Interrogation of transcriptomic changes associated with drug-induced hepatic sinusoidal dilatation in colorectal cancer

Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab’)2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients

Targeted biomarker profiling of matched primary and metastatic estrogen receptor positive breast cancers.

Patients with newly diagnosed, early stage estrogen receptor positive (ER+) breast cancer often show disease free survival in excess of five years following surgery and systemic adjuvant therapy. An important question is whether diagnostic tumor tissue from the primary lesion offers an accurate molecular portrait of the cancer post recurrence and thus may be used for predictive diagnostic purposes for patients with relapsed, metastatic disease. As the class I phosphatidylinositol 3' kinase (PI3K) pathway is frequently activated in ER+ breast cancer and has been linked to acquired resistance to hormonal therapy, we hypothesized pathway status could evolve over time and treatment. Biomarker analyses were conducted on matched, asynchronous primary and metastatic tumors from 77 patients with ER+ breast cancer. We examined whether PIK3CA and AKT1 alterations or PTEN and Ki67 levels showed differences between primary and metastatic samples. We also sought to look more broadly at gene expression markers reflective of proliferation, molecular subtype, and key receptors and signaling pathways using an mRNA analysis platform developed on the Fluidigm BioMark™ microfluidics system to measure the relative expression of 90 breast cancer related genes in formalin-fixed paraffin-embedded (FFPE) tissue. Application of this panel of biomarker assays to matched tumor pairs showed a high concordance between primary and metastatic tissue, with generally few changes in mutation status, proliferative markers, or gene expression between matched samples. The collection of assays described here has been optimized for FFPE tissue and may have utility in exploratory analyses to identify patient subsets responsive to targeted therapies

Biological validation of the breast cancer gene expression assay using samples of known immunohistochemical subtype.

<p>(A) Hierarchical clustering of thirty FFPE breast cancer tumor samples with known ER, PR and HER2 status run on the breast cancer gene expression assay. Blue =  triple negative, Pink  =  ER+, Yellow = HER2+ (red = high expression, green = low expression) (B) Box-plots indicating genes that showed statistically significant differential expression in the ER+ subtype, (C) ER+ and HER2+ subtype, (D) HER2+ subtype and (E) triple negative subtype samples (3N) (p-values indicated).</p

PI3K pathway alterations in primary and metastatic ER+ breast cancers.

<p>(A) Distribution of alterations in <i>PIK3CA</i>, <i>AKT1</i> and PTEN across 75 matched primary and metastatic ER+ breast cancers. PTEN null status denotes total absence of PTEN protein in neoplastic cells determined by immunohistochemistry. Arrows indicate patients with alterations in both <i>PIK3CA</i> and PTEN. (B) Frequency and overlap of PI3K pathway alterations in ER+ breast cancer samples. Biomarker frequencies calculated only from patients where tissue was evaluable for all biomarker assays. The data from the single <i>PIK3CA</i> exon 4 mutant sample was pooled with the exon 9 data, and the data from the exon 9/20 double mutant samples were pooled with exon 20 data. (C) Scatterplot of PTEN protein levels indicated by H-score in primary and metastatic samples. The solid diagonal line (y = x) and the dashed lines (y = x±50, y = x±100) are shown to highlight the magnitude of the absolute differences between x and y axes.</p

PIK3CA, AKT1, PTEN and Ki67 status across a collection of 77 matched ER+ breast cancers.

<p>MND - Mutation not detected; E545X - E545A, G, D, K; Q546X - Q546E, K, R, L; Positive - PTEN positive, unable to determine H-Score; NA - Not available.</p

Ki67 protein and gene expression analysis.

<p>(A) <i>MKI67</i> mRNA expression levels and relationship to Ki67 protein staining levels as determined by IHC. (B) Differentially expressed genes associated with Ki67 high or low protein staining levels (p-values indicated). Ki67 ≤ 15%, N = 16, Ki67 > 15%, N = 48.</p

Gene expression correlations between matched primary and metastatic ER+ breast cancer tumor samples.

<p>(A) Gene expression correlations between 61 matched primary and metastatic tumor samples for 90 genes from the breast cancer gene expression assay. Each dot represents the mean fold change between primary and metastatic samples for a single gene. (B) Correlation plots of genes that showed a greater than 1.5 fold difference in expression between matched primary and metastatic samples (FDR-adjusted P<0.05). Each dot represents the fold change between primary and metastatic samples for a single patient. The solid diagonal line (y = x) and the dashed lines (y = x±1) are shown to highlight the magnitude of the absolute differences between x and y axes.</p

Box-plots indicating genes affected by an overall treatment effect (oxaliplatin (n = 38) vs. oxaliplatin + bev (n = 23)).

<p>Two-way ANOVA with treatment and SD status as factors was used. P-value and FDR < 0.05 are indicated in each box plot graph <b>(A-C)</b>. OX = oxaliplatin, Bev = bevacizumab.</p

Box-plots indicating genes affected by treatment effect among CRML patients with no or mild SD only (oxaliplatin (n = 26) vs. oxaliplatin + bev (n = 22)).

<p>Two-way ANOVA with treatment and SD status as factors was used. P-value and FDR < 0.05 are indicated in each box plot graph <b>(A-E)</b>. OX = oxaliplatin, Bev = bevacizumab.</p

PCA and hierarchical clustering of NHP FFPE LCM hepatic regions and whole liver samples with or without anti-DLL4-associated SD based on their gene expression with FDR cut off at 0.25.

<p>Results from microfluidic high-throughput RT-qPCR were normalized by calculating–ΔCt using the average of reference genes. PCA <b>(A)</b> and hierarchical clustering based on Pearson correlation <b>(B)</b> were undertaken using genes with differential expression (FDR<0.25) between the LCM hepatic regions derived from liver samples of NHPs with SD present (moderate SD severity score 2 (anti-DLL4 15 mg/kg) n = 6)) and absent (SD severity score 0 (vehicle) (n = 6)) LCM hepatic regions. PCA <b>(C)</b> and hierarchical clustering based on Pearson correlation <b>(D)</b> were undertaken using genes with differential expression (FDR<0.25) between whole liver samples of NHPs with SD present (mild to severe SD severity scores 1–3 (anti-DLL4 5–50 mg/kg) (n = 17)) and absent (SD severity score 0 (vehicle or anti-DLL4 5 mg/kg) (n = 7)). PCA data are presented as spheres in PC1, PC2 and PC3 3 dimensional (3-D) space. The axes have been rotated to highlight the separation of the distinct clusters. The blue spheres surrounded with blue line represent the samples without SD and the red spheres surrounded with red line represent the samples with SD. The silver, blue, and pink axes represent principal components 1, 2 and 3 respectively <b>(A and C)</b>. In hierarchical clustering, each row represents a gene and each column represents a sample. Red squares indicate high gene expression; green squares indicate low gene expression <b>(B and D)</b>.</p