SMAD4 loss in colorectal cancer: Correlation with recurrence, chemoresistance, and immune infiltrate

587 Background: Few markers reliably identify colorectal cancer (CRC) patients at risk of recurrence and death. SMAD4 loss occurs in 10-20% of cases and has shown promise in identifying high-risk stage II/III patients. We examined SMAD4 status and association with clinical/pathologic features in 446 stage I-IV CRC patients at Memorial Sloan Kettering (MSK). Methods: Patients undergoing curative resection were included (1981-2010). Familial polyposis syndrome patients and those with inadequate tissue were excluded. Tissue microarrays were constructed (n=364). Immunohistochemistry for SMAD4 and mismatch repair (MMR) proteins was completed. SMAD4 nuclear stain intensity was scored (scale=0-3; 0=loss). On whole sections, MMR proteins (present or absent), tumor-infiltrating lymphocytes (TILs) and peritumoral lymphocyte aggregates (PLAs) were scored (scale=0-3). Associations between clinical/pathologic features and SMAD4 loss vs. retention were analyzed. Kaplan-Meier estimates and log-rank test were used for recurrence-free and overall survival analyses (RFS and OS). Results: SMAD4 loss was noted in 13%. Median age at diagnosis was 53 years, and 51% were male. The cohort consisted of 61% hindgut tumors and 62% stage II/III patients. With up to 33 years of follow-up, the mean was 6 years. SMAD4 loss correlated with higher tumor and nodal stage, adjuvant therapy use, and lower TIL and PLA scores (pmedian) were noted to have worse OS with SMAD4 loss (p<0.01). SMAD4 loss did correlate with worse RFS (p=0.02), persisting even when excluding MMR-deficient patients. Additionally, SMAD4 loss was associated with worse RFS in both the adjuvant chemotherapy group (median RFS=3.8 vs. 13 years; p=0.06) and the resection-only group (median RFS=4.2 years vs. not yet reached; p< 0.01). Conclusions: SMAD4 loss correlates with worse RFS and resistance to adjuvant therapy. SMAD4 loss also correlates with lower TIL and PLA scores. Future work will address chemoresistance mechanisms, relevance to adjuvant therapy use, and loss of immune infiltrate in SMAD4-null tumors

SMAD4 Loss in Colorectal Cancer Patients Correlates with Recurrence, Loss of Immune Infiltrate, and Chemoresistance

SMAD4 has shown promise in identifying patients with colorectal cancer at high risk of recurrence or death. A discovery cohort and independent validation cohort were classified by SMAD4 status. SMAD4 status and immune infiltrate measurements were tested for association with recurrence-free survival (RFS). Patient-derived xenografts from SMAD4-deficient and SMAD4-retained tumors were used to examine chemoresistance. The discovery cohort consisted of 364 patients with stage I-IV colorectal cancer. Median age at diagnosis was 53 years. The cohort consisted of 61% left-sided tumors and 62% stage II/III patients. Median follow-up was 5.4 years (interquartile range, 2.3-8.2). SMAD4 loss, noted in 13% of tumors, was associated with higher tumor and nodal stage, adjuvant therapy use, fewer tumor-infiltrating lymphocytes (TIL), and lower peritumoral lymphocyte aggregate (PLA) scores (all < 0.04). SMAD4 loss was associated with worse RFS ( = 0.02). When stratified by SMAD4 and immune infiltrate status, patients with SMAD4 loss and low TIL or PLA had worse RFS ( = 0.002 and = 0.006, respectively). Among patients receiving 5-fluorouracil (5-FU)-based systemic chemotherapy, those with SMAD4 loss had a median RFS of 3.8 years compared with 13 years for patients with SMAD4 retained. In xenografted mice, the SMAD4-lost tumors displayed resistance to 5-FU. An independent cohort replicated our findings, in particular, the association of SMAD4 loss with decreased immune infiltrate, as well as worse disease-specific survival. Our data show SMAD4 loss correlates with worse clinical outcome, resistance to chemotherapy, and decreased immune infiltrate, supporting its use as a prognostic marker in patients with colorectal cancer

A rectal cancer organoid platform to study individual responses to chemoradiation

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients' tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals