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`CLINICAL CANCER RESEARCH I CLINICAL TRIALS: IMMUNOTHERAPY
`
`Neoadjuvant and Adjuvant Pembrolizumab in Resectable
`Locally Advanced, Human Pap illomavirus-Unrelated
`Head and Neck Cancer: A Multicenter, Phase II Trial
`Ravindra Uppaluri 1'2, Katie M. Campbell 3", Ann Marie Eglofft, Paul Zolkind 5, Zachary L. Skidmore 4,
`Brian Nussenbaum 5'6, Randal C. Paniello5'6, Jason T. Rich 5'6, Ryan Jackson 5,6, Patrik Pipkorn 5'6,
`Loren S. Michel 6'7, Jessica Ley 7, Peter 0ppe1t 6'7, Gavin P. Dunn 6'8, Erica K. Barnell 3'4, Nicholas C. Spies4,
`Tianxiang Lin 5, Tiantian Li 9, David T. Mulder°, Youstina Hanna °, lulia Cirlan 9, Trevor J. Pugh °'10'11 ,
`Tenny Mudianto 2, Rachel Riley 2, Liye Zhou 2, Vickie Y. Jo 1, Matthew D. Stachlert2, Glenn J. Hann a2,
`Jason Kasst2, Robert Haddad 1,2, Jonathan D. Schoenfeld 2'13, Evisa Gjini 12, Ana Lako'2, Wade Thorstad 6'14,
`Hiram A. Gay 6'14, Mackenzie Daly 6'14, Scott J. Rodig 12'15, Ian S. Hagemann16, Donna Kallogjeri 5,
`Jay F. Piccirillo 5'6, Rebecca D. Chernock16, Malachi Griffith 3'4'6'7, Obi L. Griffith 3'4'6'7, and
`Douglas R. Adkins 6'7
`
`Purpose: Pembrolizumab improved survival in patients with
`recurrent or metastatic head and neck squamous-cell carcinoma
`(HNSCC). The aims of this study were to determine if pembroli-
`zumab would be safe, result in pathologic tumor response (pTR),
`and lower the relapse rate in patients with resectable human
`papifiomavirus (HPV)-unrelated HNSCC.
`Patients and Methods: Neoadjuvant pembrolizumab (200 mg)
`was administered and followed 2 to 3 weeks later by surgical tumor
`ablation. Postoperative (chemo)radiation was planned. Patients with
`high-risk pathology (positive margins and/or extranodal extension)
`received adjuvant pembrolizumab. pTR was quantified as the pro-
`portion of the resection bed with tumor necrosis, keratinous debris,
`and giant cells/histiocytes: pTR-0 (<10%), pTR-1 (10%-49%), and
`pTR-2 (≥50%). Coprimary endpoints were pTR-2 among all patients
`and 1-year relapse rate in patients with high-risk pathology (histor-
`icali 35%). Correlations of baseline PD-Ll and T-cell infiltration with
`
`pTR were assessed. Tumor clonal dynamics were evaluated (Gin-
`icalTrials.gov NCT02296684).
`Results: Thirty-six patients enrolled. After neoadjuvant pembro-
`lizurnah, serious (grades 3-4) adverse events and unexpected surgical
`delays/complications did not occur. pTR-2 occurred in eight patients
`(22%), and pTR-1 in eight other patients (22%). One-year relapse
`rate among 18 patients with high-risk pathology was 16.7% (95%
`confidence interval, 3.6%-41.4%). pTR ≥10% correlated with base-
`line tumor PD-Li. immune infiltrate, and IFNy activity. Matched
`samples showed upregulation of inhibitory checkpoints in patients
`with pTR-0 and confirmed clonal loss in some patients.
`Conclusions: Among patients with locally advanced, HPV-
`unrelated HNSCC, pembrolizumab was safe, and any pathologic
`response was observed in 44% of patients with 0% pathologic
`complete responses. The 1-year relapse rate in patients with
`high-risk pathology was lower than historical.
