Dose optimization for cancer treatments with considerations for late-onset toxicities.

Given that novel anticancer therapies have different toxicity profiles and mechanisms of action, it is important to reconsider the current approaches for dose selection. In an effort to move away from considering the maximum tolerated dose as the optimal dose, the Food and Drug Administration Project Optimus points to the need of incorporating long-term toxicity evaluation, given that many of these novel agents lead to late-onset or cumulative toxicities and there are no guidelines on how to handle them. Numerous methods have been proposed to handle late-onset toxicities in dose-finding clinical trials. A summary and comparison of these methods are provided. Moreover, using PI3K inhibitors as a case study, we show how late-onset toxicity can be integrated into the dose-optimization strategy using current available approaches. We illustrate a re-design of this trial to compare the approach to those that only consider early toxicity outcomes and disregard late-onset toxicities. We also provide proposals going forward for dose optimization in early development of novel anticancer agents with considerations for late-onset toxicities.

Incorporating patient-reported outcomes in dose-finding clinical trials with continuous patient enrollment.

Dose-finding clinical trials in oncology estimate the maximum tolerated dose (MTD), based on toxicity obtained from the clinician's perspective. While the collection of patient-reported outcomes (PROs) has been advocated to better inform treatment tolerability, there is a lack of guidance and methods on how to use PROs for dose assignments and recommendations. The PRO continual reassessment method (PRO-CRM) has been proposed to formally incorporate PROs into dose-finding trials. In this paper, we propose two extensions of the PRO-CRM, which allow continuous enrollment of patients and longer toxicity observation windows to capture late-onset or cumulative toxicities by using a weighted likelihood to include the partial toxicity follow-up information. The TITE-PRO-CRM uses both the PRO and the clinician's information during the trial for dose assignment decisions and at the end of the trial to estimate the MTD. The TITE-CRM + PRO uses clinician's information solely to inform dose assignments during the trial and incorporates PRO at the end of the trial for the estimation of the MTD. Simulation studies show that the TITE-PRO-CRM performs similarly to the PRO-CRM in terms of dose recommendation and assignments during the trial while almost halving trial duration in case of an accrual of two patients per observation window. The TITE-CRM + PRO slightly underperforms compared to the TITE-PRO-CRM, but similar performance can be attained by requiring larger sample sizes. We also show that the performance of the proposed methods is robust to higher accrual rates, different toxicity hazards, and correlated time-to-clinician toxicity and time-to-patient toxicity data.

Surv-CRM-12: A Bayesian phase I/II survival CRM for right-censored toxicity endpoints with competing disease progression.

The growing interest in new classes of anti-cancer agents, such as molecularly-targeted therapies and immunotherapies with modes of action different from those of cytotoxic chemotherapies, has changed the dose-finding paradigm. In this setting, the observation of late-onset toxicity endpoints may be precluded by treatment and trial discontinuation due to disease progression, defining a competing event to toxicity. Trial designs where dose-finding is modeled in the framework of a survival competing risks model appear particularly well-suited. We aim to provide a phase I/II dose-finding design that allows dose-limiting toxicity (DLT) outcomes to be delayed or unobserved due to competing progression within the possibly long observation window. The proposed design named the Survival-continual reassessment method-12, uses survival models for right-censored DLT and progression endpoints. In this competing risks framework, cause-specific hazards for DLT and progression-free of DLT were considered, with model parameters estimated using Bayesian inference. It aims to identify the optimal dose (OD), by minimizing the cumulative incidence of disease progression, given an acceptable toxicity threshold. In a simulation study, design operating characteristics were evaluated and compared to the TITE-BOIN-ET design and a nonparametric benchmark approach. The performance of the proposed method was consistent with the complexity of scenarios as assessed by the nonparametric benchmark. We found that the proposed design presents satisfying operating characteristics in selecting the OD and safety.

Diffuse optical tomography breast imaging measurements are modifiable with pre-surgical targeted and endocrine therapies among women with early stage breast cancer.

