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PDF file - 680K, Overall Survival and Bcl-xL H Score.

To examine the potential of combining Bcl-2 family inhibitors with chemotherapy in ovarian cancer, we evaluated a panel of 27 ovarian cancer cell lines for response to the combination of navitoclax (formerly ABT-263) and paclitaxel or gemcitabine. The majority of cell lines exhibited a greater than additive response to either combination, as determined by the Bliss independence model, and more than 50% of the ovarian cell lines exhibited strong synergy for the navitoclax/paclitaxel combination. To identify biomarkers for tumors likely to respond to this combination, we evaluated the protein levels of intrinsic apoptosis pathway components. Bcl-xL seems necessary, but not sufficient, for navitoclax/paclitaxel synergy in vitro, suggesting that exclusion of patients whose tumors have low or undetectable Bcl-xL would enrich for patients responsive to the combination. We evaluated Bcl-xL levels in ovarian cancer tumor tissue from 40 patients (20 taxane responsive and 20 with poor response to taxane) and found that patients with high Bcl-xL were less sensitive to taxane treatment (10 of 12) Bcl-xL positive patients, P = 0.014). These data support the use of navitoclax in combination with taxane-based therapy in ovarian cancer patients with high levels of Bcl-xL. Mol Cancer Ther; 11(4); 1026–35. ©2012 AACR.

PDF file - 575K, ROC for Bcl-xL H Score.

PDF file - 69K, H Score and Clinical Features.

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Purpose: Chk1 inhibition potentiates DNA-damaging chemotherapy by overriding cell-cycle arrest and genome repair. This phase I study evaluated the Chk1 inhibitor GDC-0425 given in combination with gemcitabine to patients with advanced solid tumors.Experimental Design: Patients received GDC-0425 alone for a 1-week lead-in followed by 21-day cycles of gemcitabine plus GDC-0425. Gemcitabine was initially administered at 750 mg/m2 (Arm A), then increased to 1,000 mg/m2 (Arm B), on days 1 and 8 in a 3 + 3 + 3 dose escalation to establish maximum tolerated dose (MTD). GDC-0425 was initially administered daily for three consecutive days; however, dosing was abbreviated to a single day on the basis of pharmacokinetics and tolerability. TP53 mutations were evaluated in archival tumor tissue. On-treatment tumor biopsies underwent pharmacodynamic biomarker analyses.Results: Forty patients were treated with GDC-0425. The MTD of GDC-0425 was 60 mg when administered approximately 24 hours after gemcitabine 1,000 mg/m2. Dose-limiting toxicities included thrombocytopenia (n = 5), neutropenia (n = 4), dyspnea, nausea, pyrexia, syncope, and increased alanine aminotransferase (n = 1 each). Common related adverse events were nausea (48%); anemia, neutropenia, vomiting (45% each); fatigue (43%); pyrexia (40%); and thrombocytopenia (35%). The GDC-0425 half-life was approximately 15 hours. There were two confirmed partial responses in patients with triple-negative breast cancer (TP53-mutated) and melanoma (n = 1 each) and one unconfirmed partial response in a patient with cancer of unknown primary origin.Conclusions: Chk1 inhibition with GDC-0425 in combination with gemcitabine was tolerated with manageable bone marrow suppression. The observed preliminary clinical activity warrants further investigation of this chemopotentiation strategy. Clin Cancer Res; 23(10); 2423–32. ©2016 AACR.

Supplementary Data from Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging

Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRα ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRα was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRα-mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRα in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.

Supplemental Figure 3. Clinical trial tumor immunohistochemistry. Representative images from tumor biopsies were analyzed for pCDK1/2 and Ki-67expression. Representative images from a patient with NSCLC and unknown p53 mutation status were obtained 24 hours after administration of GDC-0425 alone (A, B), 48 hours after gemcitabine alone (C, D) and at a time point that was both 24 hours after GDC-0425 and 48 hours after gemcitabine (E, F). Quantitation of percent tumor cell Ki-67 positivity varied minimally between biopsies (G); pCDK1/2 increased from baseline after gemcitabine treatment, whereas the combination of GDC-0425 given 24 hours after gemcitabine suppressed the increase in pCDK1/2. Scale bar=40 µm.

CCR Translation for the Article from Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials.Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab.Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12.Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)

Purpose: Multiple endocrine neoplasia type 1 (MEN1) is defined clinically by the combined occurrence of multiple tumors, typically of the parathyroid glands, pancreatic islet cells, and anterior pituitary gland. A mouse model with a heterozygous deletion of the Men1 gene recapitulates the tumorigenesis of MEN1. We wished to determine the role of vascular endothelial growth factor (VEGF)-A in the vascularization and growth of MEN1-associated tumors, with an emphasis on pituitary adenomas.Experimental Design: To investigate whether tumor growth in Men1+/− mice is mediated by VEGF-A dependent angiogenesis, we carried out a monotherapy with the anti–VEGF-A monoclonal antibody (mAb) G6-31. We evaluated tumor growth by magnetic resonance imaging and assessed vascular density in tissue sections. We also measured hormone levels in the serum.Results: During the treatment with mAb G6-31, a significant inhibition of the pituitary adenoma growth was observed, leading to an increased mean tumor doubling-free survival compared with mice treated with a control antibody. Similarly, the growth of s.c. pituitary adenoma transplants was effectively inhibited by administration of anti–VEGF-A mAb. Serum prolactin was lowered by mAb G6-31 treatment but not by control antibody, potentially providing a new therapeutic approach for treating the hormonal excess in MEN1 patients. Additionally, the vascular density in pancreatic islet tumors was significantly reduced by the treatment.Conclusions: These results suggest that VEGF-A blockade may represent a nonsurgical treatment for benign tumors of the endocrine system.

