Dalpicilib combined with cetuximab in patients with HPV-negative, anti-PD-1-resistant recurrent or metastatic head and neck squamous cell carcinoma: A phase II trial

Domain: neuroscience
Published: 2026-01-28
Editorial outcome: minor_revision

Abstract
Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is characterised by hyperactivation of the cyclin-dependent kinase 4/6 (CDK4/6) pathway. As immunotherapy has become the first-line treatment for HNSCC, resistance to anti-programmed death-1 (PD-1) agents has emerged as a pivotal challenge. This prospective, single arm, phase II study (NCT05721443) evaluated the efficacy and safety of dalpiciclib, a CDK4/6 inhibitor, combined with cetuximab in patients with anti-PD-1-resistant, HPV-negative recurrent and/or metastatic HNSCC. Patients diagnosed with p16-negative R/M HNSCC resistant to first-line anti-PD-1 therapy without prior cetuximab treatment were enroled. Patients received oral dalpiciclib (150 mg daily on days 1-21 of each 28-day cycle) and intravenous cetuximab (400 mg/m2 on day 1 of cycle 1, followed by 250 mg/m2 weekly in each cycle). The primary endpoint was objective response rate (ORR), secondary endpoints were overall survival, progression-free survival, duration of response, and safety. Between March 2023 and November 2024, a total of 28 patients were enroled. The ORR was 67.9% (19/28; 95% confidence interval [CI], 49.0%-82.0%), which met our primary endpoint. With a median follow-up of 9.2 months (interquartile range [IQR], 5.98-14.12), the median progression-free survival was 7.0 months (95% CI, 4.13-not reached [NR]), and the median overall survival was 17.0 months (95% CI, 10.75-23.25). Treatment-related adverse events (TRAEs) occurred in all patients, predominantly grade 1-2. Grade 3 TRAEs included neutrophil count decreased (9/28, 32.1%) and white blood cell count decreased (9/28, 32.1%). No grade 4 or 5 TRAEs were observed. Dalpiciclib combined with cetuximab was well-tolerated and showed promising efficacy in patients with anti-PD-1-resistant, HPV-negative recurrent and/or metastatic HNSCC.

Introduction
Patients diagnosed with recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) have poor outcomes, with a median overall survival (OS) of 8–10 months1,2. In recent years, immunotherapy has significantly improved clinical outcomes in patients with R/M HNSCC and has increasingly emerged as a first-line treatment for HNSCC. However, pembrolizumab combined with chemotherapy results in an objective response rate (ORR) of 36% and a median progression-free survival (PFS) of 4.9 months, revealing that a considerable number of patients rarely benefit from immunotherapy3.

The incidence of human papillomavirus (HPV)-negative HNSCC is greater than that of HPV-related HNSCC. However, compared with HPV-related HNSCC, HPV-negative HNSCC is often associated with a lower response rate and poorer survival with immunotherapy4,5. HPV-negative HNSCC is characterised by hyperactivation of the cyclin-dependent kinase 4/6 (CDK4/6) pathway, accompanied by CCND1 amplification and CDKN2A deficiency. Epidermal growth factor receptor (EGFR) is highly expressed in more than 90% of HNSCC cases and can activate the CDK4/6-cyclin D complex and ultimately drive cancer cell growth by promoting the progression of the cell cycle through the G1 phase, facilitating the malignant transformation of cancer cells6. This provides a rationale for the combination of CDK4/6 and EGFR inhibitors.

Cetuximab, an IgG1 monoclonal antibody against EGFR, binds to the extracellular domain of EGFR and thereby inhibits the tyrosine kinase activity of cancer cells7. Cetuximab is the standard salvage therapy for HNSCC patients refractory to first-line immunotherapy. However, the efficacy of cetuximab monotherapy as a second-line treatment was modest, with an objective response rate of 13% and a median PFS of 2.3 months1. A previous study reported that palbociclib, a selective CDK4/6 inhibitor, resulted in promising outcomes when combined with cetuximab in patients with platinum-resistant or cetuximab-resistant, HPV-negative R/M HNSCC8. However, the anti-tumour activity of the combination of CDK4/6 and EGFR inhibitors is unknown in patients with immunotherapy-resistant, HPV-negative R/M HNSCC.

Here, we report that dalpiciclib, a selective CDK4/6 inhibitor, combined with cetuximab has a promising objective response and considerable toxicity in patients with anti-PD-1-resistant, HPV-negative HNSCC.

Results
Between March 2023 and November 2024, a total of 31 patients were screened, and 28 patients were ultimately enrolled in this study (Fig. 1B). Baseline demographics and disease characteristics are presented in Table 1. The median patient age was 58 years (range, 30–75). The primary site of the tumour mainly originated from the oral cavity. The majority of patients (26/28, 92.9%) experienced locoregional recurrence, and only two patients (7.1%) experienced locoregional recurrence with metastases. Fourteen patients (50.0%) had previously received PD-1 inhibitors with chemotherapy, and ten patients (35.7%) had previously received PD-1 inhibitors with vascular epidermal growth factor receptor inhibitors in the first-line setting. Tissue specimens were available for 21 (75.0%) patients.

By the data cut-off date on March 3rd, 2025, the median follow-up duration was 9.2 months (IQR 5.98–14.12). Ten patients were still on treatment. The median number of treatment cycles was 4.5 (range, 1–14) for cetuximab and 4.5 (range, 1–14) for dalpiciclib. During the study period, two patients (7.1%) discontinued the study treatment because they could undergo surgery after effective treatment, and one of them achieved a pathological complete response.

