Patient Selection:
Patients with previously untreated gastric or gastroesophageal junction adenocarcinoma were enrolled in this National Cancer Institute-sponsored single-arm pilot clinical trial from October 2017 to June 2018 (NCT03279237). This study was approved by the XXXX/XXXX Institutional Review Board. All patients provided written informed consent. Prior to study enrollment, all patients underwent the following evaluation: 1) clinical staging, including EGD +/- EUS, computed tomographic (CT) scan of the chest and either a CT scan of the abdomen/pelvis or a magnetic resonance imaging (MRI) scan of the abdomen with intravenous (IV) contrast within 42 days of enrollment, 2) physical examination within 14 days of enrollment, and 3) baseline laboratory studies, including urine HCG for women of childbearing potential. Staging laparoscopy was not required prior to study treatment and was at the discretion of the treating investigator.
Study eligibility criteria were as follows: histologically or cytologically confirmed T3/4 or lymph node positive disease (defined as > 1 cm in size and/or FDG-avid), centralized pathologic confirmation; age ≥18 years; ECOG PS ≤ 1; life expectancy > 3 months; and adequate organ and marrow function as defined as absolute neutrophil count ≥ 1,500 cells/mm3; platelets ≥ 75,000 cells/mm3; AST(SGOT) and ALT (SGPT) ≤ 2.5 × upper limit of normal, and creatinine ≤ 1.5 mg/dL, or creatinine clearance ≥ 30 mL/min/1.73 m2 for participants with creatinine levels above institutional normal; and the ability to understand and the willingness to sign a written informed consent document. The full protocol is provided in supplemental data (Supplemental Appendix 1).
Exclusion criteria included: evidence of metastatic disease within six weeks of study entry, prior treatment for the participant’s gastric or GE junction cancer; treatment of other invasive carcinomas within the last five years with greater than 5% risk of recurrence at time of eligibility screening; receipt of any other investigational agents within 4 weeks preceding the start of study treatment; serious concomitant systemic disorders; pregnancy; major surgery (excluding laparoscopy) within 4 weeks of start of study treatment; prior systemic fluoropyrimidine therapy or known hypersensitivity to 5-fluorouracil or known dihydropyrimidine dehydrogenase (DPD) deficiency; and a history of allergic reaction(s) attributed to compounds of similar chemical or biologic composition to 5-fluorouracil, irinotecan, or oxaliplatin. Distant nodal disease was allowed if it was deemed to be covered within a radiation treatment field and curative therapy was planned.
Study Design:
This is a single-arm pilot study of the neoadjuvant administration of the FOLFIRINOX regimen and pre-operative radiation therapy followed by surgery in patients with locally advanced (T 3/4 or N+) gastric or GEA. The primary study objective was to determine the completion rate of neoadjuvant FOLFIRINOX followed by consolidative chemoradiation with concurrent carboplatin/paclitaxel delivered in the preoperative setting. Secondary objectives included: 1) acute toxicity; 2) pCR rate; 3) progression-free survival (PFS); 4) overall survival (OS). Pre-planned correlative studies to explore the relationship between ctDNA and clinical outcomes were performed. Exploratory analyses were performed to evaluate tumor microenvironment and molecular profiling.
FOLFIRINOX
FOLFIRINOX was planned for 8 cycles on a 14-day cycle. 5-Fluorouracil was administered as 400 mg/m2 bolus on day 1, then as a 2400 mg/m2 continuous infusion for 46 hours. Leucovorin calcium, 400 mg/m2; oxaliplatin 85 mg/m2, and irinotecan hydrochloride 180 mg/m2, were administered on day 1, as previously described14. Pegfilgrastim, 6 mg, was administered on day 4. Dose adjustments for toxicity were predefined in the protocol (Supplemental Appendix 2). Patients were restaged with CT scans of the chest, abdomen, and pelvis after 4 and 8 cycles of FOLFIRINOX.
Chemoradiation
Chemoradiation treatments were scheduled to begin within 4 weeks after completion of FOLFIRINOX. Carboplatin and paclitaxel were administered concurrently with the radiation therapy. Carboplatin (area under the curve [AUC]=2) was administered weekly over 1 hour. Paclitaxel 50 mg/m2 was administered as an IV infusion over 30-60 minutes weekly as previously described9.
All radiation treatments were administered at the XXXXX. All patients underwent 4D-CT simulation with oral and IV contrast in a supine position. For photons, all patients received IMRT or VMAT. Daily cone beam CT was performed pre-treatment for set up verification. Tumor volume was defined on the basis of CT, PET/CT and MRI. The Gross Tumor Volume (GTV) was defined as the gross primary tumor and any lymph nodes ≥1 cm. For participants with diffuse type gastric cancer, where the GTV could not be visualized, the entire stomach was identified as GTV. When the GTV was clearly identified, the clinical target volume (CTV) included a longitudinal mucosal margin of 3.5-4 cm proximally and distally to the GTV. A 5 mm CTV expansion was generated based on all grossly enlarged lymph nodes. Elective lymph node coverage included celiac and gastrohepatic lymph nodes for all patients. For GEJ and cardia lesions, coverage of paraesophageal lymph nodes within 4 cm of the tumor was recommended; for distal gastric lesions, porta hepatis lymph node coverage was recommended. Coverage of splenic lymph nodes (for GE junction/cardia lesions) and peripancreatic lymph nodes (for distal gastric lesions) were at the discretion of the treating physician. The Planning Target Volume (PTV) was customized based on the 4D CT scan; however, generally, a 5 mm expansion was used, except for 7 mm superiorly/inferiorly. The prescribed dose to the PTV for gastric tumors was 4500 cGy delivered in 180 cGy/day over 25 fractions. For gastroesophageal junction tumors, a 540 cGy boost was included to the GTV + 1 cm. Normal dose constraints were predefined in the protocol (Supplemental Appendix 3).