`
`Introduction
`
`Patients with head and neck squamous-cell carcinoma (HNSCC)
`usually present with locally advanced disease. Surgery or definitive
`(chemo)radiation is the initial treatment in many of these patients. A
`
`large subset of surgically treated patients have high-risk pathology
`features (positive margin and/or extranodal extension) that are best
`treated with intensive postoperative adjuvant cisplatin and radiation
`therapy (POACRT). However, 35% of patients, particularly those with
`human papifiomavirus (HPV)-unrelated HNSCC, will develop
`
`Department of Surgery, Brigham and Women's Hospital, Boston, Massachu-
`setts. °Department of Medical Oncology, Dana-Farber Cancer Institute, Boston,
`Massachusetts. 3Department of Genetics, Washington University School of
`Medicine, St. Louis, Missouri. 5McDonnell Genome Institute, Washington Uni-
`versity School of Medicine, St, Louis, Missouri, "Department of Otolaryngology,
`Washington University School of Medicine, St. Louis, Missouri. 6Alvin J. Siteman
`Cancer Center, Washington University School of Medicine, St. Louis, Missouri.
`7Department of Medicine/Medical Oncology, Washington University School of
`Medicine, St. Louis, Missouri. "Department of Neurological Surgery, Washington
`University School of Medicine, St. Louis, Missouri. 'Princess Margaret Cancer
`Center, University Health Network, Toronto, Ontario, Canada 15Department of
`Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. "Ontario
`Institute for Cancer Research, Toronto, Ontario, Canada. 12 Department of
`Pathology, Brigham and Women's Hospital, Boston, Massachusetts. `°Depart-
`rnent of Radiation-Oncology, Brigham and Women's Hospital, Boston, Massa-
`chusetts. 'Department of Radiation-Oncology, Washington University School
`of Medicine, St. Louis, Massachusetts. 15Center for Immuno-Oncology, Brigham
`and Women's Hospital, Boston, Massachusetts. "Department of Pathology and
`Immunology, Washington University School of Medicine, St. Louis, Missouri.
`
`Note: Supplementary data for this article are available at Clinical Cancer
`Research Online (http://clincancerres.aacrtournals,org/).
`
`Corrected online November 25, 2020.
`
`R. Uppaluri and K.M. Campbell contributed equally to this article.
`
`R. Uppaluri, O.L. Griffith, and D.R. Adkins contributed equally as the co-senior
`authors of this article.
`
`Current address for KM. Campbell: Division of Hematology-Oncology, Depart-
`ment of Medicine, University of California, Los Angeles, Los Angeles, California.
`
`Corresponding Author: Ravindra Uppaluri, Dana-Farber/Brigham and Women's
`Cancer Center, 450 Brookline Avenue, Boston, MA 02215. Phone: 6t7-632-3091;
`Fax: 617-632-5786; E-mail: Ravindra_Uppaluri%DFCl.Haraard.edu
`
`Clin Cancer Res 2020:26:5140-52
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`dol: lO.1158/1078-0432.CCR-20-1695
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`02020 American Association for Cancer Research.
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`AAC-R American Association
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`for Cancer Research
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`AACRJournals.org I 5140
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`0001
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`TwinStrand EX1095
`TwinStrand Biosciences v. Guardant Health
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`Pembrolizumab In Surgically Resectable HPV-Negative HNSCC
`
`resistance to immune checkpoint inhibitors. HNSCC is ideally suited
`for testing the effect of administration of immune checkpoint inhi-
`bitors in the neoadjuvant interval. Tumor is readily accessible to
`obtain matched baseline and surgical tissue and to perform visual
`assessments of tumor response (10). Although reported in a few other
`cancers (11-20), the safety and biologic and clinical effects of admin-
`istration of immune checkpoint inhibitors have not been reported in a
`patient cohort with resectable locally advanced HPV-unrelated
`HNSCC.
`In this multicenter, phase II trial, we aimed to determine if admin-
`istration of neoadjuvant pembrolizumab to patients with resectable
`locally advanced, HP V-unrelated HNSCC would be safe and result in
`pathologic tumor responses (pTR). We evaluated the immunologic
`correlates to pTR and assessed tumor clonal dynamics in matched
`tumor samples obtained before and after neoadjuvant pembrolizumab.
`Among patients with high-risk pathology, we aimed to determine if
`administration of neoadjuvant and adjuvant pembrolizumab would
`result in a relapse rate lower than historical. Herein, we report the
`results of our trial.