PURPOSE: Diffuse optical tomography breast imaging system (DOTBIS) non-invasively measures tissue concentration of hemoglobin, which is a potential biomarker of short-term response to neoadjuvant chemotherapy. We evaluated whether DOTBIS-derived measurements are modifiable with targeted therapies, including AKT inhibition and endocrine therapy. METHODS: We conducted a proof of principle study in seven postmenopausal women with stage I-III breast cancer who were enrolled in pre-surgical studies of the AKT inhibitor MK-2206 (n = 4) or the aromatase inhibitors exemestane (n = 2) and letrozole (n = 1). We performed DOTBIS at baseline (before initiation of therapy) and post-therapy in the affected breast (tumor volume) and contralateral, unaffected breast, and measured tissue concentrations (in µM) of total hemoglobin (ctTHb), oxyhemoglobin (ctO2Hb), and deoxyhemoglobin (ctHHb), as well as water fraction (%). RESULTS: We found consistent decreases in DOTBIS-measured hemoglobin concentrations in tumor volume, with median percent changes for ctTHb, ctHHb, ctO2Hb, and water fraction for the entire cohort of - 27.1% (interquartile range [IQR] 37.5%), - 49.8% (IQR 29.3%), - 33.5% (IQR 47.4%), and - 3.6% (IQR 10.6%), respectively. In the contralateral breast, median percent changes for ctTHb, ctHHb, ctO2Hb, and water fraction were + 1.8% (IQR 26.7%), - 8.6% (IQR 29.3%), + 6.2% (IQR 29.5%), and + 1.9% (IQR 30.7%), respectively. CONCLUSION: We demonstrated that DOTBIS-derived measurements are modifiable with pre-surgical AKT inhibition and endocrine therapy, supporting further investigation of DOTBIS as a potential imaging assessment of response to neoadjuvant targeted therapies in early stage breast cancer.

Seamless phase I/II design for novel anticancer agents with competing disease progression.

Molecularly targeted agents and immunotherapies have prolonged administration and complicated toxicity and efficacy profiles requiring longer toxicity observation windows and the inclusion of efficacy information to identify the optimal dose. Methods have been proposed to either jointly model toxicity and efficacy, or for prolonged observation windows. However, it is inappropriate to address these issues individually in the setting of dose-finding because longer toxicity windows increase the risk of patients experiencing disease progression and discontinuing the trial, with progression defining a competing event to toxicity, and progression-free survival being a commonly used efficacy endpoint. No method has been proposed to address this issue in a competing risk framework. We propose a seamless phase I/II design, namely the competing risks continual reassessment method (CR-CRM). Given an observation window, the objective is to recommend doses that minimize the progression probability, among a set of tolerable doses in terms of toxicity risk. In toxicity-centered stage of the design, doses are assigned based on toxicity alone, and in optimization stage of the design, doses are assigned integrating both toxicity and progression information. Design operating characteristics were examined in a simulation study compared with benchmark performances, including sensitivity to time-varying hazards and correlated events. The method performs well in selecting doses with acceptable toxicity risk and minimum progression risk across a wide range of scenarios.

A simulation study of approaches for handling disease progression in dose-finding clinical trials.

In traditional dose-finding studies, dose-limiting toxicity (DLT) is determined within a fixed time observation window where DLT is often defined as a binary outcome. In the setting of oncology dose-finding trials, often patients in advanced stage of diseases are enrolled. Therefore, disease progression may occur within the DLT observation window leading to treatment discontinuation and rendering the patient unevaluable for DLT assessment. As a result, additional patients have to be enrolled, increasing the sample size. We propose and compare several practical approaches for handling disease progression which occurs within the DLT observation window, while in the framework of the time-to-event continual reassessment method (TITE-CRM) which allows using partial observations. The approaches differ on the way they define an evaluable patient and in the way incomplete observations are included. The practical approaches, which we call strategies A, B and C, are illustrated and contrasted in the context of a single simulated trial, and compared via simulations under various scenarios of dose-progression relationship, in the setting of advanced soft-tissue sarcoma.