In the first stage, among the 14 evaluable participants, 3 patients experienced disease progression, 1 had stable disease, and 9 achieved a PR. In the final analysis, among 28 evaluable patients, 19 achieved a PR, 6 had stable disease, and 3 experienced disease progression. The ORR was 67.9% (95% CI, 49.0%–82.0%; Fig. 2A), which met the primary endpoint. The median DOR was 9.0 months (95% CI, 3.35–14.59; Fig. 2B). A decrease in the sum of the target lesions was observed in 25 patients (89.3%).

At the time of analysis, 16 PFS events occurred. The median PFS was 7.0 months (95% CI, 4.13-NR; Fig. 3A), and the 6-month PFS rate was 53.6%. Ten (32.1%) patients died (9 due to disease progression and one due to haemorrhage). The median OS was 17.0 months (95% CI, 10.75–23.25; Fig. 3B), and the 12-month OS rate was 67.9%.

Baseline tumour tissues from 22 patients were eligible for PD-L1 CPS examination. Among these patients, 19 had a PD-L1 CPS ≥ 1, and 6 had a PD-L1 CPS ≥ 20. In populations with a PD-L1 CPS ≥ 1, the ORR was 68.4% (95% CI, 45.8%-84.8%), the median PFS was 5.3 months (95% CI, 1.52–9.08), and the median OS was 17.0 months (95% CI, 7.21–26.79). In populations with a PD-L1 CPS ≥ 20, the ORR was 66.7% (95% CI, 29.6%–90.7%), the median PFS was 5.3 months (95% CI, 1.89–8.71), and the median OS was not reached (Supplementary Fig. 1).

The TRAEs across all cycles are shown in Table 2. TRAEs occurred in all patients, predominantly grades 1–2. The most common TRAEs were decreased neutrophil count (25/28, 89.3%), decreased white blood cell count (25/28, 89.3%), and acneiform rash (16/28, 57.1%). Grade 3 TRAEs occurred in 35.7% (10/28) of the patients, including a decreased neutrophil count (9/28, 32.1%) and a decreased white blood cell count (9/28, 32.1%). The patients rapidly recovered after symptomatic treatment within one week, except for one patient with a grade oral ulcer who recovered after nearly three weeks. No grade 4 or 5 TRAEs were observed.

During the study period, 85.7% (24/28) of the patients received a dose reduction of dalpiciclib, and no patients received a dose reduction of cetuximab. No patients discontinued the treatment because of intolerable toxicity.

Baseline tumour tissues from 20 patients were eligible for next-generation sequencing. The most frequently mutated gene was TP53 (17/20, 85%), followed by TERT (14/20, 70%), TTN (9/20, 45%), FAT1 (6/20, 30%), and CDNK2A (6/20, 30%). The most common gene amplifications were CCND1 (5/20, 25%), FGF4 (5/20, 25%), FGF3 (5/20, 25%), and FGF9 (5/20, 25%; Fig. 4A). Among the 20 patients, 9 had abnormal mutations of CDK4-related genes (4 had CDKN2A deletion, 3 had CCND1 amplification, and 2 had both CDKN2A deletion and CCND1 amplification). The ORR was 81.8% vs. 33.3% (Z = −2.20; P = 0.028), the median PFS was 14.3 vs. 4.3 months (HR 0.26 [95% CI 0.07−0.91]; P = 0.035), and the median OS was not reached vs. 8.5 months (HR 0.23 [95% CI 0.05−1.06]; P = 0.060) in populations without abnormal mutations of CDK4-related genes compared with those with abnormal CDK4-related genes (Supplementary Fig. 2A, B). For the CCND1 amplification analysis, the ORR was 60.0% vs. 60.0% (Z = 0.00, P = 1.000), and the median PFS was 14.3 vs. 3.3 months (HR 0.22 [95% CI 0.01−0.45], P = 0.007), and the median OS was not reached vs. 8.5 months (HR 0.21 [95% CI 0.02−0.75], P = 0.024) in the population without CCND1 amplification compared with the population with CCND1 amplification (Supplementary Fig. 2C, D). With respect to the results of the TP53 mutation analysis, the ORR was 66.7% vs. 58.8% (Z = −0.26, P = 0.798), the median PFS was 14.3 vs. 7.0 months (HR 0.28 [95% CI 0.08−1.11], P = 0.127), and the median OS was not reached vs. 17.7 months (HR unevaluated, P = 0.208), respectively, in the population without TP53 mutation compared with the population with TP53 mutation (Supplementary Fig. 2E, F).

The median TMB was 2.5 per Mb (range, 0.4–8.7), and all patients had microsatellite-stable disease. Substitution of KCNMA1 and TNXB and amplification of TPCN2 were more frequent in nonresponders than in responders (CR+PR; Supplementary Data 1 and 2), while no significant difference in TMB was observed (Fig. 4B). The percentage of lymphocytes significantly increased in responders at the time they achieved the best response compared with baseline (Fig. 4C). However, no significant change in the percentage of lymphocytes was observed in nonresponders (Fig. 4D).

Four patients received subsequent anti-PD-1 therapy after treatment failed with dalpiciclib plus cetuximab. Two patients who responded to dalpiciclib plus cetuximab achieved a PR to subsequent anti-PD-1 rechallenge therapy, whereas the other two patients who did not respond to dalpiciclib plus cetuximab did not respond to subsequent anti-PD-1 therapy (Fig. 4E and Supplementary Fig. 3). For tumour immune microenvironment analysis, we observed that neither baseline CD8+ T cells, Tregs, natural killer (NK) cells nor the CD8+/Treg and CD8+/CD56 ratios were associated with response, PFS or OS in participants receiving dalpiciclib with cetuximab therapy (Supplementary Fig. 4).

Methods
The trial protocol was approved by the ethics committee of Shanghai Ninth People’s Hospital. The trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. All the participants signed written informed consent prior to enrolment (the first participant was enroled on March 7th, 2023, and the last participant was enroled on November 1st, 2024). The study was registered at ClinicalTrials.gov (NCT05721443, https://register.clinicaltrials.gov/, first posted on February 10th, 2023). In our trial, dalpiciclib and imaging examinations were provided free of charge for all enrolled patients.