`
`Patients and Methods
`Study design and participants
`We did a multicenter, phase II trial at two university sites in the
`United States: Washington University, St. Louis, MO, and Dana-
`Farber/Brigham and Women's Cancer Center, Boston, MA. The
`protocol is included in the Supplementary Materials. The trial is a
`nonrandomized two-group study. Patient selection criteria were the
`same for both groups. Group 1 completed enrollment before group 2
`accrued patients. Group 1 is reported here. In group 1, patients were
`treated with one dose of neoadjuvant pembrolizumab, followed by
`surgery. Patients with high-risk pathology were scheduled to be treated
`with POACRT followed by adjuvant pembrolizumab; patients without
`high-risk pathology were scheduled to be treated with POART or
`observation but not adjuvant pembrolizumab. In group 2, patients
`were treated with two doses of neoadjuvant pembrolizumab, followed
`by surgery, then POACRT if high-risk pathology or POART (or
`observation) if without high-risk pathology. Group 2 did not receive
`adjuvant pembrolizumab. Group 2 is ongoing, having accrued 25 of the
`planned sample size of 31 patients; the data are not yet mature. The
`results of group 2 will be reported at a later date.
`Eligible patients had resectable clinical stage III-IVb (AJCC, 7th
`Edition), HPV-unrelated (oral cavity, larynx, hypopharynx or p16-
`negative oropharynx) HNSCC, measurable disease per RECIST vl.l,
`Eastern Cooperative Oncology Group performance status 0-1, and
`adequate marrow and organ function [absolute neutrophil count
`≥t,500/mcL platelets ≥100,000/mcL, hemoglobin ≥98/dL; total bili-
`ruhin ≤i.5x upper limits ofnornial (ULN), AST and ALT ≤2.5x ULN;
`serum creatinine ≤ i.5x ULN or creatinine clearance ≥30 mL/min] . Key
`exclusion criteria included HPV-related oropharynx SCC, active
`autoimmune disease, or immunodeficiency.
`Tests required to determine eligibility included complete blood
`count, metabolic panel, pregnancy test (women), coagulation and
`thyroid panels, urinalysis, and CT scans of the neck and chest.
`
`Procedures
`In group 1, patients received one dose of pembrollzumab (200 mg,
`IV) 13 to 22 days before (neoadjuvant) surgery. Surgery included
`resection of all gross disease at the primary site, ipsilateral (and
`contralateral, in some patients) therapeutic/prophylactic neck dissec-
`tion, and reconstruction using pedided or free-flap procedures as
`
`Translational Relevance
`
`Many patients with locally advanced, human papiiomavirus-
`unrelated head and neck squamous-cell carcinoma (HNSCC)
`undergo multimodality therapy, including surgery followed by
`adjuvant (chemo)radiation therapy. Outcomes are suboptimal,
`especially for high-risk patients (positive margins and/or extra-
`nodal extension). Novel treatment intensification approaches are
`needed. As blocking mAbs to the programmed death-1 pathway
`have improved patient outcomes across several cancer types, their
`integration into definitive surgical management is a logical next
`step. In this study, we completed a phase II clinical trial with
`neoadjuvant and adjuvant pembrolizumab in patients with
`HNSCC undergoing standard-of-care surgical therapy. We found
`this approach was safe in the surgical setting, identified pathologic
`changes induced by neoadjuvant pembrolizumab, and defined
`pathologic and genomic hiomarkers of response and potential for
`clinical impact Together, these findings highlight pembrolizumab
`integration into the definitive surgical management of locally
`advanced HNSCC as a rational approach that warrants testing in
`the phase III setting.
`
`relapse of disease (1, 2). Attempts to improve this outcome have been
`unsuccessful (3). Novel treatment strategies are needed for these
`patients.
`Immune checkpoint inhibitors could reduce the risk of disease
`relapse in resectable locally advanced, HPV-negative HNSCC. Ran-
`domized trials showed that pembrolizumab and nivolumab, inhibitors
`of the programmed death receptor-i (PD-1), improved overall survival
`(OS) of patients with platinum-resistant HNSCC (4, 5). Pembrolizu-
`mab given alone or with chemotherapy improved the OS of patients
`with untreated recurrent or metastatic disease (6). The results of these
`trials provide strong rationale for evaluating the clinical impact of
`immune checkpoint inhibitors in patients with resectable locally
`advanced, HPV-unrelated HNSCC.
