Spatial Transcriptomics of Vaccine Therapy With or Without Cyclophosphamide in Treating Patients Undergoing Chemotherapy and Radiation Therapy for Stage I or Stage II Pancreatic Cancer That Can Be Removed by Surgery

Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. Here, spatial transcriptomics data were generated from 16 pancreatic cancer patients treated with GVAX, whose tumors presented with induced intratumoral TLS. Resected tumor specimens were embedded in OCT media and freshly frozen. Sections were cut into 6.5 x 6.5 mm dimensions compatible with Visium spatial transcriptomics slides. TLS-positive tumor sections from 14 patients (14 tumor samples in total) and lymph nodes from remaining patients (2 lymph node samples in total) were selected by an expert pathologist for profiling. We used 4 Visium slides and covered the 16 capture areas with sections cut in a cryostat at 10 µm. The fresh frozen Visium protocol was followed according to manufacturer's recommendations (10x Genomics Spatial 3' v1). Tissue permeabilization and library construction was carried out according to the manufacturer's protocols. Permeabilization time was optimized to 30 minutes. The captured tissue sections were permeabilized to allow RNA to be captured onto the slide where it reverse-transcribed to cDNA. The cDNA was then amplified, and sequencing libraries were prepared using the 10x Genomics Visium Spatial Gene Expression Reagent Kit for Library Preparation. The libraries were then sequenced using an Illumina NovaSeq sequencer. The raw data obtained from the sequencing were processed using the SpaceRanger (spaceranger-1.2.0) pipeline for alignment onto the GRCh38-2020-A reference transcriptome quantification of gene expression. Unsupervised learning generated a TLS-specific spatial gene expression signature that significantly associates with improved survival in PDAC patients. These analyses demonstrate TLS-associated intratumoral B cell maturation.]]>
DISEASE CHARACTERISTICS: Newly diagnosed adenocarcinoma of the head, neck, or uncinate process of the pancreas Stage I or II disease Surgically resectable disease (R0 or R1) by spiral CT scan No distant metastases A clear fat plane is present around the celiac and superior mesenteric arteries Patent superior mesenteric and portal veins Candidate for a pancreaticoduodenectomy PATIENT CHARACTERISTICS: ECOG performance status 0-1 Hemoglobin ≥ 9 g/dL ANC ≥ 1,500/mm³ Platelet count ≥ 100,000/mm³ Serum creatinine ≤ 2 mg/dL AST and ALT ≤ 2 times upper limit of normal (ULN) Amylase ≤ 2 times ULN Alkaline phosphatase ≤ 5 times ULN Amylase ≤ 2 times ULN Alkaline phosphatase ≤ 5 times ULN Hyperbilirubinemia secondary to tumor-associated extrahepatic biliary obstruction allowed Not pregnant or nursing Negative pregnancy test Fertile patients must use effective contraception during and for ≥ 4 weeks after the completion of study treatment No history of autoimmune disease, including systemic lupus erythematosus, sarcoidosis, rheumatoid arthritis, glomerulonephritis, or vasculitis No uncontrolled medical problems No active infections No other cancer within the past 5 years except for superficial bladder cancer, nonmelanoma skin cancer, or low-grade prostate cancer not requiring therapy PRIOR CONCURRENT THERAPY: More than 28 days since prior anticancer therapy No prior cancer immunotherapy, including the same pancreatic cancer vaccine used in this study More than 28 days since prior systemic steroid therapy or immunosuppressive therapy No systemic steroid therapy or immunosuppressive therapy during and within 28 days after vaccine administration No other concurrent immunotherapy, chemotherapy, radiotherapy, gene therapy, biologic therapy, or investigational therapy for the treatment of pancreatic cancer No distant metastases A clear fat plane is present around the celiac and superior mesenteric arteries Patent superior mesenteric and portal veins Candidate for a pancreaticoduodenectomy]]>