Dose-finding designs for cumulative toxicities using multiple constraints.

This article addresses the concern regarding late-onset dose-limiting toxicities (DLT), moderate toxicities below the threshold of a DLT and cumulative toxicities that may lead to a DLT, which are mostly disregarded or handled in an ad hoc manner when determining the maximum tolerated dose (MTD) in dose-finding cancer clinical trials. An extension of the Time-to-Event Continual Reassessment Method (TITE-CRM) which allows for the specification of toxicity constraints on both DLT and moderate toxicities, and can account for partial information is proposed. The method is illustrated in the context of an Erlotinib dose-finding trial with low DLT rates, but a significant number of moderate toxicities leading to treatment discontinuation in later cycles. Based on simulations, our method performs well at selecting the dose level that satisfies both the DLT and moderate-toxicity constraints. Moreover, it has similar probability of correct selection compared to the TITE-CRM when the true MTD based on DLT only and the true MTD based on grade 2 or higher toxicities alone coincide, but reduces the probability of recommending a dose above the MTD.

A Phase I Trial of Intravesical Cabazitaxel, Gemcitabine and Cisplatin for the Treatment of Nonmuscle Invasive bacillus Calmette-Guérin Unresponsive or Recurrent/Relapsing Urothelial Carcinoma of the Bladder.

PURPOSE: For patients with bacillus Calmette-Guérin unresponsive or recurrent/relapsing nonmuscle invasive bladder cancer, multi-agent intravesical trials have been limited. In this study we investigate the safety of intravesical cabazitaxel, gemcitabine and cisplatin in the salvage setting. MATERIALS AND METHODS: This was a dose escalation, drug escalation trial for patients with bacillus Calmette-Guérin unresponsive or recurrent/relapsing nonmuscle invasive bladder cancer who declined or were ineligible for radical cystectomy. All patients underwent a 6-week induction regimen of sequentially administered cabazitaxel, gemcitabine and cisplatin. Complete response was defined as no cancer on post-induction transurethral bladder tumor resection and negative urine cytology, while partial response allowed for positive cytology. Responders continued with maintenance cabazitaxel and gemcitabine monthly for the first year and bimonthly for the second year. RESULTS: A total of 18 patients were enrolled. Mean age was 71 years, median followup was 27.8 months (range 16.3 to 46.9) and mean number of previous rounds of intravesical therapies before trial enrollment was 3.7. Nine patients (50%) had received intravesical chemotherapy after bacillus Calmette-Guérin and 7 (39%) were previously treated in a phase I clinical trial setting. At enrollment 6 (33%) subjects had T1 disease and 13 (72%) had carcinoma in situ. There were no dose limiting toxicities. Initial partial and complete response rates were 94% and 89%, respectively. At 1 year recurrence-free survival was 0.83 (range 0.57 to 0.94) and at 2 years estimated recurrence-free survival was 0.64 (0.32 to 0.84). CONCLUSIONS: In this high risk and highly pretreated cohort of bacillus Calmette-Guérin unresponsive or recurrent/relapsing nonmuscle invasive bladder cancer cases combination intravesical cabazitaxel, gemcitabine and cisplatin was a well tolerated and potentially effective regimen.

Incorporating patient-reported outcomes in dose-finding clinical trials.

Oncology dose-finding clinical trials determine the maximum tolerated dose (MTD) based on toxicity outcomes captured by clinicians. With the availability of more rigorous instruments for measuring toxicity directly from patients, there is a growing interest to incorporate patient-reported outcomes (PRO) in clinical trials to inform patient tolerability. This is particularly important for dose-finding trials to ensure the identification of a well-tolerated dose. In this paper, we propose three extensions of the continual reassessment method (CRM), termed PRO-CRMs, that incorporate both clinician and patient outcomes. The first method is a marginal modeling approach whereby clinician and patient toxicity outcomes are modeled separately. The other two methods impose a constraint using a joint outcome defined based on both clinician and patient toxicities and model them either jointly or marginally. Simulation studies show that while all three PRO-CRMs select well-tolerated doses based on clinician's and patient's perspectives, the methods using a joint outcome perform better and have similar performance. We also show that the proposed PRO-CRMs are consistent under robust model assumptions.

Dose-finding design and benchmark for a right censored endpoint.

Dose-finding trials aim to determine a safe dose to be tested in larger trials for efficacy. In oncology, designs generally assume conventional monotonic increasing dose-toxicity relationships, mostly with binary outcomes (e.g., dose-limiting toxicity or not), measured in the first cycle of therapy or for a fixed number of cycles. However, with new anti-cancer agents such as molecularly targeted therapies and immunotherapies, late-onset toxicities have become more frequent. Designs with prolonged observation windows and censored endpoints analyzed using survival models, appear particularly suited to these settings. Moreover, in this setting, the observation of the late-onset toxicity endpoint could be precluded by trial discontinuation due to death, progression, patient withdrawal, or physician discretion, defining a competing event to toxicity. We propose extensions of the Continual Reassessment Method (CRM) dose-finding design using survival working models for right-censored endpoints and for handling treatment discontinuation in the toxicity observation window, namely the Survival-CRM (Surv-CRM) and the informative survival-CRM (iSurv-CRM). We also developed a benchmark approach for its evaluation. In a simulation study, we compared the performance of the Surv-CRM and iSurv-CRM, to those of the Time-to-event (TITE)-CRM and the nonparametric benchmark. The performance of the proposed methods was consistent with the complexity of scenarios as assessed by the nonparametric benchmark. Without treatment discontinuations, the Surv-CRM provides proportions of correct dose selection close to those of the TITE-CRM with fewer observed toxicities and patients assigned to overtoxic dose levels. In the presence of treatment discontinuation, the iSurv-CRM outperforms the TITE-CRM in identifying the correct dose level.

A comparison of nurses' and physicians' perception of cancer treatment burden based on reported adverse events.

BACKGROUND: Cancer treatments are associated with a multitude of adverse events (AEs). While both nurses and physicians are involved in patient care delivery and AE assessment, very few studies have examined the differences between nurses' and physicians' reporting and perception of AEs. An approach was recently proposed to assess treatment burden based on reported AEs from the physician's perspective. In this paper, we use this approach to evaluate nurses' perception of burden, and compare nurses' and physicians' assessment of the overall and relative burden of AEs. METHODS: AE records for 334 cancer patients from a randomized clinical trial conducted by the SWOG Cancer Research Network were evaluated by 14 nurses at Columbia University Medical Center. Two nurses were randomly selected to assign a burden score from 0 to 10 based on their impression of the global burden of the captured AEs. These nurses did not interact directly with the patients. Scores were compared to previously obtained physicians scores using paired T-test and Kappa statistic. Severity scores for individual AEs were obtained using mixed-effects models with nurses assessments, and were qualitatively compared to physicians'. RESULTS: Given the same AEs, nurses' and physicians' perception of the burden of AEs differed. While nurses generally perceived the overall burden of AEs to be only slightly worse compared to physicians (mean average VAS score of 5.44 versus 5.14), there was poor agreement in the perception of AEs that were in mild to severe range. The percent agreement for a moderate or worse AE was 64% with a Kappa of 0.34. Nurses also assigned higher severity scores to symptomatic AEs compared to physicians (p < 0.05), such as gastrointestinal (4.77 versus 4.14), hemorrhage (5.07 versus 4.14), and pain (5.17 versus 4.14). CONCLUSIONS: These differences in the perception of burden of AEs can lead to different treatment decisions and symptom management strategies. Thus, having provider consistency, training, or a collaborative approach in follow-up care between nurses and physicians is important to ensure continuity in care delivery. Moreover, estimating overall burden from both physicians' and nurses' perspective, and comparing them may be useful for deciding when collaborations are warranted.