This study was an investigator-initiated, single-arm, single-centre phase II trial conducted at Shanghai Ninth People’s Hospital. The eligible participants were aged 18–75 years and had histologically confirmed R/M HNSCC; had received at least one cycle of anti-PD-1 therapy in the R/M setting; had at least one measurable lesion according to the Response Evaluation Criteria In Solid Tumours (RECIST) version 1.135; had radiographic confirmation of disease progression or clinical judgement indicating no further benefit from anti-PD-1 therapy; had confirmed HPV-negative disease by immunohistochemistry (IHC); had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; and had acceptable bone marrow and organ function.

Participants were excluded if they had previously received cetuximab or tyrosine kinase inhibitors targeting EGFR; were known to be hypersensitive to recombinant humanised EGFR monoclonal antibodies or CDK4/6 inhibitors; had other types of cancer (excluding cured skin basal cell carcinoma, skin squamous cell carcinoma, carcinoma in situ of the cervix, and early prostate cancer) within 5 years; had received haematopoietic growth factors within one week prior to the study treatment; had active human immunodeficiency virus or treponema pallidum infection; were unable to swallow the drugs; or had abnormal coagulation function.

Participants received cetuximab intravenously at an initial dose of 400 mg/m² followed by a weekly maintenance dose of 250 mg/m2. Dalpiciclib was administered orally at a dose of 150 mg once daily for 21 consecutive days, followed by a 7-day break. Each 28-day period was considered a treatment cycle. Treatment was continued until disease progression, unacceptable toxicity, or patient withdrawal, whichever occurred first.

Dalpiciclib treatment was stopped if a grade 2 treatment-related adverse event (TRAE) occurred and was resumed if the TRAE recovered to grade 0 or 1 within 12 weeks. If a patient experienced a grade 3 or higher TRAE, the dose was reduced to 125 mg/day. If the patient was still unable to tolerate this dose, the dose was reduced stepwise to 100 mg/day or to 75 mg/day on the basis of the investigator’s assessment. Dalpiciclib was permanently discontinued if grade 3 or higher TRAEs occurred and failed to recover to grade 0 or 1 within 12 weeks.

Imaging examinations were conducted by computed tomography or magnetic resonance imaging at baseline, every two cycles through the first year, and every 3 months thereafter. The response to treatment was determined according to RECIST 1.1, assessed by a specialised radiologist and confirmed by two other masking radiologists. Survival information was obtained from participants every 3 months during follow-up. TRAEs were collected during the treatment period and 1 month (3 months if serious adverse events occurred) after treatment withdrawal and were graded according to the Common Terminology Criteria for Adverse Events version 5.0. The detailed study protocol is included in the Supplementary Information file.

The primary endpoint was the ORR, defined as the proportion of patients with a partial response (PR) or complete response (CR) according to RECIST 1.1. Secondary endpoints included OS (defined as the time from enrolment to death from any cause), PFS (defined as the time from enrolment to the first disease progression or death from any cause, whichever occurred first), duration of response (DOR; defined as the interval from the first time that patients achieved a CR or PR to the first disease progression or death from any cause, whichever occurred first), and safety. Exploratory endpoints included the ORR, PFS, and OS in populations with PD-L1 CPSs ≥ 1 and 20 and the ORR, PFS, and OS in individuals with abnormal mutations of CDK4-related genes (CDK4 amplification, CCND1 amplification, or CDKN2A deletion).

Tumour tissue samples (archival paraffin-embedded specimens from the last 2 years or fresh tumour biopsies) and blood samples were collected from the enrolled patients. HPV status was confirmed on the basis of p16 expression detected by IHC (CINtec p16 Histology [Ventana, Tucson, AZ]) using formalin-fixed paraffin-embedded samples. A targeted sequencing library was generated using a next-generation sequencing panel (YouSuTM, OrigiMed) according to the established standard operating protocol. DNA (50–250 ng) from each sample was used for DNA library preparation as follows: first, DNA was fragmented using the M220 focused ultrasonicator Covaris (Covaris, Woburn, MA, USA) to 150–220 bp; second, the DNA fragments were end-polished A-tailed and ligated with adaptors; then, the exons of 242 cancer-related genes and the introns of 43 genes were captured and amplified by polymerase chain reaction. After quality control and quantification using a LabChip GXII Touch HT (PerkinElmer) and a Qubit 3 Fluorometer (Thermo Fisher), the DNA libraries were sequenced on the Illumina-Novaseq 6000 Platform (Illumina Incorporated, San Diego, CA) by paired-end 150-bp sequencing to a mean unique depth of 1000× coverage.

Single-nucleotide variations were identified by MuTect (version 1.7; Broad Institute, Cambridge, MA, USA). Insertion deletions were identified using PINDEL (version 0.2.5). The functional effects of the GAs were annotated using SnpEff 3.0. Copy number variation regions were identified using Control-FREEC (version 9.7; Institute Cochin, Paris, France) with the following parameters: window=50000 and step=10000. Gene fusions were detected using an in-house developed pipeline. Gene rearrangements were assessed using the Integrative Genomics Viewer. The tumour mutation burden (TMB) was estimated by counting the number of coding somatic mutations, including single-nucleotide variations and insertions deletions, per kilobase of sequence examined for each patient. The visual analysis of cancer genomics was performed with the “maftools” and “ggplot2” R packages. Biomarker analysis for tissue samples was approved by the Institutional Review Board of Shanghai Ninth People’s Hospital, and written informed consent to obtain these samples for analysis was obtained from all participants.

