Mirvetuximab soravtansine plus pembrolizumab in recurrent folate receptor alpha-positive uterine serous carcinoma: a phase II trial

Domain: biology
Published: 2026-03-26
Editorial outcome: minor_revision

Abstract
Immune checkpoint inhibitors (ICI) synergize preclinically with antibody drug conjugates (ADC),
harboring anti-tubulin maytansinoid payloads. We conducted an investigator-initiated, single-arm,
phase 2 trial of mirvetuximab soravtansine (MIRV), a folate receptor alpha (FOLR1/FRα)-targeting
ADC with the maytansinoid payload, DM4, combined with pembrolizumab in female patients with
recurrent FOLR1-expressing serous endometrial cancer (EC, NCT03835819). Co-primary
objectives include objective response rate (ORR) and rate of progression-free survival at 6
months (PFS6); secondary objectives include PFS, overall survival, duration of response and
safety. Exploratory objectives include correlation of tumor genomics and immunoprofiling with
clinical activity. Eighteen patients initiated protocol therapy [MIRV 6 mg/kg adjusted ideal body
weight IV and pembrolizumab 200 mg IV every 3 weeks]. Confirmed ORR is 28% (1 complete
and 4 partial responses, 95% CI:10-53%), Kaplan Meier estimate of PFS6 is 24.4% (95% CI:7.7-
46.1%) with 4 patients progression free at 6 months; trial was closed early for feasibility (planned
sample size of 35 patients not reached) and hence these results are considered preliminary. G3
treatment-related adverse effects were rare with no grade ≥4 toxicities. We report a population of
high FOLR1-expressing tumor-associated macrophages (CD163+FOLR1+), suggesting potential
on-target, off-tumor immune editing by MIRV. A composite biomarker score derived in this cohort
correlates with objective response to MIRV and pembrolizumab.

Introduction
Endometrial cancer (EC) is the most common gynecologic malignancy in the United States
and has a rising incidence and mortality1. Approximately one third of EC patients present with
locally advanced or metastatic disease, for which the five-year survival rates remain poor at 69%
and <20%, respectively2. Immune checkpoint inhibitors (ICI) have demonstrated activity in
advanced endometrial cancer, both for recurrent disease3-6 and in combination with chemotherapy
and continued as maintenance in the first line setting7-12. However, ICI are less effective in
mismatch repair proficient (pMMR) recurrent EC, with single agent ICI exhibiting response rates
ranging from 3%-14.1% in the pMMR cohorts across several clinical trials4-6,13, highlighting an
urgent need for alternative treatment regimens for recurrent EC.

ADCs represent a promising class of therapies showing impressive activity in numerous
tumor types, including in EC14. At present, trastuzumab deruxtecan remains the only ADC
approved for clinical use in recurrent HER2-expressing EC patients, with multiple other ADCs in
development14. Mirvetuximab soravtansine (MIRV) is an ADC comprised of a folate receptor alpha
(FOLR1/FRα)-targeted monoclonal antibody and the anti-tubulin maytansinoid payload, DM4,
joined by a cleavable linker with a drug to antibody ratio (DAR) of 3.5:115. MIRV received Food
and Drug Administration (FDA) approval for the treatment of adult patients with FOLR1-positive,
platinum resistant epithelial ovarian, fallopian tube and primary peritoneal cancers16 based on the
initial favorable ORR17 seen in the phase 2 SORAYA trial, as well as a superior progression free
(PFS) and overall survival (OS) compared with chemotherapy in the confirmatory phase 3
MIRASOL trial18. In PROC, high FOLR1 expression is defined via the PS2+ scoring method using
the VENTANA FOLR1 RxDx assay19, whereby ≥ 75% of tumor cells must display at least 2+
staining intensity by immunohistochemical (IHC) analysis. FOLR1 is also overexpressed and
associated with poor prognosis in EC20,21, suggesting its potential as a therapeutic target in this
disease. In a Phase 1 dose expansion study of MIRV monotherapy in patients with FOLR1-
positive advanced/recurrent EC (NCT01609556), the highest FOLR1 expression was reported in
tumors of serous histology, and objective responses were observed in 2 of 24 (8%) patients and
11 patients (46%) achieved stable disease22. MIRV monotherapy was well-tolerated, potentially
lending itself well to combinatorial strategies aimed at improving the modest single agent activity
in EC.

ADCs can modulate the immune microenvironment through multiple mechanisms
including induction of immunogenic cell death, activation and maturation of dendritic cells and
recruitment of other immune cells to the TME, stimulating both innate and adaptive immunity23-28.
The combination of ADC and ICI have demonstrated synergism in preclinical studies27,29 and
promising anti-tumor activity in patients30-33. Moreover, in addition to target-mediated cytotoxicity,
preclinical data has shown that MIRV activates monocytes and promotes phagocytosis of tumor
cells through Fc-FcγR interactions34. Additionally, anti-tubulin payloads like DM4 promote DC
maturation and migration to the draining lymph nodes, where they can activate naïve T cells35,36.
Therefore, we hypothesized that MIRV combined with anti-PD-1 therapy would improve the
response to ICI in patients with recurrent pMMR serous EC.

Here, we report the results of a two-stage, phase 2, single cohort study evaluating the
activity of MIRV in combination with pembrolizumab in patients with recurrent/persistent EC
(NCT03835819).

Results
FOLR1 expression in prescreening population
In the current study, a total of 144 eligible participants signed consent for pre-screening of
archival tumor tissue for FOLR1 expression by central IHC using the anti-FOLR1 2.1 antibody
developed by Ventana Medical Systems and following the PS2 scoring methodology of
quantifying the percentage of tumor cells staining at ≥2+ intensity. As shown in Table 1, of 137
patients with evaluable FOLR1 IHC results, 45 (32.8%), met the positivity cut off for trial eligibility
with ≥50% of cells exhibiting at least 2+ membrane staining intensity (PS2+). The median
percentage of cells per tumor sample expressing PS2+ staining was 30.0% (range: 0-100).
Notably, 20 (14.6%) tumors were found to have PS2+ staining in ≥75% of tumor cells, the cut off
for FOLR1-high expression selected in high grade serous ovarian cancer.

Study population
Between December 30, 2019, and March 15, 2024, 18 patients were enrolled to the main
study and received at least 1 dose of MIRV and pembrolizumab (Figure 1). All patients were
female and had histologically confirmed uterine serous adenocarcinoma with intact MMR protein
expression by IHC. Additional baseline patient characteristics are summarized in Table 2; 8
patients (44.4%) had received prior ICI therapy whereas 10 patients (55.6%) were ICI naïve. The
median FOLR1 expression based on percentage of cells with at least PS2+ expression was
72.5% (range: 50.0, 98.0); 50% of enrolled patients had FOLR1 ≥75%. At the data cutoff of July
05, 2024, the median follow-up for overall survival was 11.3 months (IQR: 3.3-NR months). No
patients were remaining on study.