`Immunotherapy maybe administered before (neoadjuvant) or after
`(adjuvant) surgery. Precinical experiments with several cancer types
`showed that administration of immunotherapy in the neoadjuvant
`interval provided greater benefit than when given in the adjuvant
`interval (7-9). In mouse models of spontaneously metastatic breast
`cancer, neoadjuvant immunotherapy and surgery were more effective
`in generating tumor-specific CD8+ T cells and preventing develop-
`ment of lethal metastases than surgery alone (7, 8). Importantly, the
`benefit of immunotherapy was dependent on resection of the primary
`tumor. In syngeneic mouse models of HPV-unrelated oral cavity
`carcinoma with defined T-cell antigens, administration of PD-1
`inhibitor before, but not after, surgery reversed functional immuno-
`dominance and induced effector T-cell immunity that resulted in
`rejection of tumor rechallenge after surgery (9). Collectively, these data
`support the critical importance of coordinating administration of
`immunotherapy before surgery to achieve optimal disease control
`with this novel strategy.
`In addition to improved disease control, administration of immune
`checkpoint inhibitors in the neoadjuvant interval could result in other
`clinical benefits, including reduction of the rate of high-risk pathology
`and downstaging of the cancer. These outcomes could alter the
`selection of adjuvant therapy, resulting in less intense treatment.
`Finally, matched tumor tissue obtained at baseline and at surgery can
`be evaluated to define biomarkers that predict tumor response and
`
`AACRiournals.org
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`Clin Cancer Res: 26(19) October 1, 2020
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`Uppalurl et al.
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`deemed appropriate. Surgical plan and extent of surgical tumor
`resection was defined by baseline assessments obtained before neoad-
`juvant pembrolizumab and did not change if treatment responses were
`observed. Patients with high-risk pathology were scheduled to be
`treated with POACRT, if they had adequate organ function and had
`recovered from surgery (1, 2). Upon resolution of POACRT-related
`adverse events (AEs) to ≤grade one and after 3 months from surgery
`date, patients with high-risk pathology were scheduled to be treated
`with adjuvant pembrolizumab (200 mg) every 3 weeks for six doses.
`Dose delays of pembrolizumab and treatment of immune-related AEs
`were performed per protocol. Patients without high-risk pathology
`(low/intermediate-risk) were scheduled to be treated with POART (or
`observation), but not adjuvant pembrolizumab. Physician discretion
`in selection of standard-of-care adjuvant therapy was permitted in
`patients with intermediate-risk pathology, such that some patients
`were treated with POACRT even though they lacked traditional high-
`risk-pathology. Administration of POA(C)RT was performed per
`protocol.
`Before neoadjuvant pembrolizumab, patients underwent baseline
`assessment by physical examination and CT scan of the neck, and a
`biopsy of the primary tumor and collection of peripheral blood for
`correlative studies. Patients were assessed by physical examination the
`day before or day of surgery. After patient 20, the trial was amended to
`perform a CT scan of the neck within 10 days prior to surgery. On the
`day of surgery, tissue from the primary tumor and peripheral blood
`were collected for correlative studies. AEs were assessed using revised
`Common Terminology Criteria for Adverse Events v4.0. Perisurgical
`AEs were assessed using Clavien-Dindo Classification of Surgical
`Complications (21). Tumor response was assessed using REC1STv1.l.
`Patients were monitored for 30 days after surgery for AEs and surgical/
`wound-healing complications. During the administration of adjuvant
`pembrolizumab, patients were assessed every 3 weeks beginning with
`the first dose of pembrolizumab by physical examination, complete
`blood count, and metabolic panel, and every 6 weeks by thyroid panel
`and urinalysis. In these patients, AEs were monitored for 90 days after
`the last dose of adjuvant pembrolizumab. Patients were monitored for
`relapse every 3 months after surgery by physical examination and CT
`scans.
`Details of PD-Li lHC and multiplex immunofluorescence (MIF)
`are described in the Supplementary Appendix. Details regarding
`whole-exome sequencing (WES), performed on tumor samples and
`matched normal blood, RNA sequencing (RNA-seq) on tumor sam-
`ples, T-cell receptor sequencing (CapTCR-seq; ref. 22) on peripheral
`blood, multisector targeted genome sequencing on tumor samples, and
`analyses (expression, deconvolution, TCR repertoire, etc.) are provid-
`ed in the Supplementary Appendix. DNA and RNA data were pro-
`cessed using the Genonie Modeling System (23, 24). Sequencing data
`have been deposited in dbGaP (phs001623). Patient HLA haplotypes
`and mutational profiles were used to predict putative neoantigens with
`pVACtools (25).