Estimating global treatment toxicity burden from adverse-event data.

BACKGROUND: A summary measure that reflects the global toxicity burden of a treatment is essential for comparing therapies. Current toxicity summaries are ad hoc and do not distinguish among the severities and types of toxicities. Here a clinically feasible method for estimating the toxicity burden, based on a prospective evaluation of the toxicity profile of a randomized clinical trial of 746 prostate cancer patients conducted by SWOG, is proposed. METHODS: For 308 patients who experienced severe toxicities, 2 physicians randomly selected from 14 physicians evaluated each toxicity profile and assigned a visual analogue scale score (0-10) based on their impression of the global burden of toxicities. With mixed-effects models, severity scores and a 10-point toxicity burden score (TBS) were derived from 27 predictors accounting for severe (grade 3) and life-threatening (grade 4) toxicities for each organ class of the Common Terminology Criteria for Adverse Events. RESULTS: For most organ classes, grade 3 toxicities had a TBS of 4.14 (95% confidence interval [CI], 3.65-4.63), but infections, cardiovascular events, and pulmonary events had a higher TBS with differences of 0.87 (95% CI, 0.53-1.21), 0.88 (95% CI, 0.51-1.25), and 0.73 (95% CI, 0.22-1.24), respectively. Moreover, most grade 4 events had a higher TBS than grade 3 events, except for hemorrhaging, pain, metabolic events, and musculoskeletal events. The intrarater and interrater correlations were 0.91 and 0.59, respectively. CONCLUSIONS: The burden of toxicity grades differs with toxicity types. A TBS provides a toxicity burden summary that incorporates physicians' perspectives and differentiates between severe and life-threatening toxicities and organ classes. Cancer 2018;124:858-64. © 2017 American Cancer Society.

Obesity and survival in the neoadjuvant breast cancer setting: role of tumor subtype in an ethnically diverse population.

BACKGROUND: Obesity may negatively affect survival in breast cancer (BC), but studies are conflicting, and associations may vary by tumor subtypes and race/ethnicity groups. METHODS: In a retrospective review, we identified 273 women with invasive BC administered Adriamycin/Taxane-based neoadjuvant chemotherapy from 2004 to 2016 with body mass index (BMI) data at diagnosis. Obesity was defined as BMI ≥30. Associations between obesity and event-free survival (EFS), using STEEP events, and overall survival (OS), using all-cause mortality, were assessed overall and stratified by tumor subtype [[Hormone Receptor Positive (HR+)/HER2-, HER2+, and Triple-Negative Breast Cancer (TNBC])] in our diverse population. RESULTS: Median follow-up was 32.6 months (range 5.7-137.8 months). Overall, obesity was associated with worse EFS (HR 1.71, 95% CI 1.03-2.84, p = 0.04) and a trend towards worse OS (p = 0.13). In HR+/HER2- disease (n = 135), there was an interaction between obesity and hormonal therapy with respect to OS but not EFS. In those receiving tamoxifen (n = 33), obesity was associated with worse OS (HR 9.27, 95% CI 0.96-89.3, p = 0.05). In those receiving an aromatase inhibitor (n = 89), there was no association between obesity and OS. In TNBC (n = 44), obesity was associated with worse EFS (HR 2.62, 95% CI 1.03-6.66, p = 0.04) and a trend towards worse OS (p = 0.06). In HER2+ disease (n = 94), obesity was associated with a trend towards worse EFS (HR 3.37, 95% CI 0.97-11.72, p = 0.06) but not OS. Race/ethnicity was not associated with survival in any subtype, and there were no interactions with obesity on survival. CONCLUSIONS: Obesity may negatively impact survival, with differences among tumor subtypes.

Using Delayed Toxicities to Re-evaluate Tolerability in Phase 2 Trials: A Case Example using Bortezomib.