Tumour-infiltrating lymphocytes (TILs) were analysed via multiplex immunofluorescence using the following primary antibodies: rabbit anti-human CD4 (Servicebio, cat. no. GB150062; 1:1000), mouse anti-human CD8 (Servicebio, cat. no. GB12068; 1:500), rabbit anti-human CD56 (Servicebio, cat. no. GB112671; 1:500), and rabbit anti-human FOXP3 (Servicebio, cat. no. GB152325; 1:500). Briefly, tissue sections underwent deparaffinization and rehydration, followed by antigen retrieval. The sections were incubated in 3% H₂O₂ in an enclosed humid chamber for 25 min to block endogenous peroxidase activity, after which they were washed on a decolourising shaker. The sections were blocked with 10% goat anti-rabbit serum for 30 min and then incubated with the primary antibody at 4°C overnight. After the cells were washed, an HRP-conjugated secondary antibody was applied for 50 min at room temperature, followed by incubation at room temperature for 10 mins. Antibody stripping preceded repetition of the blocking-primary-secondary-TSA-stripping cycle for the remaining three primary antibodies. Finally, the sections were counterstained with DAPI, quenched with an autofluorescence quencher, mounted with anti-fluorescence quenching medium, and imaged via spectral-based systems with panoramic section scanning.

Historical data revealed that the ORR for second-line treatment with cetuximab monotherapy was 13%. The study adopted a Simon’s two-stage design to minimise the number of subjects exposed to ineffective treatment (Fig. 1A). On the basis of the expected ORR of 33% for dalpiciclib combined with cetuximab, assuming an α of 0.05 (two-sided) and a power of 80%, we aimed to enrol 14 participants in the first stage. If the number of responders (CR+PR) was no more than 1, the study was terminated because of insufficient efficacy. If more than one participant achieved a CR or PR, another 12 participants were recruited in the second stage. A total of 26 participants were enrolled. If the total number of responders (CR+PR) across both stages was more than 6, the combination therapy was considered effective.