`
`Outcomes
`The coprimaiy endpoints were 1-year relapse rate (the absolute
`proportion of patients who developed local-regional and/or distant
`relapse within 1 year of surgery) in patients with high-risk pathology,
`and the proportion of all patients with pTR-2 in the surgical specimen
`after administration of one dose of neoadjuvant pembrolizumab. pTR
`was defined as the presence of tumor cell necrosis and keratinous
`debris with giant cell/histiocytic reaction, quantified as a percentage of
`the overall tumor bed (area pathologic response/area pathologic
`response plus viable tumor): pTR-0 (<10%), pTR-1 (10%-49%), and
`
`pTR-2 (≥50%). Two pathologists with head and neck expertise (R.D.
`Chemock and I.S. Hagemann) independently evaluated all slides from
`baseline and surgery specimens and quantified pTR in increments of
`10%. Primary tumor and lymph node metastases were scored sepa-
`rately. Overall pTR was classified based on the best pTR observed in
`either primary tumor or lymph node. Joint review consensus was
`reached when discrepancies occurred. Current standardized defini-
`tions of immune checkpoint inhibitor-induced pTR were not available
`when the study was begun, and were not used in the analysis.
`Secondary endpoints included safety of administration ofneoadjuvant
`pembrolizumab and clinical tumor response to neoadjuvant pembro-
`lizumab assessed by physical examination and, in some patients, by
`RECISTvl.l. Correlative endpoints assessed on matched tumor speci-
`mens obtained before and after (on day of surgery) neoadjuvant
`pembrolizumab included PD-Li expression, histologic, immunologic,
`genomic, and tumor clonal dynamic changes. T-cell donality was
`performed on peripheral blood obtained before and after neoadjuvant
`pembrolizumab (Supplementary Table St).
`No major protocol deviations occurred. The protocol was amended
`six times over the course of the study. Amendments 1 to 3 included
`updates of the risk profile and dose modifications of study drug,
`clarified eligibility criteria, and added Dana-Farber/Brigham and
`Women's Cancer Center, Boston, MA, as a secondary site. Amend-
`ment 4 added correlative studies. Amendment 5 added an unplanned
`interim analysis after the first 20 patients enrolled into group 1 due to
`the lower-than-expected rate of patients with high-risk pathology, and
`added CT scan of the neck to be performed after neoadjuvant
`pembrolizumab and prior to surgery. Amendment 6 closed accrual
`to group 1, and added group 2.
`
`Statistical analysis
`Relapse rate at 1 year in patients with high-risk pathology was the
`initial primary endpoint. Historic data showed that the 1-year relapse
`rate after surgery and POACRT in patients with high-risk pathology
`was 35% (1, 2). Relapse rate at 1 year was selected because the majority
`(>90%) of relapse events in these patients occurred within i year of
`surgery (i, 2). A sample size of 31 evaluable patients was required to
`detect a reduction in the 1-year relapse rate to ≤20%, with a power of
`80%, using a one-sided alpha of 0.05. Evaluable patients for this
`endpoint were those who had high-risk pathology in the surgical
`specimen after one dose of neoadjuvant pembrolizumab. Assuming a
`rate of high-risk pathology of 80% in patients with clinical stage III/IV
`HPV-unrelated HNSCC, and a 20% drop-out rate, we planned to
`accrue a total of 46 patients to group 1. However, after enrollment of
`the first 20 patients, the proportion of patients with high-risk pathol-
`ogy after one dose of neoadjuvant pembrolizumab and surgery was
`lower than expected (35%), prompting an unplanned interim analysis
`to assess the feasibility to achieve the initial primary endpoint.
`Enrollment continued during the interim analysis. The results of the
`interim analysis confirmed the unexpectedly lower rate of high-risk
`pathology. The trial was amended to (i) close enrollment of group 1
`based on inability to accrue the required number of patients with high-
`risk pathology in a practical interval, (ii) addition of pTR-2 after
`neoadjuvant pembrolizumab in all patients as a coprimary endpoint,
`and (iii) addition ofgroup 2, as previously described. In this report, the
`analysis of 1-year relapse rate in patients with high-risk pathology was
`performed as intention to treat.