In advanced stage patients enrolled in dose-finding trials, it is difficult to assess delayed toxicities because frequently patients discontinue after one or two cycles of treatment. Patients enrolled in phase 2 trials are typically followed longer to assess efficacy. Thus, their data may be useful for evaluating long-term tolerability. We illustrate this using as example two phase 2 bortezomib trials (total N = 172) conducted by SWOG. While treatment-related severe toxicity rates based on cycle 1 were acceptable (23% and 31%), they were notably higher over extended administration (37% and 70%). This additional information should be considered when designing subsequent trials.

Modelling semi-attributable toxicity in dual-agent phase I trials with non-concurrent drug administration.

In oncology, combinations of drugs are often used to improve treatment efficacy and/or reduce harmful side effects. Dual-agent phase I clinical trials assess drug safety and aim to discover a maximum tolerated dose combination via dose-escalation; cohorts of patients are given set doses of both drugs and monitored to see if toxic reactions occur. Dose-escalation decisions for subsequent cohorts are based on the number and severity of observed toxic reactions, and an escalation rule. In a combination trial, drugs may be administered concurrently or non-concurrently over a treatment cycle. For two drugs given non-concurrently with overlapping toxicities, toxicities occurring after administration of the first drug yet before administration of the second may be attributed directly to the first drug, whereas toxicities occurring after both drugs have been given some present ambiguity; toxicities may be attributable to the first drug only, the second drug only or the synergistic combination of both. We call this mixture of attributable and non-attributable toxicity semi-attributable toxicity. Most published methods assume drugs are given concurrently, which may not be reflective of trials with non-concurrent drug administration. We incorporate semi-attributable toxicity into Bayesian modelling for dual-agent phase I trials with non-concurrent drug administration and compare the operating characteristics to an approach where this detail is not considered. Simulations based on a trial for non-concurrent administration of intravesical Cabazitaxel and Cisplatin in early-stage bladder cancer patients are presented for several scenarios and show that including semi-attributable toxicity data reduces the number of patients given overly toxic combinations. © 2016 The Authors. Statistics in Medicine Published by John Wiley & Sons Ltd.

Selection of the initial design for the two-stage continual reassessment method.

In the two-stage continual reassessment method (CRM), model-based dose escalation is preceded by a pre-specified escalating sequence starting from the lowest dose level. This is appealing to clinicians because it allows a sufficient number of patients to be assigned to each of the lower dose levels before escalating to higher dose levels. While a theoretical framework to build the two-stage CRM has been proposed, the selection of the initial dose-escalating sequence, generally referred to as the initial design, remains arbitrary, either by specifying cohorts of three patients or by trial and error through extensive simulations. Motivated by a currently ongoing oncology dose-finding study for which clinicians explicitly stated their desire to assign at least one patient to each of the lower dose levels, we proposed a systematic approach for selecting the initial design for the two-stage CRM. The initial design obtained using the proposed algorithm yields better operating characteristics compared to using a cohort of three initial design with a calibrated CRM. The proposed algorithm simplifies the selection of initial design for the two-stage CRM. Moreover, initial designs to be used as reference for planning a two-stage CRM are provided.

Dose-finding designs for trials of molecularly targeted agents and immunotherapies.

Recently, there has been a surge of early phase trials of molecularly targeted agents (MTAs) and immunotherapies. These new therapies have different toxicity profiles compared to cytotoxic therapies. MTAs can benefit from new trial designs that allow inclusion of low-grade toxicities, late-onset toxicities, addition of an efficacy endpoint, and flexibility in the specification of a target toxicity probability. To study the degree of adoption of these methods, we conducted a Web of Science search of articles published between 2008 and 2014 that describe phase 1 oncology trials. Trials were categorized based on the dose-finding design used and the type of drug studied. Out of 1,712 dose-finding trials that met our criteria, 1,591 (92.9%) utilized a rule-based design, and 92 (5.4%; range 2.3% in 2009 to 9.7% in 2014) utilized a model-based or novel design. Over half of the trials tested an MTA or immunotherapy. Among the MTA and immunotherapy trials, 5.8% used model-based methods, compared to 3.9% and 8.3% of the chemotherapy or radiotherapy trials, respectively. While the percentage of trials using novel dose-finding designs has tripled since 2007, the adoption of these designs continues to remain low.

