Pembrolizumab Plus Ultrasound-Induced Microbubble Cavitation in Head and Neck Cancer

The proposed study is a feasibility trial of Pembrolizumab plus Definity Perflutren Lipid Microsphere (Lantheus Medical Imaging), followed by ultrasound-induced microbubble cavitation (UC), which will target patients with R/M HNSCC, for which Pembrolizumab is already indicated as standard of care treatment. Patients will undergo up to 3 cycles of therapy. Participants may continue study treatment for up to 3 cycles, 3 weeks apart, from the time of initiating treatment, with 3-month safety follow-up.

Participant characteristics and endpoints will be summarized through appropriate descriptive statistics-the mean and standard deviation (or median and interquartile range, if appropriate) for continuous variables, and frequency counts and percentages for categorical variables.

Primary Analysis Among all patients enrolled, feasibility will be assessed, defined as enrollment of 6 participants within 1 year of study initiation, and completion of all trial procedures, including 3 cycles of PD treatment and follow-up visits, among at least 80 percent of enrolled patients.

Recruitment feasibility (primary objective secondary endpoint) will be assessed as the number of participants enrolled during the first 12 months from site activation. Success is defined as 6 participants enrolled within this window. The investigators will summarize the 6-month enrollment count and accrual rate descriptively. Protocol completion feasibility (primary objective primary endpoint) will be assessed as the proportion of enrolled participants who complete all pre-specified trial procedures (all scheduled visits/assessments through end-of-treatment and the primary follow-up time point). The denominator is all enrolled participants. Unknown or missing completion status will be counted as not complete. Success is defined as at least 80 percent completion. The investigators will report the completion proportion with 95 percent exact binomial (Clopper-Pearson) confidence intervals.

Secondary Analysis Assessment of the following secondary metrics is anticipated using descriptive statistics; no hypothesis tests are planned. Where applicable, 95% exact binomial CIs (Clopper-Pearson) will be provided for proportions.

• Proportion of serious adverse events (according to CTCAEv5.0)
• Objective Response Rate (Clinical/Radiographic Response)
• Progression Free Survival
• Overall Survival

Exploratory/Correlative Analysis/Assessments Assessment of the following correlative metrics through descriptive statistics is anticipated. No hypothesis tests are planned.

• IHC measures of the prevalence of intratumoral immune cell populations including but not limited to CD8, CD45, FoxP3, PD-L1
• IHC-based measures of myeloid lineage differentiation markers:CD3, CD4, CD8, CD11b, CD11c, CD14, CD33, CD163, CD204, and HLA-DR, also analyzed with flow cytometry
• Luminex panel of inflammatory markers with the Milliplex MAP Human Cytokine/chemokine kit
• Variant files (VCFs) including point mutations, insertions, and deletions, as well as HLA-type, identified from whole exome sequencing
• Epitope analysis of neoantigens and HLA typing using RNAseq
• Spatially-resolved, digital quantitation of mRNA using Nanostring GeoMx DSP
• Quantitative-CEUS parameters collected during treatment infusion, correlated with treatment efficacy and TME parameters

Interim Analyses and Stopping Rules The study will be discontinued if there are more than 2 "study-limiting adverse events" at any point during the trial, with a "study-limiting adverse event" being defined as any grade 3 or higher cardiac, endocrine, or immunologic adverse event, or any grade 4 or higher adverse event.

Inclusion criteria

• Individuals must meet all of the following inclusion criteria in order to be eligible to participate in the study:

• Pathologically confirmed, HPV-negative, R/M HNSCC per American Joint Committee on Cancer (AJCC) 8th Edition Staging Criteria, with CPS score > 1
• The target lesion is ultrasound-accessible, biopsy-accessible, and measurable in at least one dimension, based on RECISTv1.1 criteria.
• Male or female, aged ≥18 years of age
• ECOG Performance status 0-2.
• Must have a life expectancy of at least 6 months as judged by the treating physician.
• Adequate organ function:

• Absolute neutrophil count 1500/μl or more;
• Platelets 100,000/μl or more,
• Hemoglobin 8 g/dl or more;
• Bilirubin less than or equal to 1.5 x the upper limit of normal (except subjects with Gilbert syndrome, who can have total bilirubin <3 mg/dl);
• AST and ALT less than or equal to 2.5x the upper limit of normal
• Women of reproductive potential should have a negative serum or urine pregnancy test (minimum sensitivity 25 IU/L or equivalent units of HCG), which must also be confirmed as negative within 28 days of the start of study drugs.
• Women of reproductive potential must use highly effective contraception methods to avoid pregnancy for 90 days after the last dose of study drugs. "Women of reproductive potential" is defined as any female who has experienced menarche and who has not undergone surgical sterilization (hysterectomy, tubal ligation, or bilateral oophorectomy) or who is not postmenopausal. Menopause is defined clinically as 12 months of amenorrhea in a woman over 45 in the absence of other biological or physiological causes. In addition, women under the age of 55 must have a documented serum follicle stimulating hormone (FSH) level less than 40 mIU/mL.
• Men of reproductive potential who are sexually active with women of reproductive potential must use any contraceptive method with a failure rate of less than 1% per year. Men who are receiving the study medications will be instructed to adhere to contraception for 90 days after the last dose of study drugs. Men who are azoospermic do not require contraception.
• Informed Consent: All subjects must be able to comprehend and sign a written informed consent document.

Exclusion criteria

• An individual who meets any of the following criteria will be excluded from participation in this study:

• Patients with HPV-positive or p16-positive SCC
• Prior treatment with an anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, or antiCTLA-4 antibody
• Any history of allergy to the study drug components, including to polyethyleneglycol or medications containing polyethylene glycol.
• Patients with the following cardiac conditions or history:

• Patients with a known history of anatomic right-to-left, bi-directional, or transient right-to-left cardiac shunts
• Patients with NYHA class III or greater heart failure, unstable anginal syndrome, or experiencing active chest pain.
• Uncontrolled arterial hypertension (defined as systolic blood pressure ≥ 200 mmHg or diastolic blood pressure ≥ 110 mmHg) or arterial hypotension (defined as systolic blood pressure ≤ 90 mmHg)
• Patients who have experienced a STEMI or NSTEMI within the last 6 months
• QTc >500 as determined by Fridericia correction
• Patients with a history of ventricular arrhythmia that has not been corrected by placement of a PPM or ICD
• Any concurrent malignancies: exceptions include- cutaneous basal cell carcinoma, chronic lymphocytic leukemia, melanoma in situ, squamous cell carcinoma of the skin of a secondary location, superficial bladder cancer or in situ cervical cancer that has undergone potentially curative therapy. Patients with a history of other prior malignancy must have been treated with curative intent and must have remained disease-free for 2 years post-diagnosis.
• Any unresolved toxicity CTCAEv5.0 Grade ≥2 from previous anticancer therapy with the exception of alopecia, vitiligo, and the laboratory values defined in the inclusion criteria. Patients with Grade ≥2 neuropathy will be evaluated on a caseby-case basis after consultation with the Study Physician. Patients with irreversible toxicity not reasonably expected to be exacerbated by treatment with anti-PD-1 therapy may be included only after consultation with the Study Physician.
• Any subjects with a condition requiring systemic treatment with either corticosteroids (> 10 mg daily prednisone equivalents) or other immunosuppressive medications within 28 days of study drug administration., or a prior history of allogenic organ transplantation.
• Any active autoimmune disease that has required systemic treatment in the past 2 years (i.e., with use of disease modifying agents, corticosteroids, or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic treatment.
• Patients must not be receiving any other investigational agents; must not have participated in a study of an investigational agent or be using an investigational device within 4 weeks of the first dose of Pembrolizumab plus Definity.
• Receipt of a live attenuated vaccine within 30 days prior to the first dose of drug on trial.
• Uncontrolled intercurrent illness, including but not limited to, ongoing or active infection, symptomatic congestive heart failure, uncontrolled hypertension, unstable angina pectoris, cardiac arrhythmia, interstitial lung disease, serious chronic gastrointestinal conditions associated with diarrhea, or psychiatric illness/social situations that would limit compliance with study requirement, substantially increase risk of incurring AEs or compromise the ability of the patient to give written informed consent.
• Patients must not be pregnant or breastfeeding.
• Active infection including tuberculosis (clinical evaluation that includes clinical history, physical examination and radiographic findings, and TB testing in line with local practice), hepatitis B (known positive HBV surface antigen (HBsAg) result), hepatitis C, or human immunodeficiency virus (positive HIV 1/2 antibodies). Patients with a past or resolved HBV infection (defined as the presence of hepatitis B core antibody [anti-HBc]and absence of HBsAg) are eligible. Patients positive for hepatitis C (HCV) antibody are eligible only if polymerase chain reaction is negative for HCVRNA.
• Any untreated metastasis(es) to the brain that may be considered active.
• History of pneumonitis within the past 5 years.