Efficacy and safety were analysed in patients who received at least one dose of the study drug. Continuous variables are expressed as medians (ranges), and categorical variables are expressed as frequencies and percentages. The 95% confidence interval (CI) of the ORR was estimated using the Clopper–Pearson method. The median PFS, OS, and DOR were estimated using the Kaplan–Meier method, and their 95% CIs were estimated using the Brookmeyer–Crowley method (IBM SPSS Statistics 27.0). Comparisons of the TMB and gene mutation frequency between responders and nonresponders were performed using the Mann–Whitney test and Fisher’s exact test, respectively. Comparisons of the percentage of lymphocytes during treatment were performed using the paired t test method. A P-value < 0.05 was considered to indicate statistical significance.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study addresses a significant clinical need and presents encouraging efficacy and safety data for dalpiciclib plus cetuximab in a challenging patient population. While the single-arm design and limited biomarker sample size restrict definitive conclusions, the results are hypothesis-generating and relevant for future research. I recommend revision, with a focus on:\n1. Deeper correlative biomarker analyses (linking genomic features to outcomes):\n• I recommend the authors perform detailed subgroup analyses to determine if specific genomic alterations—such as TP53 mutation, CCND1 amplification, or their combination—are associated with differential response rates, PFS, or OS. Given the established roles of these alterations in HNSCC biology and therapy resistance, such analyses would provide valuable insight into potential biomarkers of response to dalpiciclib plus cetuximab.\n• Additionally, please explore whether patients with CCND1 amplification or other cell cycle pathway alterations had higher response rates to this regimen, as this could support a precision medicine approach for future studies.\n2. More detailed reporting and interpretation of immune cell changes:\n• The authors should, if possible, provide more granular data on immune cell subsets—such as CD8+ T cells, Tregs, and natural killer (NK) cells—rather than reporting only total lymphocyte percentages. These subsets play distinct and sometimes opposing roles in anti-tumor immunity and are well-established as prognostic and predictive biomarkers in HNSCC.\n• Please clarify whether changes in immune infiltration (such as the CD8+/Treg ratio or NK cell abundance) were associated with clinical outcomes in your cohort.\n3. Clearer discussion of the limitations and the exploratory nature of biomarker findings:\n• The authors should highlight the exploratory nature and potential significance of findings such as KCNMA1, TNXB, and TPCN2 alterations in nonresponders. While these genes may plausibly contribute to resistance mechanisms through known signaling pathways, their roles as biomarkers in HNSCC or in the context of CDK4/6 inhibitor therapy are not established in the current literature.\n• Please also discuss limitations such as the small biomarker cohort, lack of functional validation, and the need for independent confirmation.\n4. Improved contextualization with existing literature and justification of study design choices:\n• The manuscript would benefit from more direct comparison to outcomes with other regimens (e.g., cetuximab monotherapy, other CDK4/6 inhibitors), as well as a rationale for the single-arm design and chosen dosing strategy.\nIf these revisions are addressed, the manuscript will make a valuable contribution to the field and provide a strong foundation for future research in this area.", "reviewer_2": "This paper explore Dalpicilib in combination with cetuximab after failure of anti-PD1.\nThis is a small monoarm study well conducted.\nWe observe a clear signal of efficacy with high response rate and promising PFS and OS even if it is difficult to be confident in a mOS of 17 months with only 9.2 months of median PFS.\nThis drug remains toxic with 85% of patinets who need to reduce the dose: in further studies perhaps the schedule/dosage should be modified.\nIn the discussion the response rate to cetuximab alo,e after anti-PD1 is 20% in the interlink study and this level should probably be indicated. But clearly the combination alpicilib/cetuximab seems better.", "reviewer_3": "1. Although the protocol stated that the full analysis set (FAS) is to consist of \"all enrolled subjects who have taken the study drug at least once\", the study's analysis population apparently excludes 2 withdrawals (Figure 1B). Unless they withdrew consent, they should be included in the analyses. They can be used for OS analyses regardless.\n2. The statement in the Introduction, \"The incidence of human papillomaviruses (HPV)-negative HNSCC is higher than that of HPV-related HNSCC, which is often associated with lower response rate and poorer survival with immunotherapy.\", seems reversed.\n3. The 95% confidence interval for PFS extends down to 0. This seems unlikely and should be checked.\n4. The legend of Fig. 4A) should include descriptions of the two marginal bar charts.\nCongratulations on these important results.\nR. Chappell, FASA, FSCT\nProfessor, Dept. of Biostatistics and Medical Informatics\nUniversity of Wisconsin School of Medicine and Public Health\nchappell@stat.wisc.edu"}, "author_response": "Response to Reviewers\nReviewer(s)' Comments to Author:\nReviewer #1 (Remarks to the Author):\nThe study addresses a significant clinical need and presents encouraging efficacy and\nsafety data for dalpiciclib plus cetuximab in a challenging patient population. While the\nsingle-arm design and limited biomarker sample size restrict definitive conclusions, the\nresults are hypothesis-generating and relevant for future research. I recommend\nrevision, with a focus on:\n1. Deeper correlative biomarker analyses (linking genomic features to outcomes):\n• I recommend the authors perform detailed subgroup analyses to determine if specific\ngenomic alterations — such as TP53 mutation, CCND1 amplification, or their\ncombination—are associated with differential response rates, PFS, or OS. Given the\nestablished roles of these alterations in HNSCC biology and therapy resistance, such\nanalyses would provide valuable insight into potential biomarkers of response to\ndalpiciclib plus cetuximab.\n• Additionally, please explore whether patients with CCND1 amplification or other\ncell cycle pathway alterations had higher response rates to this regimen, as this could\nsupport a precision medicine approach for future studies.