate. The threshold and timeframe are specified, yet the response count, denominator, and uncertainty are not reported in the registry record. A reader can understand what the endpoint was meant to capture, but cannot evaluate the observed response or its precision. The non-randomized design, response-dependent parts, and absent analysis details further limit comparison and generalization. These are statistical interpretive issues, not evidence for or against the biological hypothesis.

15. 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 principal limitation is that the registry record does not report results for the primary endpoint. The response count, endpoint denominator, continuous SI distribution, and uncertainty estimates are all not reported in the registry record. Without these, neither the frequency nor precision of the primary response can be evaluated. The listed secondary outcomes likewise have no reported results in the registry record.

The non-randomized design and response-guided treatment sequence limit comparisons among groups. The combination group was conditional on Part A findings, and Part B depended on those findings as well. The groups should not be treated as exchangeable control and treatment populations. The registry record does not provide baseline comparability data or a comparative analysis to address these concerns.

Enrollment was 19, while safety denominators are reported separately by group. The endpoint-specific population and participant flow are not reported in the registry record, so it is unclear how many enrolled participants contributed to the primary endpoint or each secondary measure. The named intent-to-treat population does not resolve the absence of endpoint-specific denominators or missing-data methods.

The full Part B intervention description is incomplete in the registry record. Details of procedures beyond the recorded feeding schedule and the statement that participants were then immunized are not reported in the registry record. The reason for termination, the trial’s own sample-size justification, the statistical analysis plan, multiplicity handling, interim analyses, and missing-data procedures are also not reported in the registry record. These gaps restrict assessment of the intended analyses, conduct, and completeness of follow-up.

16. Related Tutorials

17. Related Statistical Calculators

18. Sources

• ClinicalTrials.gov: A Study to Determine the Immunogenicity and Oral Tolerance to Keyhole Limpet Hemocyanin (KLH) — NCT01489956.
• Registry record last updated 2016-04-12; status last verified 2016-03.
• Results first posted 2016-04-12.
• 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.

19. Record Summary

NCT01489956 is an early phase 1, non-randomized, unmasked, single-group study titled A Study to Determine the Immunogenicity and Oral Tolerance to Keyhole Limpet Hemocyanin (KLH). The official title describes a pilot study of Immucothel immunogenicity and oral tolerance induction with native KLH in healthy subjects. Enrollment was 19; the registry lists status as terminated.

The primary endpoint was the count of Part A participants with a positive T-cell stimulation index after in vitro KLH stimulation on Day 16, with an SI of 3 or higher defining a positive response. The registry record does not report the primary endpoint results. It describes conditional treatment procedures and secondary mechanistic outcomes, but detailed results and several analysis details are not reported in the registry record. Safety information lists zero serious adverse events in the groups with participants at risk, with the group-specific denominators described in the safety information.