Study design.

Supplemental Figure 2. Preclinical xenograft immunohistochemistry. HT-29 xenografts were analyzed for pCDK1/2 and Ki-67 expression. Representative images from 2 unique animals in each treatment group (n=5 animals per group) are shown. Ki-67 expression in surviving tumor cells is relatively consistent among treatment groups. pCDK1/2 expression increases from baseline (A, B) after gemcitabine treatment (I, J), whereas GDC-0425 alone (E, F) had little effect. In contrast, GDC-0425 given 16 hours after gemcitabine caused marked inhibition of the gemcitabine-induced expression of pCDK1/2. Scale bar=90 µm.

Supplementary Figures S1-S2 from Blocking Vascular Endothelial Growth Factor-A Inhibits the Growth of Pituitary Adenomas and Lowers Serum Prolactin Level in a Mouse Model of Multiple Endocrine Neoplasia Type 1

Supplementary Figure 1 from Tumor-Driven Paracrine Platelet-Derived Growth Factor Receptor α Signaling Is a Key Determinant of Stromal Cell Recruitment in a Model of Human Lung Carcinoma

Members of the TGF β , Wnt and FGF families act in concert to induce and pattern the mesoderm of gastrulating embryos. Downstream effectors for these growth factors include homeobox proteins, which also feed back to activate and repress upstream signaling pathways (e.g. Fainsod, A., Steinbeisser, H., De Robertis, E.M. 1994. On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13, 5015–5025; Carnac, G., Kodjabachian, L., Gurdon, J.B., Lemaire, P. 1996. The homeobox gene Siamois is a target of the Wnt dorsalization pathway and triggers organizer activity in the absence of mesoderm. Development 122, 3055–3065). As well as having interwoven upstream and downstream regulatory pathways Mix.1 , siamois and goosecoid , all paired- type homeobox genes, may physically interact with each other as heterodimers to regulate dorsal-ventral polarity (Mead, P.E., Brivanlou, I.H., Kelley, C.M., Zon, L.I. 1996. BMP-4 responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature 382, 357–360). We report here a chicken paired- type homeobox gene, CMIX , with a homeodomain having 72% aa identity to its nearest homolog, Xenopus Mix.1 . CMIX is expressed in the epiblast of the posterior marginal zone of early chick embryos, and later along the entire anterior-posterior axis of the primitive streak in cells of the medial ectoderm, in nascent mesoderm, but not in endoderm. Coincident with formation of prechordal mesoderm, CMIX mRNA levels rapidly decline throughout the embryo.

Imaging of tumor microvasculature has become an important tool for studying angiogenesis and monitoring antiangiogenic therapies. Ultrasmall paramagnetic iron oxide contrast agents for indirect imaging of vasculature offer a method for quantitative measurements of vascular biomarkers such as vessel size index, blood volume, and vessel density (Q). Here, this technique is validated with direct comparisons to ex vivo micro-computed tomography angiography and histologic vessel measurements, showing significant correlations between in vivo vascular MRI measurements and ex vivo structural vessel measurements. The sensitivity of the MRI vascular parameters is also demonstrated, in combination with a multispectral analysis technique for segmenting tumor tissue to restrict the analysis to viable tumor tissue and exclude regions of necrosis. It is shown that this viable tumor segmentation increases sensitivity for detection of significant effects on blood volume and Q by two antiangiogenic therapeutics [anti-vascular endothelial growth factor (anti-VEGF) and anti-neuropilin-1] on an HM7 colorectal tumor model. Anti-vascular endothelial growth factor reduced blood volume by 36±3% (p0.0001) and Q by 52±3% (p0.0001) at 48 h post-treatment; the effects of anti-neuropilin-1 were roughly half as strong with a reduction in blood volume of 18±6% (p0.05) and a reduction in Q of 33±5% (p0.05) at 48 h post-treatment.

Tissue microarrays enable the rapid histological localization of gene expression in hundreds of archival samples by in situ hybridization. However, the scoring of tissue microarray data may be influenced by intra- and inter-observer variations, and categorizing continuous variables risks discarding potentially meaningful information. Quantitation imposes a greater degree of objectivity, is more reproducible than subjective discriminations, and facilitates the communication and clarity of definitions. Phosphorimaging has been successfully used to quantitate the hybridization signal intensity from arrayed tissues. The process is rapid and has a wide dynamic range, surpassing the densitometric analysis of autoradiograms. This paper presents a detailed method for quantitative isotopic in situ hybridization on formalin-fixed paraffin-embedded tissue microarrays. In addition, the method includes a protocol for the development of synthetic agarose cores to control for the specificity and sensitivity of hybridization.

We present a technique, multiplex display polymerase chain reaction (MD-PCR), that amplifies and resolves coding sequences from messenger RNAs sharing only a single moderately conserved domain encoding eight or nine amino acids. The technique, a form of single-sided PCR, allows detection of known and novel genes in a family by using one degenerate primer complementary to a gene family-specific domain. A second common primer is complementary to an oligonucleotide ligated to a nearby restriction enzyme cleavage site. Uniquely, restriction enzyme digestion of single-stranded cDNA, a technique never previously performed to useful advantage, is used to increase the specificity and sensitivity of the technique. Up to several hundred bases of coding sequence are amplified simultaneously from many (potentially from all) genes in a specific family, yielding products of different sizes from different genes, and allowing amplified products to be resolved electrophoretically. Typically, more than 50% of the amplified sequences are from the targeted gene family and many of the amplified products are novel sequences. mRNAs representing less than 1 in 100,000 messages can be detected. The method allows the focused yet open-ended examination of genes in families known to be important in both normal cellular homeostasis and the etiology of many diseases.