\nRe: Thank you very much for your valuable suggestion. We added the biomarker\nanalyses on TP53 mutation and abnormal CDK4 related genes (CDNK2A deletion\nand CCND1 amplifications). For TP53 mutation analysis, no significant\ndifferences in ORR (66.7% vs 58.8% (Z=-0.26, P=0.798)), median PFS (14.3 vs 7.0\nmonths (HR 0.28 [95% CI 0.08−1.11], P=0.127)), and OS (unreached vs 17.67\nmonths (HR unevaluated, P=0.208)) were observed (Supplemental data 2E, F).\nInterestingly, we found contrary results in abnormal CDK4 related genes\nanalysis compared to prior studies. In our study, patients without abnormal CDK4\nrelated genes had higher response and better prognosis compared with those with\nabnormal CDK4 related genes (ORR was 81.8% vs 33.3% (P=0.028), median PFS\nwas 14.3 vs 4.3 months (HR 0.26 [95% CI 0.07−0.91], P=0.035), and median OS\nwas unreached vs 8.5 months (HR 0.23 [95% CI 0.05−1.06], P=0.060)). Similar\nphenomena were also observed in patients with only CCND1 amplification\n(Supplemental data 2A-D).\nWe re-checked our NGS sequence and confirmed the grouping information was\ncorrect. We analysed that CDK4/6 has been activated since p16(INK4a) was\nupstream of CDK4/6 and inactivated in HPV negative patients, CDNK2A deletion\nor CCND1 amplifications might facilitate the progression of the cancer cell cycle\nfrom G1 to S phase through bypassing CDK4/6 pathway. In the previous study,\nClark AS et al revealed that palbociclib, a selective CDK4/6 inhibitor, was not\neffective at treating nonbreast solid tumors with either CCND1, 2, or 3\namplification.1 Oppelt PJ et al also showed that HPV-negative, locally advanced\nHNSCC patients with CCND1 amplification had a lower response (16.7% vs 50%)\ntowards palbociclib compared with those without CCND1 amplification.3\nHowever, these results need further investigation and confirmation in the future\nstudy.\nReferences\n1. Clark AS, Hong F, Finn RS, DeMichele AM, Mitchell EP, Zwiebel J, Arnaldez FI, Gray RJ,\nWang V, McShane LM, Rubinstein LV, Patton D, Williams PM, Hamilton SR, Copur MS,\nKasbari SS, Thind R, Conley BA, Arteaga CL, O'Dwyer PJ, Harris LN, Chen AP, Flaherty\nKT. Phase II Study of Palbociclib (PD-0332991) in CCND1, 2, or 3 Amplification: Results\nfrom the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol Z1B. Clin Cancer Res.\n2. Oppelt PJ, Ley JC, Puram SV, Jackson RS, Paniello RC, Rich JT, Pipkorn P, Liu J, Thorstad\nWL, Adkins DR. Palbociclib, a Selective CDK4/6 Inhibitor, Administered Before and After\nChemoradiotherapy for Human Papillomavirus-Negative, Locally Advanced Head and\nNeck Squamous Cell Carcinoma: A Single-Arm, Phase II Trial. JCO Precis Oncol. 2025\nMay;9:e2500069.\n2. More detailed reporting and interpretation of immune cell changes:\n• The authors should, if possibxle, provide more granular data on immune cell\nsubsets—such as CD8+ T cells, Tregs, and natural killer (NK) cells—rather than\nreporting only total lymphocyte percentages. These subsets play distinct and sometimes\nopposing roles in anti-tumor immunity and are well-established as prognostic and\npredictive biomarkers in HNSCC.\n• Please clarify whether changes in immune infiltration (such as the CD8+/Treg ratio\nor NK cell abundance) were associated with clinical outcomes in your cohort.\nRe: Thank you so much for your valuable advice. During this trial, we made an\nincidental observation: among patients who received dalpiciclib plus cetuximab,\nthe two who responded to this regimen subsequently achieved a partial response\n(PR) to anti-PD-1 rechallenge therapy, whereas the two non-responders to\ndalpiciclib plus cetuximab showed no response to subsequent anti-PD-1 treatment.\nWe then analyzed lymphocyte percentages in blood samples from enrolled patients\nand found preliminary evidence that responders exhibited a significant increase\nin lymphocyte percentages at the time of best response compared with baseline,\nwhile no significant changes in lymphocyte percentages were observed in non-\nresponders.\nRegrettably, due to insufficient attention to sample preservation during the trial,\nwe did not retain post-treatment blood or tissue samples. This has made it\nextremely challenging to compare pre- and post-treatment changes in the immune\nmicroenvironment—a limitation we acknowledge.\nTo address your request for more granular immune cell analysis, we have\nsupplemented the revised manuscript with an analysis of the relationship between\nbaseline immune cell subsets (CD8⁺ T cells, regulatory T cells [Tregs], natural\nkiller [NK] cells) and clinical outcomes. Specifically, we examined associations\nbetween baseline CD8⁺ T cell counts, Treg counts, NK cell counts, and the\nCD8⁺/Treg ratio with treatment response, progression-free survival (PFS), and\noverall survival (OS) in patients receiving dalpiciclib plus cetuximab. The results\nshowed no significant correlations between these baseline immune parameters and\nresponse, PFS, or OS.\nWe fully agree that assessing dynamic changes in immune infiltration would be\ncritical to validate the aforementioned observational phenomenon. Therefore, in\nour ongoing randomized controlled trial (“Dalpiciclib Combined with Cetuximab\nversus Cetuximab Alone in Patients with HPV-Negative, Anti-PD-1-Resistant\nRecurrent or Metastatic Head and Neck Squamous Cell Carcinoma: An Open-\nLabel, Randomized Controlled, Phase II Trial”, NCT06935188), we have\nproactively implemented protocols to collect both pre- and post-treatment blood\nand tissue samples. These samples will be used for single-cell sequencing and\nrelated assays to systematically characterize changes in immune cell infiltration.\nOnce again, we sincerely appreciate your valuable suggestion, which has provided\nimportant new directions and insights for our subsequent research.\n3. Clearer discussion of the limitations and the exploratory nature of biomarker findings:\n• The authors should highlight the exploratory nature and potential significance of\nfindings such as KCNMA1, TNXB, and TPCN2 alterations in nonresponders. While\nthese genes may plausibly contribute to resistance mechanisms through known\nsignaling pathways, their roles as biomarkers in HNSCC or in the context of CDK4/6\ninhibitor therapy are not established in the current literature.\n• Please also discuss limitations such as the small biomarker cohort, lack of functional\nvalidation, and the need for independent confirmation.\nRe: Thank you so much for your valuable advice. In our study, KCNMA1, TNXB,\nand TPCN2 alterations were significantly higher in nonresponders, suggesting to\nbe acted as potential biomarkers in dalpiciclib resistance. Additionally, PI3K/Akt\npathway is subsequentially activated by these alterations, activation of PI3K/Akt\nsustained CDK2/Cyclin E2 expression, rendering the cells able to bypass CDK4/6\npathway, inducing resistance to CDK4/6i.1 Prior studies observed promising\nobjective response rate in patients with hormone receptor-positive, HER2-\nnegative advanced breast cancer receiving PI3Ki and CDK4/6i combination\ntherapy.2,3 Our observations indicated PI3Ki with CDK4/6i combination therapy\nwith might achieve better efficacy in patients with R/M HNSCC.