Antitumor activity of mirvetuximab and pembrolizumab
A total of 18 patients were evaluable for efficacy analyses; one patient withdrew from the
study for non-AE reasons after receiving 3 doses of study treatment. Among the first 5 patients
enrolled, we observed 2 confirmed objective responses meeting the criteria to continue enrollment
in the two-stage design. Among all 18 patients, the confirmed ORR was 28% (95% CI: 10-53%)
including 1 confirmed complete response, 4 confirmed partial responses (Figure 2A). While the
study meets the required number of responses for ORR (≥4 objective responses) specified by the
two-stage design, and the 95% confidence interval excludes the null ORR of 5%, we did not
complete enrollment of the trial to the planned sample size of 35 patients and hence these results
should be considered preliminary. There were also 2 unconfirmed partial responses. An additional
4 patients had stable disease of any duration as best response. Depth and duration of response
by subject are shown in Fig. 2B and Supplemental Fig. 1, respectively. FOLR1 expression and
prior history of ICI (anti-PD-1 or anti-PD-L1) therapy is noted for each patient. There were 4
patients alive and progression-free at 6 months resulting in a PFS6 rate of 22% (95% CI: 6-48%).
The observed median PFS (mPFS) was 2.73 months (95% CI: 1.2-4.5 months)
(Supplemental Figure 2) and the PFS at 6 months was 24.4% (95% CI 7.7-46.1%) by the Kaplan
Meier method. Median overall survival (OS) for the overall cohort was 12.5 months (95% CI: 5.2-
NR months). The observed median duration of response (mDOR) was 8.17 months (range: 2.76
– 18.14 months) in the 5 patients with confirmed responses. Notably, in the patient with a complete
response, despite discontinuation of pembrolizumab at 2.8 months from treatment initiation for
adrenal insufficiency, she exhibited a sustained response of 18.4 months before discontinuing
mirvetuximab for pneumonitis. Another patient achieved a deep partial response (50.5% tumor
reduction as best response) that was sustained for at least 10.1 months including on last
assessment. Mirvetuximab and pembrolizumab were both discontinued in that patient at 12.2
months from treatment initiation for toxicities.

The initial design of the trial enrolled ICI naïve patients only but a subsequent protocol
amendment allowed for enrollment of up to 19 patients with prior ICI targeting the PD-1/PD-L1
pathway. In an exploratory subgroup analysis, we evaluated the efficacy of mirvetuximab and
pembrolizumab in patients that were ICI naïve (n=10) vs exposed (n=8). All 5 confirmed
responses were in patients who were ICI naïve; 2 patients with prior ICI therapy exhibited
unconfirmed responses. Among the 11 patients with either a response or stable disease, 8 were
ICI naïve while 3 had received prior ICI therapy (Supplemental Table 1). In contrast, 4 of 6
patients with progressive disease as best response had received prior ICI. All 4 patients who
achieved PFS6 were ICI naïve. The median PFS for the ICI-naïve and ICI-exposed groups were
4.5 months (95% CI: 1.15, NR) and 1.2 months (95% CI: 1.12, NR), respectively (Supplemental
Figure 3A). Prior ICI exposure was significantly associated with shorter PFS (HR = 0.16; 95% CI
0.04 – 0.63; p=0.003) and a significantly lower response rate (Fisher’s exact test p-value=0.036,
ORR is 0% [95% CI: 0, 37%] in the ICI-exposed cohort, and 50% [95% CI: 19, 81%] in the ICI-
naïve cohort), however these results are all preliminary given small sample sizes in the subgroups
of interest.

Safety
Safety and tolerability were as expected for this two-drug combination. Of the 18 patients
who initiated treatment with this regimen, 18 (100%) reported a treatment-related adverse event
(TRAE) of any grade (Supplemental Table 2). The most common TRAEs of any grade occurring
in >25% of patients were diarrhea (n=8, 44%), fatigue (n=8, 44%), AST elevation (n=8, 44%),
blurred vision (n=7, 39%), anemia (n=6, 34%) and peripheral sensory neuropathy, decreased
platelet count, and anorexia (n=8, 28% for each). Grade 3 TRAEs were rare and are presented
in Table 3; these included diarrhea, anemia, thrombocytopenia, neutropenia, peripheral sensory
neuropathy, hypertension, corneal ulcer and adrenal insufficiency, each occurring in 1 participant
(6%). There were no grade 4 or 5 toxicities reported.

A total of 4 (22.2%) and 4 (22.2%) patients discontinued mirvetuximab and
pembrolizumab, respectively, owing to treatment-related toxicities. Among patients who
discontinued study drugs for toxicities, the median number of completed cycles was 10 cycles
(IQR:6.5-15.5) for mirvetuximab and 6.5 cycles (IQR: 5.5-8.5) for pembrolizumab. Dose delays
and/or reductions were permitted for mirvetuximab according to the investigator brochure37; 8
patients (44.4%) required a dose delay, and 7 patients (38.9%) required dose reductions.
Based on the known safety profile of mirvetuximab, ocular events were expected and were
managed by prophylactic mitigation strategies, including lubricating and steroid eye drop use, and
through dose modifications as needed per protocol. Additionally, ocular surface adverse events,
including rare cases of corneal ulcer, have been reported in association with ICIs including
pembrolizumab38,39. Ocular adverse events occurred in 10 participants (55.6%); the most common
were blurred vision (39%), keratitis (11%) and dry eyes (11%). All ocular adverse events were
grade 1 or 2 except for one participant (6%) with a grade 3 corneal ulcer. Immune-related AEs
occurred as expected based on the known safety profile of pembrolizumab. There were two
participants (11%) with grade 2 pneumonitis and one participant each (6%) with grade 3 adrenal
insufficiency and grade 2 hyperthyroidism.

Biomarker analyses
Given the encouraging ORR and the prolonged duration of response in some patients, we
aimed to identify potential biomarkers of response to MIRV and pembrolizumab in this cohort of
patients. In post-hoc exploratory analyses, we first examined FOLR1 expression levels and
performed genomic and immune profiling of archival tumor tissue and assessed whether any
biomarkers were individually associated with objective response or PFS. With regard to FOLR1
expression, 4 of the 5 confirmed responses occurred in patients with FOLR1 expression of 75%
or higher; one patient with FOLR1 expression <75% achieved a confirmed PR. The confirmed
ORR in patients with FOLR1 expression 75-100% was 44% (95% CI: 14%, 79%), whereas the
confirmed ORR was 11% (95% CI: 0%, 48%) in patients with FOLR1 expression between 50%
and 74% (Supplemental Table 3). However, there was not a statistically significant association
between FOLR1 expression level and objective response (p=0.29). The median PFS for FOLR1-
moderate and FOLR1-high tumors was similar at 2.63 (95% CI: 1.12, 4.50) and 2.73 (95% CI
1.18, 8.25) months, respectively (Supplemental Figure 3B).

Tumor genomic profiling and IHC analyses (ER, HER2) were either previously available
or performed on archival FFPE specimens from 17 patients in the study. All 17 (100%) tumors
were MMR proficient (pMMR) or microsatellite stable (MSS) as assessed by IHC or PCR on next
generation sequencing (NGS) and all harbored TP53 mutations; no tumors were polymerase
epsilon (POLE) mutated. Table 4 presents the associations between specific genomic and protein
biomarkers and objective response; all missing cases were excluded when calculating the p-
value. There were 3 tumors with CCNE1 amplifications, and all of these were in patients who had
an objective response (p=0.02). There were no other statistically significant associations between
HER2 and ER protein expression by IHC, tumor mutational burden (TMB) or specific genomic
alterations, including MYC and ERBB2 amplifications, with clinical activity.

We next performed immune profiling of archival tumors to identify alternative biomarkers
of clinical activity to this ADC and ICI combination. Immune profiling of 17 tumors was performed
on archival FFPE specimens using multiplexed immunofluorescence (ImmunoProfile). Staining
with two separate panels of six markers each (Supplemental Tables 4, 5) was performed on
tumor specimens, and the count density (positive cells per mm2) and percentage of positive cells
was determined for each marker. All 17 tumors were stained for cytokeratin (CytoK, tumor cells),
CD8 (CD8+ T cells), CD163 (macrophages), PD-L1 and FOLR1 (Figure 3A), and 15 tumors were
additionally stained for FOXP3 (regulatory T cells) and PD-1. Notably, we first compared FOLR1
IF to the FOLR1 IHC performed in pre-screening for trial eligibility. Although the two assays were
performed on non-contiguous tumor sections, we observed an overall correlation between FOLR1
cell density by IF and FOLR1 PS2+ by IHC (r=0.432; Supplemental Fig 4). The immune profile
of tumors was compared between patients with confirmed objective response vs no objective
response (Table 5). While overall PD-L1 and FOLR1 cell densities in tumor sections were not
independently significantly associated with objective response, the PD-L1 cell density in tumor
cells (PD-L1+CYTOK+; p=0.035) and in FOLR1-expressing cells (PD-L1+FOLR1+; p=0.045) was
significantly associated with objective response. In addition, we observed a striking presence of
CD163+FOLR1+ cells in 3 of 5 patients with objective responses (Figure 3B), though cell density
of this marker combination was not statistically associated with objective response. No additional
statistically significant associations between any single biomarker or double positive population
of interest (e.g. CD8+PD-1+, CD163+PD-L1) with clinical responses were detected.

Based on the observations of higher FOLR1 expression and markers of an inflamed tumor
on immune profiling without strong individual associations with objective response or PFS to MIRV
and pembrolizumab, we reasoned that a combination of these markers may be predictive of
response. Therefore, we defined a composite Biomarker score (B comp), comprised of high vs
low scoring of FOLR1 IHC and CD8+, PD-L1+ and CD163+FOLR1+ cell densities. The median
B comp score was significantly higher in patients with an objective response (2; IQR: 2.00-2)
compared with non-responders (1; IQR: 0.75-2; p=0.015; Figure 3C) and Biomarker positive
status (B+), defined as tumors with 2 or more biomarkers in the High category, was positively
correlated with objective response (two-sided Fisher exact test, p=0.026; Figure 3D). Patients
with B+ tumors also demonstrated a non-significant trend toward improved PFS (p=0.061;
Supplemental Fig 5). Median overall survival was 1.28 months (95% CI: 1.18–NR) for patients
with B– scores (n=7, 6 events) compared with 6.19 months (95% CI: 2.73–NR) for patients
with B+ scores (n=8, 5 events), supporting the potential clinical significance of this hypothesis-
generating biomarker score, or others similarly describing a favorable tumor and immune
microenvironment, in predicting patients who may derive benefit from an ADC combined with
ICB.

Methods
Study design and treatment
This was a single-arm, two-stage phase 2 study conducted at 3 centers: the Dana-
Farber Cancer Institute (Boston, MA), UMass Memorial Medical Center (Worcester, MA) and
Northwell Health Center Institute (New Hyde Park, NY). The study was reviewed and approved
by the Dana-Farber and Harvard Cancer Center Institutional Review Board in 2018 and was
conducted in accordance with the study protocol, Good Clinical Practice, and the Declaration of
Helsinki. The study was preregistered on February 11, 2019 and registered at ClinicalTrials.gov
(NCT03835819). All participants provided written informed consent separately to prescreening
for FOLR1 expression in archival tumor tissue and then for treatment on study. The first and last
patients were enrolled on 2/14/2020 and 5/1/2024, respectively.

Patients with histologically confirmed recurrent or persistent MSS/pMMR, serous (or
predominantly serous mixed histology) endometrial cancer who had received at least one and
not more than four prior lines of systemic therapy were eligible for pre-screening to determine
FOLR1 expression. Initially, patients were ineligible if they had received any prior
immunotherapy including anti-PD-1 or anti-PD-L1. However, to reflect the changing treatment
landscape for endometrial cancer patients, an amendment was introduced on January 6, 2022
(after enrollment of the first 5 patients) to broaden eligibility criteria to allow prior anti-PD-1/PD-
L1 therapy so long as this was not discontinued due to therapy-related toxicities. Additional key
eligibility criteria included adults ages 18 years or older with measurable disease, an Eastern
Cooperative Oncology Group (ECOG) performance status of 0 or 1 and adequate organ
function as specified in the protocol. Additional exclusion criteria included known active CNS
disease, active or chronic corneal disorders, active autoimmune disease, history or pneumonitis
or inflammatory lung disease (ILD), and history of allergy to monoclonal antibody therapy. For
participants who signed pre-screening consent, FRα expression was assessed by central
immunohistochemistry (IHC) testing with the anti-FOLR1 2.1 antibody (Ventana Medical
Systems) using the PS2+ scoring methodology of quantifying the percentage of tumor cells with
staining at ≥2+ intensity. Eligibility for the main study required FOLR1 PS2+ staining of ≥50% of
tumor cells. The full inclusion and exclusion criteria are provided in the study protocol in the
Supplementary Information.

All participants in the study received pembrolizumab at a fixed dose of 200 mg
intravenously and mirvetuximab soravtansine at a starting dose of 6 mg/kg adjusted ideal body
weight (AIBW) intravenously every 3 weeks. Participants were required to undergo a baseline
ophthalmic exam during screening and to have repeat ophthalmic exam every 2 cycles and at
the end of treatment or at the 30-day follow-up visit. As prophylaxis for ocular symptoms,
participants were required to use preservative-free lubricating artificial tears daily throughout
treatment and corticosteroid eye drops starting the day before each dose and continuing
through day 8 of each cycle as detailed in the study protocol. The combination treatment was
continued until progression or adverse event up to 35 cycles; beyond 35 cycles, mirvetuximab
soravtansine could be continued until progression or adverse event. If either study drug was
discontinued, patients were permitted to continue the other agent as monotherapy as tolerated.
Response to treatment was assessed every 6 weeks according to Response Evaluation Criteria
in Solid Tumors (RECIST) version 1.1. Participants who experienced disease progression but
remained clinically stable were given the option to continue treatment, with repeat response
assessment after 4 weeks to confirm progression, at the discretion of the investigator.
Participants who stopped one drug due to toxicity were permitted to continue receiving the other
drug as a single agent. Subsequent therapies were not recorded as part of the conduct of the
trial.

Study endpoints and assessments
The primary objective of the study was to assess the clinical activity of mirvetuximab
soravtansine in combination with pembrolizumab in patients with recurrent or persistent
MSS/pMMR and FRα positive endometrial cancer by evaluating the co-primary endpoints of
ORR (defined as confirmed complete and partial responses according to RECIST v1.1), and
rate of progression-free survival at 6 months (PFS6) defined as having disease assessment
scans for at least 6 months or longer showing no progression per RECIST v1.1. Patients with
less than 6 months of follow-up for PFS were treated as non-PFS6. The secondary objectives
were to assess the safety and tolerability of MIRV combined with pembrolizumab, as well as
additional measures of clinical activity including PFS, OS, duration of response (DOR) immune-
related ORR (irORR), immune-related PFS (irPFS). Although irOSRR and irPFS were included as
secondary endpoints, analysis of ORR and PFS were only reported as per the standard, non
immune-related RECIST 1.1, similar to reports of other immunotherapy combination studies in
EC such as KEYNOTE-77566, nivolumab/cabozantinib67, avelumab/axitinib68 as well as the
recently reported pembrolizumab and mirvetuximab study in ovarian cancer69. Exploratory
objectives were to correlate clinical responses with tumor infiltrating lymphocytes (TILs), other
tumor infiltrating and circulating immune cell populations, expression of immune checkpoint
proteins and tumor cell somatic alterations, and to assess the activity of the combination in
patients with and without prior exposure to ICI therapy.

The analysis population for both safety and efficacy included all patients who received at
least one dose of protocol therapy (modified intent-to-treat). Efficacy endpoints, including ORR,
PFS6, PFS, DOR, irORR and irPFS, were based on investigator-assessed tumor response
according to RECIST 1.1 criteria. Radiologic tumor assessments were performed using
computerized tomography (CT) scans or magnetic resonance imaging (MRI) at baseline and
then every 6 weeks (2 cycles) while receiving study therapy until time of progression.
Participants who discontinued study drugs were followed for 3 years by phone after removal
from study or until death. Safety and tolerability of the combination were assessed by recording
adverse events using the Adverse Event Severity scale based on the NCI Common Terminology
Criteria for Adverse Events version 5.0.

FOLR1 assessment
FOLR1 status was assessed centrally at Ventana Medical Systems by
immunohistochemistry on archival tumor tissue using the anti-FOLR1 clone FOLR1-2.1 antibody
(Roche Diagnostics). Additional exploratory analyses were performed on archival tumor tissue
to assess the immune composition of tumors and correlate with response to MIRV and
pembrolizumab.

Molecular subtyping and targeted NGS
POLE mutation status, tumor mutational burden (TMB) and the presence of additional
somatic alterations were determined from archival, formalin-fixed tissues, using targeted-panel
next-generation sequencing (Oncopanel; Dana Farber Cancer Institute, Boston, MA) performed
at Dana-Farber Cancer Institute70,71. MMR status was determined by IHC, by polymerase chain
reaction (PCR) and genomically by assessment of the mutational signature as determined by
Oncopanel.

Multiplexed immunofluorescence
Multiplexed immunofluorescence staining was performed using a BOND RX fully
automated stainer (Leica Biosystems) and counterstained with Spectral DAPI (4′,6-diamidino-2-
phenylindole; Akoya Biosciences) as previously described72. The target antigens, antibody clones,
and dilutions for the two multiplex panels are listed in Supplemental Tables 1 and 2. The antibody
clones and staining conditions used for the multiplex panels are validated using chromogenic IHC
staining as the baseline73. Sample slides are stained on the autostainer in batches along with
positive control slides that verify the consistency of staining within and between batches. After
staining, slides were cover slipped and loaded into a PhenoImager HT 1.0 (Akoya Biosciences)
for whole slide scanning at a 20x magnification. For each sample, three to eight representative
regions of interest corresponding to a 20x field of view were selected under pathologist (SJR)
supervision using Phenochart 1.0 software (Akoya Biosciences). Following image acquisition,
each field of view was spectrally unmixed and analyzed with inForm 2.6 Image Analysis Software
(Akoya Biosciences). All nucleated cells within a field of view were segmented via nuclear DAPI
staining. A supervised machine-learning algorithm within the software was utilized to assign each
cell a phenotype according to biomarker expression within its nuclear and membrane
compartments. Positive cell identification was guided by a pathologist (SJR) and visually
confirmed. Cell densities (number of positive cells per mm2) and percent positive populations were
calculated for each marker.

Biomarker score calculation
Each tumor was categorized as high or low for each component of the composite
biomarker score. For FOLR1 IHC, tumors with 50-74% PS2+ staining were considered low and
75-100% were considered high. For CD8+ cell density, a cutoff of 200 positive cells/mm2 was
used for low vs high tumors. For PD-L1+ and CD163+FOLR1+ cell densities, cutoffs of 25 positive
cells/mm2 and 85 positive cells/mm2 were used, respectively.

Statistical analysis
Statistical considerations were developed for two primary objectives of objective
response rate (ORR) and rate of PFS6, using a two-stage design that allowed for early stopping
for futility. A two-stage test was constructed using the method of Sill, Rubinstein, Litwin and
Yothers with the goal of stopping early for futility to limit patient exposure to an inactive regimen
while restricting the probabilities of type I and type II errors to approximately 10% and 15%,
respectively74. A true ORR of 5% or less and a rate of PFS6 of 10% or less would not be of
clinical interest (H0 : OR ≤ 5% AND PFS6 ≤ 10%), whereas an improvement to a 20% ORR or
30% PFS6 rate would warrant further investigation of mirvetuximab and pembrolizumab. In the
first stage, 16 patients would be enrolled and accrual would pause. If we observed 2 or more
objective responses or 2 or more patients were progression-free at 6 months, accrual would
continue to the second stage where an additional 19 patients would be enrolled. With this
design and assuming independence of the endpoints, the probability of early termination for
futility under the null is 0.417. The study type I error is 9.8% and we would have 88.9% power to
reject the null when the true ORR is 20% and the true PFS6 rate is 10%, and 87.3% power to
reject the null when the true ORR is 5% and the PFS6 rate is 30%. Note that since the study
was non-blinded, if we met the criteria to continue to enrollment at any point during the first
stage, we could then open enrollment to the target accrual of 35 patients in total. Overall, if ≥4
treated patients with an objective response OR ≥8 patients who are progression-free at 6
months were observed, mirvetuximab and pembrolizumab would be considered worthy of
further study.

The study met the continuation criteria of 2 objective responses in Stage 1 after the first
5 patients were enrolled. Once these responses were confirmed and the statistical team was
consulted, the study opened to full enrollment of 35 patients. After consideration of the rapidly
changing treatment landscape in EC and slow study enrollment, completing the trial was
deemed unfeasible and the trial was closed after enrollment of 18 patients.