`Stopping rules were in place for delay of surgery or serious (grade
`3-5) AEs attributed to pembrolizumab. The study was to be stopped,
`and amended or closed, in the event of (i) neoadjuvant pembrolizu-
`mab-related AEs leading to significant delay in surgery (more than
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`CLINICAL CANCER RESEARCH
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`Pembrollzumab In Surgically Resectable HPV-Negative HNSCC
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`14 days delay in one of the first 15 patients, two of 30, or three of 45)
`or (ii) serious pembrolizumab-related AEs (occurring in one of the
`first 10 patients, two of 20, three of 30, four of 40, or five of all
`patients).
`Distribution of demographic and clinical characteristics was defined
`and compared between patients in high-risk and other (low/interme-
`diate-risk) pathology groups. Percent difference and 95% confidence
`intervals (Cis) were calculated for categorical variables; median dif-
`ference and 95% Cl were calculated for continuous variables. Spear-
`man's rank correlation coefficient was used to estimate correlations
`between tumor PD-Li staining and numbers of tumor-infiltrating T
`cells (CD 8± and CD4±) and extent of pTR Molecular correlates were
`evaluated for changes across pTR categories using the nonparametric
`test of trend. Kaplan-Meier estimates of OS, progression-free survival
`(PFS), and relapse-free survival (RFS) rates and 95% CI by pathology
`risk category or pTR category were determined and differences
`between categories assessed using the log rank test. OS was defined
`as time (months) from day of surgery to death; PFS was defined as time
`from day of surgery to first disease progression event (new primary,
`recurrence, distant metastasis, or death from disease) or death from
`any cause; RFS was defined as time from day of surgery to first relapse
`event (recurrence or distant metastasis). In gene expression analysis,
`unpaired Mann-WhitneyfWilcoxon rank-sum tests were used to
`compare the immune cell populations across groups of patients, and
`paired Wilcoxon signed-rank tests were used to compare matched
`baseline and posttreatment tumor samples. Differential gene expres-
`sion analysis was performed using the Wald test across groups.
`
`Study approval
`The study protocol was approved by the Institutional Review Board
`at each participating site and registered nationally (ClinicalTrials.gov
`NC'r02296684). All patients provided written, signed, informed con-
`sent to participate. This study followed ethical guidelines of the
`Declaration ofHelsinld, Belmont Report, and the U.S. Common Rule.
`Independent data monitoring was done by the quality assurance
`committee of Washington University (St. Louis, MO).
`
`Role of the funding source
`The funders had no role in the study design, data collection, data
`analysis, data interpretation, or writing of the report. All authors had
`full access to all data in the study. The corresponding author had final
`responsibility for the decision to submit for publication.
`
`Results
`Between June 30, 2015, and March 30, 2018, 36 patients enrolled
`into group 1 of the trial. Patient and tumor characteristics were typical
`of those observed in patients with locally advanced, HPV-unrelated
`HNSCC: mostly males with a smoking history and large tumors
`(Table 1). The trial profile is shown in Fig. IA. All patients received
`one dose of neoadjuvant pembrolizumab and underwent surgery.
`Microvascular flap reconstruction was required in 28 patients. Eigh-
`teen patients (50%) had high-risk pathology, of which 12 were treated
`with POACRT and adjuvant pembrolizumab, four with POACRT, one
`with pOART, and one was observed (Supplementary Table S2).
`Adjuvant pembrolizumab was not administered to six patients with
`high-risk pathology due to persistent toxicity of POACRT (2), patient
`decision (2), perioperative myocardial infarction (1), and interim
`development of distant metastasis (1). Eighteen patients (50%) had
`low/intermediate-risk pathology, of which 10 were treated with
`POART, four with POACRT, and four were observed.
`
`Administration of neoadjuvant pembrolizumab before surgery was
`safe. Serious immune-related AEs did not occur in the interval between
`administration of neoadjuvant pembrolizumab and through 30 days
`after surgery (Table 2). Unexpected surgical delays or complications
`were not observed (Supplementary Table S3). Delivery of POACRT
`was not compromised by prior neoadjuvant pembrolizumab. During
`administration of adjuvant pembrolizumab, one serious reversible
`immune-related AE occurred, hypothyroidism (Table 2).
`Median follow-up after surgery was 22 months (interquartile range,
`17.1-32.2); 97% had ≥1 year follow-up after surgery. In patients with
`high-risk pathology, the i-year relapse rate was 16.7% (3/18, 95% CI,
`3.6-41.4). In patients with low/intermediate-risk pathology, the 1-year
`relapse rate was 0% (Supplementary Figs. Si-S2).