\nFor biomarker analysis, biomarkers findings such as KCNMA1, TNXB, and\nTPCN2 alterations in nonresponders were analysed from a relatively small patient\ncohort. Downstream functional validation and the independent confirmation of\nthese alterations are also needed in further study. We have added the limitation of\nbiomarker analysis in the revised manuscript.\nReferences\n1. Herrera-Abreu MT, Palafox M, Asghar U, Rivas MA, Cutts RJ, Garcia-Murillas I, Pearson\nA, Guzman M, Rodriguez O, Grueso J, Bellet M, Cortés J, Elliott R, Pancholi S, Baselga\nJ, Dowsett M, Martin LA, Turner NC, Serra V. Early Adaptation and Acquired Resistance\nto CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer. Cancer Res. 2016 Apr\n15;76(8):2301-13.\n2. Layman RM, Han HS, Rugo HS, Stringer-Reasor EM, Specht JM, Dees EC, Kabos P,\nSuzuki S, Mutka SC, Sullivan BF, Gorbatchevsky I, Wesolowski R. Gedatolisib in\ncombination with palbociclib and endocrine therapy in women with hormone receptor-\npositive, HER2-negative advanced breast cancer: results from the dose expansion groups\nof an open-label, phase 1b study. Lancet Oncol. 2024 Apr;25(4):474-487.\n3. Konstantinopoulos PA, Lee EK, Xiong N, Krasner C, Campos S, Kolin DL, Liu JF,\nHorowitz N, Wright AA, Bouberhan S, Penson RT, Yeku O, Bowes B, Needham H, Hayes\nM, Sawyer H, Polak M, Shea M, Cheng SC, Castro C, Matulonis UA. A Phase II, Two-\nStage Study of Letrozole and Abemaciclib in Estrogen Receptor-Positive Recurrent\nEndometrial Cancer. J Clin Oncol. 2023 Jan 20;41(3):599-608.\n4. Improved contextualization with existing literature and justification of study design\nchoices:\n• The manuscript would benefit from more direct comparison to outcomes with other\nregimens (e.g., cetuximab monotherapy, other CDK4/6 inhibitors), as well as a rationale\nfor the single-arm design and chosen dosing strategy.\nIf these revisions are addressed, the manuscript will make a valuable contribution to the\nfield and provide a strong foundation for future research in this area.\nRe: Thank you very much for your valuable suggestion. The ORR of cetuximab\nmonotherapy as second line treatment in patients with R/M HNSCC was only 13%,\nwith 2.3 months of median PFS and 5.9 months median OS.1 In the INTERLINK-\n1 trial, cetuximab monotherapy in HPV negative R/M HNSCC patients after\nfailure of both platinum-based chemotherapy and previously treated with PD-1\ninhibitors only had an ORR of 23.9%, median PFS and OS were 3.8 months and\n8.6 months.2 In our single-arm trial, we observed the ORR, median PFS and OS\nwere 67.9%, 7.0 months, and 17 months with dalpiciclib plus cetuximab,\nindicating the promising response and survival benefit in this population.\nPalbociclib, another CDK4/6 inhibitor, was reported to have an ORR of 39% in\nplatinum-resistant, HPV-negative R/M HNSCC, the median PFS and OS were 5.4\nmonths and 9.5 months respectively.3 In our study, we observed a better response\nand survival benefit in PD-1-resistant, HPV-negative R/M HNSCC by dalpiciclib\nplus cetuximab.\nReferences\n1. Vermorken JB, Trigo J, Hitt R, Koralewski P, Diaz-Rubio E, Rolland F, Knecht R, Amellal\nN, Schueler A, Baselga J. Open-label, uncontrolled, multicenter phase II study to evaluate\nthe efficacy and toxicity of cetuximab as a single agent in patients with recurrent and/or\nmetastatic squamous cell carcinoma of the head and neck who failed to respond to\nplatinum-based therapy. J Clin Oncol. 2007 Jun 1;25(16):2171-7.\n2. J. Fayette, L.F.L. Licitra, K.J. Harrington, R. Haddad, L.L. Siu, Y.C. Liu, M. Tahara, J-P.\nMachiels, D. Rischin, T. Seiwert, R. Ferris, U. Keilholz, A. Psyrri, B. Keam, P. Bossi, R.\nMetcalf, O. Serrano, P. Kaur, D. Ruscica, R.B. Cohen, 854O INTERLINK-1: Phase III\nstudy of cetuximab (CTX) ± monalizumab (M) in participants (pts) with\nrecurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) with disease\nprogression on/after platinum chemotherapy (CT) and previously treated with an immune\ncheckpoint inhibitor (ICI). Ann Oncol. 2023;34:S554-S555.\n3. Adkins D, Ley J, Neupane P, Worden F, Sacco AG, Palka K, Grilley-Olson JE, Maggiore\nR, Salama NN, Trinkaus K, Van Tine BA, Steuer CE, Saba NF, Oppelt P. Palbociclib and\ncetuximab in platinum-resistant and in cetuximab-resistant human papillomavirus-\nunrelated head and neck cancer: a multicentre, multigroup, phase 2 trial. Lancet Oncol.\nReviewer #2 (Remarks to the Author):\nThis paper explore Dalpicilib in combination with cetuximab after failure of anti-PD1.\nThis is a small monoarm study well conducted.\nWe observe a clear signal of efficacy with high response rate and promising PFS and\nOS even if it is difficult to be confident in a mOS of 17 months with only 9.2 months\nof median PFS.\nThis drug remains toxic with 85% of patinets who need to reduce the dose: in further\nstudies perhaps the schedule/dosage should be modified.\nRe: Thank you very much for your valuable suggestion. Since CDK4/6 inhibitors\nhave not yet been explored in patients with R/M HNSCC progressed on PD-1\ninhibitors, we here designed a single-arm, small-sample study to preliminarily\ninvestigate the efficacy and safety of this regimen. Based on the potential efficacy\ndemonstrated in this study, we are currently conducting a randomized controlled\ntrial to further verify the efficacy of this regimen (NCT06935188). Considering the\nrelatively high proportion of patients who experienced dose reductions due to drug\ntoxicity, we will explore the optimal efficacy and medication schedule/dosage in\nthe future study.\nIn the discussion the response rate to cetuximab alo,e after anti-PD1 is 20% in the\ninterlink study and this level should probably be indicated. But clearly the combination\nalpicilib/cetuximab seems better.\nRe: Thank you very much for your valuable comments. In the INTERLINK-1 trial,\ncetuximab monotherapy in HPV negative R/M HNSCC patients after failure of\nboth platinum-based chemotherapy and previously treated with PD-1 inhibitors\nonly had an ORR of 23.9%, median PFS and OS were 3.8 months and 8.6 months.1\nIn our single-arm trial, we observed the ORR, median PFS and OS were 67.9%,\n7.0 months, and 17 months with dalpiciclib plus cetuximab, indicating the\npromising response and survival benefit in this population.\nReferences\n1. J. Fayette, L.F.L. Licitra, K.J. Harrington, R. Haddad, L.L. Siu, Y.C. Liu, M. Tahara, J-P.\nMachiels, D. Rischin, T. Seiwert, R. Ferris, U. Keilholz, A. Psyrri, B. Keam, P. Bossi, R.