ORR was defined as the rate of achieving either complete or partial response by RECIST
1.1 among patients who initiated protocol therapy. PFS6 was defined as a binary outcome
indicating whether or not a patient was deemed progression-free for at least 6 months. Kaplan-
Meier estimates of the progression-free survival function were also estimated, which incorporated
censored outcomes in the analysis. 95% confidence intervals (CI) are reported for events rates
at landmark times and the median using Greenwood’s formula. The statistical significance of
biomarker associations with ORR was evaluated using Fisher’s exact test, with the missing
information excluded from the significance testing. For each type of immune profile data and each
region, comparisons were made between clinical groups of interest (defined by objective
response) using Wilcoxon rank-sum tests. No adjustments for multiple comparisons were
performed. All biomarker analyses are considered exploratory.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "The authors have edited the manuscript appropriately based on the comments from the reviewers. The reviews were overall positive to begin with unfortunately with the smaller numbers the enthusiasm is a little more muted. With that being said re-reviewing the manuscript, the responses and the global story presented with the therapeutic angle and translational angle it's an excellent manuscript.", "reviewer_3": "I would like to thank the editorial office for the opportunity to review the revised manuscript submitted by Porter at al. The authors describe a single arm phase 2 trial examining the combination of mirvetuximab + pembrolizumab in patients with recurrent serous endometrial cancer.\n\nI have a few comments/considerations for the authors:\n- In the methods section, please clarify if FOLR1 expression levels were adjudicated locally, by the institutional pathologist?\nAlso, was FOLR1 assessment done using the current approved platform in patients with epithelial ovarian cancer.\nI anticipate this may be the case, but would include this results paragraph detailing FOLR1 expression in the cohort.\n- In lines 104-105 it stated that 20 tumors were found to have FOLR1 expression >/= 75% of tumor cells. In the\nsubsequent paragraph, lines 115, it states that 50% of patients had FOLR1 >/= 75%. The population references should be clarified to\navoid confusion.\n- Was the median PFS of 2.73 months equivalent to the imaging assessment interval on trial?\n- If the authors report on OS (median OS in the overall cohort was 12.5 months), it would be beneficial to understand\nsubsequent therapies. With a small cohort, this should be available and will help contextualize the reported median OS\nvalue. As an example, did the patients with HER2 2+/3+ treated with T-Dxd?\n- The authors report that the median PFS in the ICI-exposed cohort was 1.2 months. Were these patients scanned earlier\nthan anticipated per protocol for clinical symptoms of disease progression?\n- There is a typo on line 182, it reads “profiling of on..”. I believe that the word “on” needs to be removed.\n- Comparing FOLR1 by IF and IHC in non-contiguous tumor samples may limit the interpretation of the data substantially.\nAlso, were these assessments on the same tissue block, or from alternate sites of metastatic disease?\n- Although interesting, the biomarker analysis needs to be controlled for multiple testing. Did the authors divide the\nsignificance threshold by the number of tests being performed, or the number of potential biomarker combinations explored?\nAlternatively, a Hochberg correction can be considered. It is unclear to me if this was done? I am concerned that too much\nweight is being placed on this exploratory analysis in the discussion.", "reviewer_4": "Statistical / Design Review\nSummary of study design and analyses This is a single-arm, two-stage phase 2 trial evaluating mirvetuximab soravtansine (MIRV) plus pembrolizumab in 18 patients with recurrent/persistent pMMR serous endometrial cancer (FOLR1 expression ≥50%). The study utilized a dual-endpoint two-stage design (Sill et al.) with co-primary endpoints of Objective Response Rate (ORR) and 6-month Progression-Free Survival (PFS6). The trial was designed for 35 patients but closed early due to feasibility issues rather than futility.\nAmong the 18 treated patients, the confirmed ORR was 28% (95% CI 10–53%), and the Kaplan–Meier estimated PFS6 was 24.4%. All confirmed responses occurred in ICI-naïve patients. Safety was consistent with known profiles. Exploratory analyses suggested potential associations between response and CCNE1 amplification or a composite biomarker (\"B score\").\nMajor comments\n1. Early closure and two-stage design implications The trial was designed for 35 patients with specific operating characteristics (Type I ≈10%, Type II ≈15%) but closed after 18 patients. While the operational decision is reasonable, it invalidates the originally specified error control and decision boundaries.\nRecommendation: Explicitly state in the Methods/Results that while the trial met the stage 1 continuation rule (≥2 responses), the full two-stage design was not completed. Consequently, the reported results should be framed as preliminary and hypothesis-generating rather than as meeting formal phase 2 success criteria.\n2. Co-primary endpoints and success criteria The use of a dual-endpoint design is a strength. However, the specific success criteria for the co-primary endpoints need better visibility.\nRecommendation: Explicitly state the exact success criterion (e.g., \"≥4 responses OR ≥8 patients progression-free at 6 months\") to clarify how multiplicity was handled. Additionally, briefly summarize the actual operating characteristics under plausible scenarios rather than citing approximate error rates.\n3. Definition of PFS6 (Design Count vs. KM Estimate) The design utilizes a simple count of patients progression-free at 6 months, yet the Results report a Kaplan–Meier estimate (24.4%).\nRecommendation: Please distinguish between the design quantity (binary count in the n=35 design) and the KM-estimated PFS6. Clarify if all 18 patients had sufficient follow-up for the binary endpoint or how censoring was handled regarding the stopping decision.\n4. Population heterogeneity (Protocol Amendment) The amendment allowing prior ICI exposure introduces heterogeneity. Prior ICI exposure was associated with shorter PFS (HR = 0.2), complicating efficacy interpretation.\nRecommendation: Highlight that the original design assumptions implicitly assumed a homogeneous population. The subgroup findings (ICI-naïve vs. exposed) should be labeled as exploratory and unadjusted for confounders given the sample size.\n5. Biomarker analyses and overfitting The biomarker work is a strength but is statistically underpowered. The composite \"B score\" was derived and evaluated in the same small cohort, carrying a high risk of overfitting.\nRecommendation: Explicitly state that no adjustment for multiple comparisons was performed. Clearly label the B score as a hypothesis-generating composite requiring external validation. I suggest simplifying the narrative to focus on biological signals (e.g., CCNE1, TIME) rather than specific p-values or cut-points.\n6. Estimation and uncertainty Given the sample size (n=18), the precision of estimates is low.\nRecommendation: Emphasize the wide confidence intervals for ORR (10–53%) in the Discussion. Comparisons by prior ICI or FOLR1 status should be framed as descriptive patterns rather than definitive subgroup effects.\nMinor comments / suggestions\nAnalysis Population: Explicitly define the analysis population (e.g., mITT). Confirm that all treated patients, including early withdrawals, were included in the ORR denominator.\nSubgroup Visualization: A forest plot or table summarizing ORR/PFS by ICI status and FOLR1 expression levels (50–74% vs. ≥75%) would clarify the uncertainty around these subgroup effects.\nFeasibility: You note that only ~15% of prescreened tumors met the ≥75% FOLR1 cutoff. A brief comment on the implications of this for future trial feasibility and screening volume would be valuable.\nOverall assessment This trial addresses an important question using a sensible design. The primary limitations—early closure (n=18 vs. 35), small sample size, and lack of multiplicity control in exploratory analyses—do not invalidate the study but require careful framing. With the clarifications suggested above, the statistical reporting will be appropriate for a signal-seeking phase 2 study."}, "author_response": "Phase II Study of Mirvetuximab Soravtansine plus Pembrolizumab in Recurrent Folate Receptor Alpha-Positive Uterine Serous Carcinoma\nResponse to Reviewer Comments\nWe thank the reviewers for the thorough and considerate review and for including helpful comments.\nWe have carefully considered the reviewers’ remarks and have responded point-by-point below (in bold) and revised the manuscript accordingly.\nReviewer #2 (Remarks to the Author):\nThe authors have edited the manuscript appropriately based on the comments from the reviewers. The reviews were overall positive to begin with unfortunately with the smaller numbers the enthusiasm is a little more muted. With that being said re-reviewing the manuscript, the responses and the global story presented with the therapeutic angle and translational angle it's an excellent manuscript.\nWe thank Reviewer #2 for re-reviewing our manuscript and for their overall positive assessment.