`After neoadjuvant pembrolizumab, pTR-2 occurred in the surgical
`specimens of eight patients (22%) and pTR-1 occurred in eight
`additional patients (22%). Overall, pTR of ≥10% was observed in
`16 of 36 patients (44%; Fig. iB; Supplementary Table S4). The
`proportion of patients who experienced pTR was similar in the
`high-risk and low/intermediate-risk pathology groups (Fig. I B; Sup-
`plementary Table SS). Two patients had major pathologic response
`(>90%) in both the tumor and lymph nodes, but there were no
`pathologic complete responses. Of the patients with pTR and tumor
`in the primary site and lymph nodes, eight of 10 had pTR in only one of
`these sites.
`Significant clinical tumor responses occurred ins minor proportion
`of patients after neoadjuvant pembrolizumab. Patient 20 experienced
`an exceptional clinical tumor response, and tumor downstaging
`(clinical stage IV: T2N25 downstaged to pathologic stage I: T1N5),
`after neoadjuvant pembrolizumab (Fig. IC-F). The surgical specimen
`from this patient showed extensive pTR (90%), and only a small
`residual focus of SCC (Fig. lG). Most patients had stable disease
`(Fig. 1H). The proportion of patients with high-risk pathology (50%)
`was lower than estimated (80%). Downstaging of cancer (defined as
`pathologic stage lower than clinical stage) after neoadjuvant pembro-
`lizumab occurred in seven patients (19%; Supplementary Table S4).
`We explored immunologic correlates of tumor response to neoad-
`juvant pembrolizumab using IHC, MIF, and RNA-seq performed on
`baseline and surgical samples. A positive correlation existed between
`PD-Li protein expression in baseline biopsies and pTR (Fig. 2A and B,
`r = 0.42; 95% Cl, 0.08-0.67; P = 0.02). MIT showed a positive
`correlation between extent of pTR and infiltration of CD8± (r =
`0.72; 95% CI, 0.44-0.88; P <0.01) but not CD4 T cells (r = 0.27; 95%
`Cl: -0.16-0.62; P = 0.20) in baseline biopsies (Fig. 2C and D;
`Supplementary Fig. S3). These immune cell populations were queried
`by RNA-seq (26) and revealed significantly higher levels of overall
`immune infiltrate, Ml macrophages, and CD4 and CD8 T cells (P <
`0.05) at baseline in patients with PTR compared with those without
`(Fig. 3; Supplementary Table S7). Consistent with these findings,
`differential gene expression analysis revealed that patients with PTR (n
`= 6) displayed significantly increased baseline expression of immune
`and inflammatory genes (e.g., IFNG, CXCL9, CXCLIO, CXCLI1, P <
`0.01, FDR < 0.2) as well as significant enrichment of genes associated
`with these processes (P < 0.01, FDR < 0.2, Fig. 3A; Supplementary
`Table SS), compared with patients without PTR (n = 10). These
`inflammatory expression patterns were maintained in patients with
`PTR over treatment (n = 4).
`Interestingly, comparing posttreatment, surgical samples from
`patients without PTR (n = it) with matched baseline samples,
`posttreatment samples showed enrichment for inflammatory gene
`signatures (P < 0.01, FDR < 0.2) and increased expression of T-cell
`checkpoint molecules, including PDCDI. CTLA4, ICOS, TIGIT, iDOl,
`
`AACRJournals.org
`
`Chn Cancer Res; 26(19) October 1, 2020
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`5143
`
`0004
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`

`

`Uppalurl et al.
`
`Table 1. Patient characteristics by pathological risk category.
`
`ded from http:/I000rjournaIsorg/clincencerres/article-pd1126/19/514012061844/5140pd1 by guest on 31 July 2023
`
`Characteristic
`
`Age at enrollment (years)
`Median (range)
`Sex, N (%)
`Male
`Female
`Ethnicity, N (%)
`White
`Black
`Asian
`Smoking history, N (%)
`Ever
`Never
`Smoking pack-years
`Median (range)
`Alcohol use history, N (%)
`Ever
`Never
`Eastern Cooperative Oncology Group performance status, N (%)
`0
`
`Tumor site, N (%)
`Larynx/hypopharynx
`Oral cavity
`Oropharynx
`Clinical T stage, N (%)
`T1-T2
`T3
`T4
`Clinical N stage, N (%)
`NO-Ni
`N2
`M3
`Clinical disease stage, N (%)
`III
`Iv
`Days first visit to surgery
`Median (range)
`Days neoadjuvant pembrolizumab to surgery
`Median (range)
`Pathologic disease stage, N (%)
`
`lVA-IVB
`
`Patients with
`high-risk
`All patients pathology
`(N=36)
`(N=18)
`
`Patients with low!