\nMetcalf, O. Serrano, P. Kaur, D. Ruscica, R.B. Cohen, 854O INTERLINK-1: Phase III\nstudy of cetuximab (CTX) ± monalizumab (M) in participants (pts) with\nrecurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) with disease\nprogression on/after platinum chemotherapy (CT) and previously treated with an immune\ncheckpoint inhibitor (ICI). Ann Oncol. 2023;34:S554-S555.\nReviewer #3 (Remarks to the Author):\n1. Although the protocol stated that the full analysis set (FAS) is to consist of \"all\nenrolled subjects who have taken the study drug at least once\", the study's analysis\npopulation apparently excludes 2 withdrawals (Figure 1B). Unless they withdrew\nconsent, they should be included in the analyses. They can be used for OS analyses\nregardless.\nRe: Thank you very much for your carefully review. These two patients withdrew\ntheir consents prior to the administration of study drugs, therefore, we did not\ninclude these two patients in the full analysis set (FAS), we revised the\n“withdrawals” to “withdrew consents” to avoid ambiguity.\n2. The statement in the Introduction, \"The incidence of human papillomaviruses (HPV)-\nnegative HNSCC is higher than that of HPV-related HNSCC, which is often associated\nwith lower response rate and poorer survival with immunotherapy.\", seems reversed.\nRe: Thank you very much for your carefully review. Epidemiologically, the\nincidence of human papillomaviruses (HPV)-negative HNSCC is higher than that\nof HPV-related HNSCC.1 Mandal et al. reported in an analysis of TCGA found\nthat HPV-positive HNSCCs had more significant immune infiltration and had\nhigher T cell levels infiltrated in HPV positive tissues and associated with a\nfavorable prognosis,2,3 which suggests patients with HPV-positive status may\nobtain more benefit from immune checkpoints blockade. Keynote 012 study was\nthe first clinical trial to evaluate the relationship between HPV status and immune\ncheckpoints blockade response. 60 patients with PD-L1+ (> 1%) were treated with\npembrolizumab. ORR was 24% (95% CI, 13-40%) in those patients with HPV\npositive, and 16% in HPV negative patients (95% CI, 10-23%, P > 0.05).4 In the\nCheckmate 141 subgroup analysis, patients with p16 positive tumors tend to\nobtain more benefit from nivolumab than the standard treatment group (median\nOS: 9.1 months vs. 4.4 months HR: 0.56; 95% CI: 0.32 to 0.99). On the other hand,\nin patients with p16 negative disease, the median OS was 7.5 months in the\nnivolumab monotherapy group and 5.8 months in the standard treatment group\n(HR: 0.73; 95% CI: 0.42 to 1.25).5 These results indicate that HPV positive\nHNSCC is often associated with higher response rate and better survival with\nimmunotherapy. To avoid ambiguity, we changed our statement from \"The\nincidence of human papillomaviruses (HPV)-negative HNSCC is higher than that\nof HPV-related HNSCC, which is often associated with lower response rate and\npoorer survival with immunotherapy.\" to “The incidence of human\npapillomaviruses (HPV)-negative HNSCC is higher than that of HPV-related\nHNSCC. However, HPV-negative HNSCC is often associated with lower response\nrate and poorer survival with immunotherapy compared with that of HPV-related\nHNSCC.”\nReferences\n1. Sun Y, Wang Z, Qiu S, Wang R. Therapeutic strategies of different HPV status in\nHead and Neck Squamous Cell Carcinoma. Int J Biol Sci. 2021 Mar 10;17(4):1104-\n1118.\n2. Sridharan V, Margalit DN, Lynch SA, Severgnini M, Zhou J, Chau NG. et al.\nDefinitive chemoradiation alters the immunologic landscape and immune\ncheckpoints in head and neck cancer. Br J Cancer. 2016;115:252–60.\n3. Cillo AR, Kurten CHL, Tabib T, Qi Z, Onkar S, Wang T. et al. Immune Landscape\nof Viral- and Carcinogen-Driven Head and Neck Cancer. Immunity. 2020;52:183–\n99.\n4. Seiwert TY, Burtness B, Mehra R, Weiss J, Berger R, Eder JP. et al. safety and\nclinical activity of pembrolizumab for treatment of recurrent or metastatic\nsquamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label,\nmulticentre, phase 1b trial. Lancet Oncol. 2016;17:956–65.\n5. Harrington KJ, Ferris RL, Blumenschein G Jr, Colevas AD, Fayette J, Licitra L.\net al. Nivolumab versus standard, single-agent therapy of investigator's choice in\nrecurrent or metastatic squamous cell carcinoma of the head and neck (CheckMate\n141): health-related quality-of-life results from a randomised, phase 3 trial. Lancet\nOncol. 2017;18:1104–15.\n3. The 95% confidence interval for PFS extends down to 0. This seems unlikely and\nshould be checked.\nRe: Thank you very much for your carefully review. We checked our PFS raw data\nand found an error occurred when running the analysis in SPSS. The correct\nmedian PFS was 7.0 months (95% CI, 4.13-NR). The reason for the missing upper\nlimit is that a relatively high proportion of patients had no tumor progression and\nthe survival curve has not declined completely. Therefore, the maximum possible\nvalue of the median progression-free survival (PFS) time cannot be determined.\n4. The legend of Fig. 4A) should include descriptions of the two marginal bar charts.\nCongratulations on these important results.\nRe: Thank you very much for your carefully review. We have added descriptions\nof the two marginal bar charts. The bar chart on the right of the percentages\nrepresented the count of patients with mutations. The bar chart on the right of the\nmutation counts of patients represented the types of gene alternation. Bottom bars\nshow responders (marked as blue, complete or partial response [n = 12]) and non-\nresponders (marked as red, stable or progressive disease [n = 8]).\nHere, we would like to thank all the reviewers for your help to improve the quality\nof our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the previous concerns thoughtfully, and the noteworthy results are clearly presented. The work holds significance for the field and is well positioned in relation to the established literature. The conclusions are well supported by the data, and the methodology meets the expected standards, with sufficient detail to allow reproducibility.\nThere are no major flaws in the data analysis or interpretation that would prevent publication. Any questions/ suggestions previously raised have been adequately addressed.\nIn summary, I find the manuscript suitable for publication."}, "author_response": "Re: Thank you so much for your valuable advice. All of your comments are\nvaluable and helpful in revising and improving our paper, as well as providing\nimportant guidance for our research.\nRe: Thank you very much for your valuable comments.\nHere, we would like to thank all the reviewers for your help to improve the\nquality of our manuscript."}]

Decision letter