Surgery
After completion of chemoradiation, restaging CT scan was evaluated by the multidisciplinary team. Surgical resection was performed approximately 4-6 weeks after completion of chemoradiation. Surgical procedure and extent of LN dissection was performed at the discretion of the treating surgeon. For gastric cancers, an extended (D1+ or D2) lymph node dissection was performed.
Follow-Up
After treatment completion, study participants had a follow-up baseline CT scan performed 3-8 weeks postoperatively. Subsequently, follow-up visits were scheduled with laboratory evaluations every 3 months and with CT scans every 6 months for the first 2 years, visits with blood work every 3 months and annual CT scans for year 3, and visits with blood work at least every 6 months and annual CT scans up to year 5.
Pathologic Evaluation
Pathologic findings were scored per standard institutional practices, including margin status (proximal, distal, radial) and nodal status (total assessed, total positive). Tumor regression grade (TRG)18 was recorded for all patients undergoing surgery, and pathologic complete response was defined as TRG 0.
Correlative Studies:
Circulating tumor DNA
Tumor biopsies from the time of diagnosis as part of routine care were subjected to an internal next-generation sequencing platform evaluating 104 known cancer genes. Blood was drawn at pre-specified study time points (baseline, cycle 1 day 8, cycle 5 day 1, pre-chemoradiation, preoperatively, every 3 months postoperatively, and at progression) at time of draws for routine clinical care. ctDNA was extracted from plasma using QIAGEN-based protocols and amplified by digital droplet PCR (ddPCR) using primers for tumor-specific point mutations. One or more driver mutations that matched the patient’s tumor sequencing were followed longitudinally.
Tumor Microenvironment
Immunohistochemical staining to evaluate tumor microenvironment was performed with the remaining tissue available from the diagnostic biopsy after ctDNA analysis. Surgical resection samples were also obtained for comparison. Five-micron sections were cut from the selected paraffin blocks and stained by immunohistochemistry (IHC). CD8+ TIL were scored based on % of tumor cells with CD8+ T cells on them as follows19: 0-rare; 1-<5%; 2-≥5% but <25%; 3->25%. In the pretreatment specimens, 3 high power fields (HPFs) were reviewed, and the average was reported, except for PD-L1 which was reported as combined positive score (CPS). In the post-treatment cases, due to heterogeneity in immune cell infiltration appreciated, semiquantitative scoring was performed (Supplemental Appendix 4). The specific antibodies and conditions used for each immune marker were: (1) CD8 - clone 4B11, Leica Biosystem; (2) PD-1 - clone EH33, Cell Signaling Technology; (3) PD-L1 - clone E1L3N, Cell Signaling Technology; (4) CD3 - clone LN10, Leica Biosystem; (5) FOXP3 - clone 236A/7, Biocare. Due to the limited sample size, statistical analysis was not performed for paired comparison.
Tumor Molecular Analysis by SNaPshot analysis
Molecular profiling of hotspot SNP, indel and copy number variants was performed using SNaPshot analysis, an Anchored Multiplex PCR strategy previously described20. Briefly, after pathologic review to determine sample tumor enrichment, genomic DNA was isolated from FFPE- embedded tumor specimens. Genomic DNA was sheared, end-repaired, A-tailed and then modified by ligation of half-functional Illumina adapters. Two hemi-nested PCR reactions targeting genomic regions of interest (i.e., SNP/mutation, indel or copy number hotspots, Supplemental Appendix 5) were performed, and sequenced using Illumina Nextseq 2x150bp paired-end sequencing. Genome alignment to the hg19 reference sequence was performed using Novoalign (Novocraft Technologies; Selangor, Malaysia) and various methods for variant calls were employed, including use of MuTect121, LoFreq22, GATK23-25 and a laboratory-developed hotspot and copy number caller. This test is validated to detect variants with at least 5% allelic frequency, in regions with sufficient NGS read coverage.
Analysis of the Cancer Genome Atlas (TCGA) Data
Data from the TCGA26 were analyzed using cBioportal27 and Xenabrowser.net28 to assess co-mutation frequency and alteration subtype specificity.
Statistical Analysis:
The primary endpoint was neoadjuvant therapy completion, defined as the portion of patients receiving all planned induction FOLFIRINOX followed by chemoradiation. Sample size was planned for 25 patients, and if at least 18 of the 25 patients were able to complete the specified treatment plan, then the primary endpoint was met. The decision rule was associated with 89% power for declaring success if FOLFIRINOX in combination with chemoradiation were associated with an underlying completion rate of 80%. In contrast, the probability of a type I error was only 15% if 60% of patients were able to complete the intended treatment plan. Toxicity was defined per CTCAE v4.03. PFS and OS were measured from the date of treatment start and estimated by the Kaplan-Meier method. OS time was censored at the date of last follow-up for patients still alive. PFS time was defined until detection of locoregional recurrence, distant metastases, or death without documented progression, whichever date was earliest, or censored at the date of last follow-up. The distant metastasis (DM) rate was estimated as the cumulative incidence with progression or death before chemoradiation as a competing risk. Fisher’s exact test and Gray’s test were used, respectively, to analyze pCR and DM rates by ctDNA detection status, with the p-values based on a two-sided hypothesis. As the biomarker analysis was exploratory, the level of type 1 error was not pre-specified. Statistical analysis was performed using SAS version 9.4 (SAS Institute) and R version 3.3.1 (R Foundation).