`Intermediate-risk
`pathology (N = 18) P vaTs? 01ff (95% Ci)
`
`60(32-87)
`
`61.5 (37-87)
`
`58.5 (32-73)
`
`0.16
`
`5 (-2 to 13)
`
`26 (72)
`10 (28)
`
`28 (78)
`6 (17)
`2 (5)
`
`31(86)
`5 (14)
`
`13 (72)
`5 (28)
`
`15 (83)
`2 (ii)
`1(6)
`
`i6 (89)
`2 (ii)
`
`13 (72)
`5 (28)
`
`13 (72)
`4 (22)
`1(6)
`
`15 (83)
`3 (17)
`
`1.00
`
`0 (-29 to 29)
`
`0.82
`
`ii (-16 to 38)
`-ii (-35 to 13)
`0 (-15 to 15)
`
`1.00
`
`6 (-17 to 28)
`
`30(0-80)
`
`30(0-80)
`
`30(0-80)
`
`0.79
`
`0 (-15 to 10)
`
`23 (64)
`13 (36)
`
`26 (72)
`10 (28)
`
`10 (28)
`22 (61)
`4 (11)
`
`7 (19)
`4 (11)
`25 (69)
`
`13 (36)
`21(58)
`2 (6)
`
`3 (8)
`33 (92)
`
`11 (61)
`7 (39)
`
`16 (89)
`2 (ii)
`
`4 (22)
`11 (61)
`3 (17)
`
`4 (22)
`3 (17)
`11(61)
`
`2 (11)
`14 (78)
`2 (11)
`
`0
`18 (100)
`
`12 (67)
`6 (33)
`
`10 (56)
`8 (44)
`
`6 (33)
`11(61)
`1(6)
`
`3 (17)
`1(6)
`14 (78)
`
`11 (61)
`7 (39)
`0
`
`3 (17)
`15 (83)
`
`1.00
`
`-6 (-37 to 26)
`
`0.06
`
`33 (6 to 60)
`
`0.56
`
`0.59
`
`-11 (-4 to 18)
`0 (-32 to 32)
`11 (-9 to 31)
`
`5 (-20 to 31)
`U (-9 to 31)
`-17 (-46 to 31)
`
`0.005
`
`-50 (-77 to 23)
`39 (9 to 68)
`11 (-3 to 26)
`
`023
`
`-17 (-34 to 1)
`
`33(2S-76)
`
`35(25-76)
`
`33(25-58)
`
`0.81
`
`1 (-5 to Ii)
`
`16 (13-22)
`
`1603-22)
`
`18(14-22)
`
`0.23
`
`-1 (-3 to 1)
`
`3 (8)
`6 (17)
`27 (75)
`
`0
`2 (ii)
`16 (89)
`
`3 (17)
`4 (22)
`11(61)
`
`1.00
`
`-17 (-34 to 1)
`-11 (-35 to 13)
`28 (10 to 55)
`
`Wilcoxon signed-rank test used for continuous variables; Fisher exact test used for categorical variables.
`
`and TNFSF4 (P < 0.01, FDR < 0.2, n = 10, Fig. 3D; Supplementary
`Fig. S4; Supplementary Table S8).
`TCR sequencing was performed to assess the peripheral blood T-cell
`repertoire relationship to tumor-infiltrating immune-related expres-
`sion patterns (22). We summarized the TCR repertoire using several
`metrics associated with either the clonality, richness, or diversity (see
`Supplementary Methods; ref. 27). There were no significant differences
`at baseline in the diversity, clonality, and richness in TRB clones of
`peripheral blood between patients without or with PTR. However,
`patients with PTR exhibited patterns associated with increased
`TCR diversity and clonality in peripheral blood after neoadjuvant
`
`pembrolizumab, with significantly higher Shannon, Gini Simpson, and
`Inverse Simpson diversity indices and lower geometric coefficient of
`variance (P < 0.05, Supplementary Figs. S5 and S6; ref. 27). Of note,
`t