Lymphovascular invasion is an independent predictor of survival in breast cancer after neoadjuvant chemotherapy.

Various prognostic indicators have been investigated in neoadjuvant chemotherapy (NAC)-treated invasive breast cancer (BC). Our study examines if lymphovascular invasion (LVI) is an independent predictor of survival in women receiving NAC. We performed a retrospective analysis in 166 women with operable invasive BC who underwent adriamycin- and taxane-based NAC between 2000 and 2013. The presence of LVI was noted in breast excisions following NAC. Associations between progression-free and overall survival and LVI and other clinicopathologic variables were assessed. Median follow-up was 31 months (range 1.4-153 months) with a total of 56 events and 24 deaths from any cause. LVI was found in 74 of 166 patients (45 %). In univariate analysis, the presence of LVI was associated with worse progression-free survival (HR 3.37, 95 % CI 1.87-6.06, p < 0.01) and overall survival (HR 4.35, 95 % CI 1.61-11.79, p < 0.01). In multivariate models adjusting for breast cancer subtype, LVI was significantly associated with a decrease in progression-free survival (HR 3.76, 95 % CI 2.07-6.83, p < 0.01) and overall survival (HR 5.70, 95 % CI 2.08-15.64, p < 0.01). When stratified by subtype, those with hormone receptor or HER2-positive BCs with no LVI had the most favorable progression-free and overall survival. Those with both LVI and triple-negative BC had the worst progression-free and overall survival. LVI is an important prognostic marker and is associated with worse clinical outcome in breast cancer patients receiving NAC.

A comparison of prevaginal and postvaginal manipulation fetal fibronectin.

BACKGROUND: Fetal fibronectin (fFN) is used as a biomarker for preterm delivery. Currently, its use is discouraged if there has been vaginal manipulation in the previous 24 hours. OBJECTIVE: Our objective is to determine if there are differences between fFN results before and after vaginal manipulation in the form of sterile vaginal exam or transvaginal ultrasound. STUDY DESIGN: This was a prospective observational cohort study at a single center of women between 22-33 6/7 weeks at risk for preterm delivery due to: (1) a history of preterm delivery, short cervix, or multifetal gestation; or (2) symptoms of preterm labor. We excluded women with vaginal bleeding or infection, placenta previa, ruptured membranes, cervical dilation >3 cm, or any form of vaginal manipulation in the previous 24 hours. Specimen A was collected prior to planned vaginal exam or transvaginal ultrasound and specimen B was collected within 4 hours. The agreement between specimens A and B was assessed using descriptive statistics. Test characteristics of specimens A and B using the outcome of preterm delivery (<37 weeks) were calculated. RESULTS: In all, 310 specimen pairs from 237 women were collected. Specimen A was positive in 37 (12%) instances and negative in 273 (88%) while specimen B was positive in 39 (13%) and negative in 271 (87%). There were discordant results in 26 specimen pairs. Of these, 14 (5%) negative specimen A results subsequently became positive for specimen B, and 12 (32%) positive specimen A results became negative for specimen B. Overall, there was a 92% agreement between specimens A and B (confidence interval, 88-94%). The specificity of specimens A and B for preterm birth was 90% vs 89%, respectively, with a negative predictive value of 87% for both. The false-negative rate was 12.8% for specimen A and 13.3% for specimen B. CONCLUSION: There is a moderately high degree of agreement between prevaginal and postvaginal manipulation fFN results. Their test characteristics for evaluating preterm birth are similar with strong specificity and negative predictive values, and their false-negative rates are not clinically different. Consideration should be made to the utilization of postvaginal manipulation fFN when a prevaginal manipulation specimen is not available.