\nReviewer #3 (Remarks to the Author):\nI would like to thank the editorial office for the opportunity to review the revised manuscript submitted by Porter at al. The authors describe a single arm phase 2 trial examining the combination of mirvetuximab + pembrolizumab in patients with recurrent serous endometrial cancer.\nI have a few comments/considerations for the authors:\n- In the methods section, please clarify if FOLR1 expression levels were adjudicated locally, by the institutional pathologist? Also, was FOLR1 assessment done using the current approved platform in patients with epithelial ovarian cancer. I anticipate this may be the case, but would include this results paragraph detailing FOLR1 expression in the cohort.\nWe thank the reviewer for pointing out the need for further clarification on FOLR1 assessment. It is correct that the current approved Ventana platform was used in this study, and this was performed centrally at Ventana. This is stated in the Results section under “FOLR1 assessment” on line 492:\n“FOLR1 status was assessed centrally at Ventana Medical Systems by immunohistochemistry on archival tumor tissue using the anti-FOLR1 clone FOLR1-2.1 antibody (Roche Diagnostics).”\nWe have also added this clarification to the Results section as suggested on line 100: ” In the current study, a total of 144 eligible participants signed consent for pre-screening of archival tumor tissue for FOLR1 expression by central IHC using the anti-FOLR1 2.1 antibody developed by Ventana Medical Systems and following the PS2 scoring methodology of quantifying the percentage of tumor cells staining at ≥2+ intensity.”\n- In lines 104-105 it stated that 20 tumors were found to have FOLR1 expression >/= 75% of tumor cells. In the subsequent paragraph, lines 115, it states that 50% of patients had FOLR1 >/= 75%. The population references should be clarified to avoid confusion.\nWe have specified in line 117 (previously line 115) that the 50% of patients with FOLR1 >/= 75% was in the enrolled population, whereas the 20 patients (14.6%) with FOLR1 >/= 75% was in the prescreening population.\n- Was the median PFS of 2.73 months equivalent to the imaging assessment interval on trial?\nImaging assessment was performed every 6 weeks (+/- 1 week) on study. The median PFS of 2.73 months (i.e., 2.73 * 30.4 = 82.99 days = 11.86 weeks) corresponds to roughly the second imaging assessment.\n- If the authors report on OS (median OS in the overall cohort was 12.5 months), it would be beneficial to understand subsequent therapies. With a small cohort, this should be available and will help contextualize the reported median OS value. As an example, did the patients with HER2 2+/3+ treated with T-Dxd?\nWe thank the reviewer for this question. Subsequent therapies were not officially recorded as part of the conduct of the trial. However, of the 19 patients in the study, subsequent treatment information was available for 11 patients. 5 patients received one more regimen, 2 had received two regimens and 5 patients received 3 or more subsequent regimens (maximum 6). Subsequent regimens included chemotherapy (such as PLD, carbo/PLD, gemcitabine), immunotherapy/antiangiogenic therapy (pembro/lenvatinib), targeted therapies (Enhertu, PARPi, PI3K targeted therapies) and clinical trial participation. We have added in the manuscript Methods that subsequent therapies was not recorded as part of the study.\n- The authors report that the median PFS in the ICI-exposed cohort was 1.2 months. Were these patients scanned earlier than anticipated per protocol for clinical symptoms of disease progression?\nThe median PFS in the ICI-exposed cohort of 1.2 months (1.2*30.4 = 36.48 days = 5.21 weeks) corresponds to the first imaging assessment (6 weeks +/- 1 week) as specified in the protocol; no patients were scanned earlier than anticipated.\n- There is a typo on line 182, it reads “profiling of on..”. I believe that the word “on” needs to be removed.\nWe thank the reviewer for this point out; the manuscript file has been corrected.\n- Comparing FOLR1 by IF and IHC in non-contiguous tumor samples may limit the interpretation of the data substantially. Also, were these assessments on the same tissue block, or from alternate sites of metastatic disease?\nWe thank the reviewer for recognizing this potential limitation of the biomarker analyses. We agree that contiguous tumor samples would be ideal for assessing FOLR1 IHC and IF. However, contiguous slides were not available in all samples and therefore the assays were performed on the available tissue. In general, as shown in the representative images in Supplemental Fig 4, similar tumor sections were selected for IHC and IF staining and we observed good concordance between the two assays. We have acknowledged this issue in the manuscript (line 242) as well.\n- Although interesting, the biomarker analysis needs to be controlled for multiple testing. Did the authors divide the significance threshold by the number of tests being performed, or the number of potential biomarker combinations explored? Alternatively, a Hochberg correction can be considered. It is unclear to me if this was done? I am concerned that too much weight is being placed on this exploratory analysis in the discussion.\nPlease see our response to Reviewer #4 comment 5 below.\nReviewer #4 (Remarks to theAuthor):\nWe thank the statistical Reviewer for the opportunity to improve our manuscript with their very helpful comments and recommendations. We have incorporated and responded to each in a point-by-point response below.\nStatistical / Design Review\nSummary of study design and analyses This is asingle-arm, two-stage phase 2 trial evaluating mirvetuximab soravtansine (MIRV) plus pembrolizumab in 18 patients with recurrent/persistent pMMR serous endometrial cancer (FOLR1 expression ≥50%). The study utilized a dual-endpoint two-stage design (Sill et al.) with co-primary endpoints of Objective Response Rate (ORR) and 6-month Progression-Free Survival (PFS6). The trial was designed for 35 patients but closed early due to feasibility issues rather than futility.\nAmong the 18 treated patients, the confirmed ORR was 28% (95% CI 10–53%), and the Kaplan–Meier estimated PFS6 was 24.4%. All confirmed responses occurred in ICI-naïve patients. Safety was consistent with known profiles. Exploratory analyses suggested potential associations between response and CCNE1 amplification ora composite biomarker (\"B score\").\nMajor comments\n1. Early closure and two-stage design implications The trial was designed for 35 patients with specific operating characteristics (Type I ≈10%, Type II ≈15%) but closed after 18 patients. While the operational decision is reasonable, it invalidates the originally specified error controland decision boundaries.\nRecommendation: Explicitly state in the Methods/Results that while the trial met the stage 1 continuation rule (≥2 responses), the full two-stage design was not completed. Consequently, the reported results should be framed as preliminary and hypothesis-generating rather than as meeting formal phase 2 success criteria.\nIn the Statistical Analysis section on line 555, we state:\n“The study met the continuation criteria of 2 objective responses in Stage 1 after the first 5 patients were enrolled. Once these responses were confirmed and the statistical team was consulted, the study opened to full enrollment of 35 patients.”\nIn addition, we have revised the following text in the results section starting on line 123:\n“Among the first 5 patients enrolled, we observed 2 confirmed objective responses meeting the criteria to continue enrollment in the two-stage design. Among all 18 patients, the confirmed ORR was 28% (95% CI: 10-53%) including 1 confirmed complete response, 4 confirmed partial responses (Figure 2A). While the study meets the required number of responses for ORR (≥4 responses) specified by the two-stage design, and the 95% confidence interval excludes the null ORR of 5%, we did not complete enrollment of the trial to the planned sample size of 35 patients and hence these results should be considered preliminary.”\n2. Co-primary endpoints and success criteria. The use of adual-endpoint design is a strength. However, the specific success criteria for the co-primary endpoints need better visibility.\nRecommendation: Explicitly state the exact success criterion (e.g., \"≥4 responses OR ≥8 patients progression-freeat 6 months\") to clarify how multiplicity was handled. Additionally, briefly summarize theactual operating characteristics under plausible scenarios rather than citing approximate error rates.\nThe success criterion has been clearly stated on line 552 of the manuscript:\n“Overall, if ≥4 treated patients with an objective response OR ≥8 patients who are progression-free at 6 months were observed, mirvetuximab and pembrolizumab would be considered worthy of further study.”\nWe have also added the following text on line 543 to describe the full detailed operating characteristics of the design.\n“With this design, the probability of early termination for futility under the null assuming independence of the endpoints is 0.417 and 0.502 assuming dependence of the endpoints. The study would have 88.9% power to reject the null when the true ORR is 20% and the true PFS6 rate is 10%, and 87.3% power to reject the null when the true ORR is 5% and the PFS6 rate is 30% assuming independence of the endpoints. When the endpoints are assumed to be dependent, the study power is 84.6% and 86.2%, respectively. The type I error is 9.8% assuming independence of endpoints and 0.088 assuming dependence of endpoints.