A Phase II Study of Biomarker-Guided De-escalation Using Anthracycline-Free Neoadjuvant Chemoimmunotherapy in Early-Stage Triple Negative Breast Cancer (TNBC) Patients With High Tumor-Infiltrating Lymphocytes (TILs)

NeoTILs is a single-arm, single-institution phase II non-inferiority trial evaluating a 12-week neoadjuvant regimen of carboplatin, paclitaxel, and pembrolizumab (CPP) in patients with early-stage (anatomic Stage II to IIIB) triple negative breast cancer whose tumors show high tumor-infiltrating lymphocytes (TILs), defined as TILs of 30 percent or greater on a digitized hematoxylin and eosin slide from the diagnostic biopsy, assessed centrally. The trial tests whether the pathologic complete response (pCR) rate achieved with this anthracycline-free neoadjuvant regimen is not significantly inferior to the historical pCR rate of 65 percent reported with the KEYNOTE-522 regimen in a comparable TIL-enriched population.

Following pre-screening consent and confirmation of high TIL status, eligible patients are formally consented and enrolled. All patients receive 12 weeks of neoadjuvant CPP, with anthracycline omitted entirely from the neoadjuvant phase, followed by definitive surgery. Treatment after surgery is determined by pathologic response, making this a response-adapted design. Patients who achieve pCR continue pembrolizumab alone to complete a total of nine doses and never receive anthracycline chemotherapy. Patients with residual invasive disease in the breast or axillary lymph nodes receive adjuvant doxorubicin and cyclophosphamide together with continued pembrolizumab for four cycles, consistent with current standard of care, and also complete the planned nine total doses of pembrolizumab. Additional adjuvant therapy, including olaparib for patients with germline BRCA1 or BRCA2 mutations and capecitabine for residual disease, may be given at the treating physician's discretion.

The trial uses a group sequential design with a planned futility analysis at 50 percent information (n1 = 25) and a maximum sample size of 50 evaluable patients if the futility boundary is not crossed, out of an anticipated screening population of approximately 150 patients. Continuous safety monitoring begins with the sixth patient enrolled, with significant safety events defined as grade 3 or 4 adverse events or serious adverse events, or a surgical delay of more than 12 weeks, judged definitely or probably related to the neoadjuvant regimen.

The primary endpoint is pCR rate. Secondary endpoints include three-year event-free survival, three- and five-year overall survival, treatment-related toxicity, incidence of chemotherapy-induced peripheral neuropathy, and patient-reported quality of life measured with the EORTC QLQ-C30 and QLQ-CIPN20 at baseline, end of neoadjuvant therapy, and 6 and 12 months post-treatment. Exploratory endpoints include near-pCR rate (residual cancer burden 0 or 1), radiographic response, and correlation of baseline and on-treatment TIL levels, PD-L1 expression, and circulating tumor DNA dynamics with pathologic response and long-term survival outcomes. Patients are followed for a minimum of five years for event-free and overall survival.

Inclusion criteria

Pre-Screening Phase:

• Age ≥ 18 years at the time of informed consent.
• Ability to understand and willingness to sign a written informed consent document in accordance with institutional and federal guidelines.
• Histologically confirmed diagnosis of triple-negative breast cancer (TNBC) or hormone receptor-low invasive breast carcinoma, with clinical anatomic Stage II or Stage IIIA/B as defined by the AJCC 8th Edition Anatomic Breast Cancer Staging System.

a. Invasive tumor must be estrogen receptor (ER) and/or progesterone receptor (PR) negative or low, defined as ≤10% positive staining by immunohistochemistry (IHC).

b. HER2-negative disease, defined in accordance with current ASCO-CAP HER2 testing guidelines.

• Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2.

Screening and Treatment Phases:

General Eligibility

• Individuals of childbearing potential must be willing and able to use highly effective contraception from the time of informed consent, throughout study treatment, and for at least 6 months after the last dose of trial therapy.

a. NOTE: Highly effective contraception is defined as methods with a failure rate <1% per year when used consistently and correctly, and include: copper intrauterine device (IUD); bilateral tubal ligation/occlusion or other documented surgical sterilization; vasectomized partner with documented azoospermia, provided this is the sole sexual partner; or true sexual abstinence, defined as complete abstinence from heterosexual intercourse, when this is the participant's usual and preferred lifestyle. Use of hormonal contraceptive methods (including combined oral contraceptives, progestin-only pills, injectables, implants, hormonal IUDs, patches, or vaginal rings) is not permitted during the study and for at least 6 months after the last dose of study treatment.

• Willingness and ability to comply with all study procedures, including scheduled visits, treatment plans, laboratory tests, and other protocol-specified requirements.
• Willingness and ability to sign and date written informed consent prior to initiation of any study-specific procedures.

Disease Characteristics

• Breast and axillary imaging (mammogram, ultrasound, or MRI) must have been completed within 45 days prior to registration.
• Patients with abnormal axillary lymph nodes (identified clinically and/or radiographically) must undergo routine pathological confirmation via image-guided core biopsy or fine needle aspiration.
• Presence of:

• Measurable disease in the breast measuring at least 1cm with or without nodal involvement; or
• Clinical T0 disease with biopsy-proven regional lymph node involvement (cT0N1-2M0), consistent with an overall anatomic stage II-IIIB classification.

i. For patients with clinical T0 disease confirmed by mammogram/US and MRI, nodal involvement must be documented by core needle biopsy or fine needle aspiration of an axillary or other regional lymph node demonstrating invasive breast carcinoma prior to initiation of neoadjuvant systemic therapy.

ii. Patients must not have undergone prior surgical excision of an invasive breast primary tumor that would account for the T0 designation (i.e., T0 must not be the result of complete prior excision of the primary breast lesion).