\n3. Definition of PFS6 (Design Count vs. KM Estimate) The design utilizes asimple count of patients progression-free at 6 months, yet the Results report a Kaplan–Meier estimate (24.4%).\nRecommendation: Please distinguish between the design quantity (binary count in the n=35 design) and the KM-estimated PFS6. Clarify if all 18 patients had sufficient follow-up for the binary endpoint or how censoring was handled regarding the stopping decision.\nWe have added the definition of PFS6 to manuscript on line 470:\n“(PFS6) defined as having disease assessment scans for at least 6 months or longer showing no progression per RECIST v1.1. Patients with less than 6 months of follow-up for PFS were treated as non-PFS6.”\nWe have now separated the binary PFS6 estimate from the Kaplan-Meier method estimate more clearly in the Results section on line 134:\n“There were 4 patients alive and progression-free at 6 months resulting in a PFS6 rate of 22% (95% CI: 6-48%).”\n“The observed median PFS (mPFS) was 2.73 months (95% CI: 1.2-4.5 months) (Supplemental Figure 2) and the PFS at 6 months was 24.4% (95% CI 7.7-46.1%) by the Kaplan-Meier method.”\n4. Population heterogeneity (ProtocolAmendment) Theamendment allowing prior ICI exposure introduces heterogeneity. Prior ICI exposure was associated with shorter PFS (HR = 0.2), complicating efficacy interpretation.\nRecommendation: Highlight that the original design assumptions implicitly assumed ahomogeneous population. The subgroup findings (ICI-naïve vs. exposed) should be labeled as exploratory and unadjusted for confounders given the sample size.\nWe have modified this section of the results on line 147 and added the following text prior to reporting these results:\n“The initial design of the trial enrolled ICI naïve patients only but a subsequent protocol amendment allowed for enrollment of up to 19 patients with prior ICI targeting the PD-1/PD-L1 pathway. In an exploratory subgroup analysis, we evaluated the efficacy of mirvetuximab and pembrolizumab in patients that were ICI naïve (n=10) versus exposed (n=8).”\n5. Biomarker analysesand overfitting The biomarker work is astrength but is statistically underpowered. The composite \"B score\" was derived and evaluated in the same small cohort, carrying ahigh risk of overfitting.\nRecommendation: Explicitly state that no adjustment for multiple comparisons was performed. Clearly label the B scoreas ahypothesis-generating composite requiring external validation. I suggest simplifying the narrative to focus on biological signals (e.g., CCNE1, TIME) rather than specific p-values or cut-points.\nIn the Statistical methods section on line 569 we have added the following text:\n“No adjustments for multiple comparisons were performed. All biomarker analyses are considered exploratory.”\nIn the Discussion section on line 342, we have added the following text when discussing the B score:\n“However, this score was derived and evaluated in this small cohort and should therefore be considered hypothesis-generating. Validation in external cohorts of patients in the future will be required to draw conclusions on the clinical relevance of this B score.”\nWe also expanded the summary statement regarding the B score on line 264 to focus more on the TIME rather than specific score, as suggested:\n“Median overall survival was 1.28 months(95% CI: 1.18–NR) for patients with B–scores (n=7, 6 events) compared with6.19 months (95% CI: 2.73–NR) for patients with B+ scores (n=8, 5 events), supporting the potential clinical significance of this hypothesis-generating biomarker score, or others similarly describing a favorable tumor and immune microenvironment, in predicting patients who may derive benefit from an ADC combined with ICB.”\n6. Estimationand uncertainty Given the sample size (n=18), the precision of estimates is low.\nRecommendation: Emphasize the wide confidence intervals for ORR (10–53%) in the Discussion. Comparisons by prior ICI or FOLR1 status should be framed as descriptive patterns rather than definitive subgroup effects.\nWe have addressed this on line 304 in the discussion of comparisons by prior ICI or FOLR1 status:\n“Given the smaller size of this study, associations between clinical activity and FOLR1 expression and/or prior ICI exposure will need to be confirmed in future studies.”\nAnd have additionally added a limitations paragraph at the end of the discussion (line 388) to more explicitly state these limitations:\n“The small sample size and early closure of this trial are clear limitations of the study. Given this, the exploratory subgroup analyses are viewed as descriptive patterns that can inform future hypotheses but will require evaluation in larger cohorts. Similarly, the lack of adjustment for multiple comparisons in the biomarker score analyses makes the composite B score also hypothesis-generating and requires external validation. However, given the broad interest in ADC and ICI combinations in many cancer types, these analyses may serve as a foundation for developing similar composite methods to score the overall favorability of the tumor immune microenvironment for ADC and ICI response.”\nMinor comments / suggestions\nAnalysis Population: Explicitly define theanalysis population (e.g., mITT). Confirm that all treated patients, including early withdrawals, were included in the ORR denominator.\nYes, we used a modified intent to treat analysis population where all patients that received any protocol therapy were included in the safety and efficacy populations. Of note, one patient was incorrectly assigned a study ID (#18) but did not enroll in the trial and hence they were not included in the analysis population.\nSubgroup Visualization:A forest plot or table summarizing ORR/PFS by ICI status and FOLR1 expression levels (50–74% vs. ≥75%) would clarify the uncertainty around these subgroup effects.\nSupplementary Tables 2 and 4 summarize the ORR/median PFS by ICI status and FOLR1 expression levels (50–74% vs. ≥75%), respectively. These have been modified to include ORR and associated confidence intervals and HR and associated confidence intervals to be comprehensive per the reviewer’s suggestion.\nFeasibility: You note that only ~15% of prescreened tumors met the ≥75% FOLR1 cutoff. A brief comment on the implications of this for future trial feasibility and screening volume would be valuable.\nThe 75% reference in the discussion around FOLR1 expression refers to the cutoff for clinical use of mirvetuximab in ovarian cancer, not in endometrial cancer. We were raising the point that activity was seen in our lower cut-off of 50% which may allow for improved access to the drug in patients with endometrial cancer. To make the reference to ovarian cancer clearer, we have spelled out PROC to state “ovarian cancer”.\nOverall assessment This trial addresses an important question using asensible design. The primary limitations—early closure (n=18 vs. 35), small sample size, and lack of multiplicity control in exploratory analyses—do not invalidate the study but require careful framing. With the clarifications suggested above, the statistical reporting will be appropriate for asignal-seeking phase 2 study."}, {"round": 2, "reviewer_comments": {"reviewer_3": "The revised manuscript was reviewed. The authors/study team have thoughtfully and comprehensively addressed all concerns. I find the manuscript suitable for publication in the current format.", "reviewer_4": "The authors carefully addressed all of my questions and concerns. I have no further major concerns.\nOne minor suggestion relates to the power and Type I error calculation under endpoint dependence. The authors state:\n'When the endpoints are assumed to be dependent, the study power is 84.6% and 86.2%, respectively. The type I error is 9.8% assuming independence of endpoints and 0.088 assuming dependence of endpoints.\nStatistical Clarification: Since dependence is determined by the correlation ($\\rho$), the dependence scenario is not unique.\nCould the authors please specify the assumed correlation coefficient ($\\rho$) between the co-primary endpoints that resulted in the stated power (86.2%) and Type I error (0.088)?\nAlternatively, to simplify the discussion, the authors may consider removing the dependence scenario and focusing solely on the operating characteristics calculated under the independence assumption."}, "author_response": "Reviewer #4 (Remarks to the Author):\nOne minor suggestion relates to the power and Type I error calculation under endpoint dependence. The authors state: 'When the endpoints are assumed to be dependent, the study power is 84.6% and 86.2%, respectively. The type I error is 9.8% assuming independence of endpoints and 0.088 assuming dependence of endpoints.\nStatistical Clarification: Since dependence is determined by the correlation ($\\rho$), the dependence scenario is not unique. Could the authors please specify the assumed correlation coefficient ($\\rho$) between the co-primary endpoints that resulted in the stated power (86.2%) and Type I error (0.088)?\nAlternatively, to simplify the discussion, the authors may consider removing the dependence scenario and focusing solely on the operating characteristics calculated under the independence assumption.\nWe thank the reviewer for this suggestion. We agree that the dependence scenario is not unique and have removed it from the manuscript and only present the operating characteristics calculated under the independence assumption."}]

Decision letter