• Patients with multifocal or multicentric disease are allowed if the dominant tumor is confirmed ER and/or PR ≤10%, and HER2-negative.
• Patients with bilateral breast cancer are eligible if both tumors are HER2-negative.
• Staging scans (e.g., CT chest/abdomen/pelvis with a nuclear bone scan) must be performed to rule out metastatic disease under any of the following conditions:

a. Two or more abnormal axillary lymph nodes on imaging, or b. Clinical suspicion of metastatic disease, or c. At the discretion of the treating physician.

Clinical and Laboratory Requirements

• Peripheral neuropathy must be Grade ≤1 per CTCAE v5.0.
• Complete history and physical examination performed within 45 days prior to registration.
• Adequate hematologic and organ function as defined by the following:

a. Hematologic i. Hemoglobin ≥9.0 g/dL (without transfusion or erythropoietin support within 14 days prior to testing) ii. Leukocytes ≥3,000/μL iii. Absolute neutrophil count (ANC) ≥1,500/μL iv. Platelet count ≥100,000/μL b. Hepatic i. AST (SGOT) and ALT (SGPT) ≤3 × ULN ii. For participants without a history of Gilbert's syndrome, total bilirubin ≤1.5 × upper limit of normal (ULN) iii. For participants with a history of Gilbert's syndrome, total bilirubin ≤5 × ULN c. Renal i. Serum creatinine ≤1.5 mg/dL or creatinine clearance ≥50 mL/min/1.73 m² by Cockcroft-Gault formula.

ii. Note: Patients with creatinine clearance 30-50 mL/min may be enrolled at the discretion of the Principal Investigator, given that paclitaxel is primarily hepatically metabolized and carboplatin dosing can be adjusted to renal function.

• Cardiac function must be within acceptable limits, as follows: left ventricular ejection fraction (LVEF) ≥50% by

a. Echocardiogram (ECHO), or b. Multi-gated acquisition (MUGA) scan.

• Participants with known human immunodeficiency virus (HIV)-infection must be on effective antiretroviral therapy (ART) at randomization and have an undetectable viral load test on the most recent test results obtained within six (6) months prior to randomization.
• Participants with evidence of chronic hepatitis B virus (HBV) infection must have an undetectable HBV viral load while on suppressive therapy on the most recent test results obtained within six (6) months prior to randomization, if indicated.

a. NOTE: No testing for HBV is required unless mandated by local health authority.

• Participants with a history of hepatitis C virus (HCV) infection must have been treated and cured. Participants currently being treated for HCV infection must have an undetectable HCV viral load test on the most recent test results obtained within six (6) months prior to randomization, if indicated.

• NOTE: No testing for HCV is required unless mandated by local health authority.

Exclusion criteria

Participants meeting any of the following criteria will be excluded from the study:

Pre-Screening Phase:

• Presence of tumor-infiltrating lymphocytes (TILs) <30% based on central pathology review of hematoxylin and eosin (H&E) stained slides.

Screening and Treatment Phases:

Disease-Related Exclusions

• Presence of N3, inflammatory, or metastatic (M1) breast cancer.
• History of other malignancies within the past 3 years, with the exception of:

• Adequately treated non-melanoma skin cancer, or
• Cervical carcinoma in situ, or
• Other malignancies with a ≥3-year disease-free interval. Prior and Concurrent Therapy
• Prior systemic therapy, radiation therapy, or definitive surgery for current breast cancer.
• Prior treatment with immune checkpoint inhibitors, including anti-PD-1, anti-PD-L1, or any other T-cell co-inhibitory or co-stimulatory agents.
• Use of investigational agents or devices within 28 days prior to registration
• Participant is planning to participate, currently participating in or has participated in a study of an investigational agent or has used an investigational device within 4 weeks prior to the first dose of study treatment.

Medical Conditions

• History of severe (Grade ≥3) allergic reactions or hypersensitivity to study drugs or their components.
• Uncontrolled diabetes mellitus or hypertension.
• Has a diagnosis of immunodeficiency or is receiving chronic systemic steroid therapy (in dosing exceeding 10 mg daily of prednisone equivalent) or any other form of immunosuppressive therapy (7-day clearance period for immunosuppressant therapy prior to starting study treatment, if applicable).
• History of solid organ transplant.
• Active autoimmune disease requiring systemic treatment within the past 1 year.
• Recent (within 12 weeks) or active non-infectious pneumonitis requiring corticosteroid therapy.
• Major surgical procedure or active/severe infection within 14 days prior to registration.
• Subject is a WOCBP who has had a positive pregnancy test within 24 hours prior to initiation of study treatment. Females will be determined to be not of child-bearing potential with a history of hysterectomy or with postmenopausal status of >12 months.
• Pregnant or breastfeeding, or expecting to conceive within the projected duration of the study, starting with the screening visit through 180 days after the last dose of trial treatment.
• History of hypersensitivity to compounds that are similar to carboplatin and paclitaxel.
• Has received major surgery and has not recovered adequately from the toxicity and/or complications before starting study treatment.
• Has a history of non-infectious pneumonitis that required high-dose steroids and/or has current pneumonitis.
• Has an active bacterial infection requiring systemic therapy.
• Known psychiatric or substance abuse disorders that would interfere with the requirements of the trial.

Vaccination

• Administration of live vaccines within 30 days prior to registration. Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella/zoster (chicken pox), yellow fever, rabies, Bacillus Calmette-Guérin (BCG), and typhoid vaccine. Seasonal influenza or COVID vaccines for injection are generally killed virus vaccines and are allowed; however, intranasal influenza vaccines (e.g., FluMist®) are live attenuated vaccines and are not allowed.