Steve Anderson

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or SrcK, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src-/- mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.

Constitutive expression of microRNA-150 in mammary epithelium suppresses secretory activation and impairs de novo lipogenesis

Profiling of RNA from mouse mammary epithelial cells (MECs) isolated on pregnancy day (P)14 and lactation day (L)2 revealed that the majority of differentially expressed microRNA declined precipitously between late pregnancy and lactation. The decline in miR-150, which exhibited the greatest fold-decrease, was verified quantitatively and qualitatively. To test the hypothesis that the decline in miR-150 is crucial for lactation, MEC-specific constitutive miR-150 was achieved by crossing ROSA26-lox-STOP-lox-miR-150 mice with WAP-driven Cre recombinase mice. Both biological and foster pups nursed by bitransgenic dams exhibited a dramatic decrease in survival compared with offspring nursed by littermate control dams. Protein products of predicted miR-150 targets Fasn, Olah, Acaca, and Stat5B were significantly suppressed in MECs of bitransgenic mice with constitutive miR-150 expression as compared with control mice at L2. Lipid profiling revealed a significant reduction in fatty acids synthesized by the de novo pathway in L2 MECs of bitransgenic versus control mice. Collectively, these data support the hypothesis that a synchronized decrease in miRNAs, such as miR-150, at late pregnancy serves to allow translation of targets crucial for lactation. Summary: A decline in miR-150 is critical for lactation, as pups nursed by dams in which this decline is prevented exhibit a dramatic decrease in survival compared with littermate controls.

Abstract 1549: Development and validation of a biomarker for prospective selection of Notch1 activation in patients with certain advanced solid tumors in a first-in-human phase1 study of the cancer stem cell targeting antibody OMP-52M51 (anti-Notch1)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The Notch pathway plays a key role in embryonic development, the regulation of stem and progenitor cells, and is implicated centrally in many forms of human cancer. Notch1 is known to be frequently activated in certain solid tumor types. OMP-52M51 is a humanized IgG2 antibody that inhibits the signaling function of the Notch1 receptor. Mouse xenograft studies using minimally-passaged, patient-derived xenografts have shown that OMP-52M51 impedes tumor growth and selectively eliminates CSCs in a range of tumor types particularly in tumors with activated Notch1 signaling. We previously reported the frequency of Notch pathway activation across a large panel of human tumors (n>600) by an Immunohistochemistry (IHC) assay that detects the activated form of Notch1 using an antibody that specifically recognizes the Notch1 intracellular domain (ICD). Using this test and a rigorous H-score cut-off, we found elevated Notch1 ICD in 7-53% of the following cancers: chemo-resistant breast (29%), gastric (13%), cholangiocarcinoma (20%), esophageal (27%), hepatacellular carcinoma (HCC, 7%), small cell lung cancer (SCLC, 12%), pancreatic (12%) and colorectal cancer (53%). Here we developed a specific CLIA-validated IHC assay capable of identifying patients with Notch1 activation. The assay has a rapid turn-around time and shows robust precision, reproducibility and accuracy. The sensitivity of the method was confirmed by comparing results obtained on xenograft tumor tissue samples with known levels of Notch1 expression and response to anti-Notch1 therapy. From these results, the cut-off of the assay was set. This predictive biomarker was specifically designed to screen patients for prospective selection in the first-in-human Phase1 study of OMP-52M51 (anti-Notch1) in certain advanced solid tumors ([NCT01778439][1]). Detailed assay validation data and its application to the analysis of clinical trial samples will be highlighted. Citation Format: Belinda Cancilla, Raymond Tam, Chun Zhang, Steve Anderson, John Lewicki, Tim Hoey, Bryan McCune, Lori Johnson, Esohe Idusogie, Ann M. Kapoun. Development and validation of a biomarker for prospective selection of Notch1 activation in patients with certain advanced solid tumors in a first-in-human phase1 study of the cancer stem cell targeting antibody OMP-52M51 (anti-Notch1). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1549. doi:10.1158/1538-7445.AM2015-1549 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01778439&atom=%2Fcanres%2F75%2F15_Supplement%2F1549.atom

Abstract 2757: Using liquid biopsies and NGS as tools to analyze mutation burden and copy number variation in the blood of a patient with triple negative breast cancer to better inform therapeutic targets

The ability to characterize molecular features of cancer from liquid biopsies is resulting in the development of innovative health care for patients. Longitudinal changes in the mutational profiles of DNA isolated from liquid biopsies are being used to better understand and monitor the development, progression, and evolution of therapy resistance in cancer patients. To define changes in the mutational landscape and predict drug susceptibilities in Triple Negative Breast Cancer (TNBC) patients, we used whole exome analysis to profile circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from eight selected time points of a patient enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001). The patient initially received weekly cisplatin infusions followed by additional targeted therapy. Peripheral blood samples were collected at specific time points over a period of 272 days following enrollment in the clinical trial. Our data indicates that the identified mutations in genomic DNA isolated from CTCs and ctDNA can be used to understand and mitigate the impact of tumor heterogeneity in addition to identifying clinically relevant mutations at these selected time points. To further increase the resolution of our analysis, we profiled ctDNA from these samples to a higher depth targeting only clinically relevant genes. These analyses increased the sensitivity of detection and identified additional targets that could have been used for therapeutic intervention. In addition to sequence variants, copy number variations (CNVs) have also been significantly associated with the development of metastasis and changes in CNVs have been used to monitor disease progression. We performed a bioinformatics analysis of genomic instability and CNVs across 32 different time points from ctDNA from the same patient throughout the treatment period. The genomic instability number (GIN) calculated for each of the 32 time points seems to mirror the overall CTC burden in the patient at each time point tested. CNV analysis is ongoing and these data sets are being further analyzed in combination with TCGA data to define possible cancer driver genes for the functional prediction of significant TNBC candidate alterations and the results of these analyses will be presented. Citation Format: Kellie Howard, Kimberly Kruse, Brianna Greenwood, Elliott Swanson, Mathias Ehrich, Christopher K. Ellison, Taylor Jensen, Sharon Austin, Arturo Ramirez, Debbie Boles, John Pruitt, Elisabeth Mahen, Jackie L. Stilwell, Eric P. Kaldjian, Michael Dorschner, Sibel Blau, Marcia Eisenberg, Steve Anderson, Anup Madan. Using liquid biopsies and NGS as tools to analyze mutation burden and copy number variation in the blood of a patient with triple negative breast cancer to better inform therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2757. doi:10.1158/1538-7445.AM2017-2757

Abstract 1394: RT-PCR and NGS comparison: detection of known EGFR mutations in non-small-cell lung cancer clinical samples with routine laboratory testing

Next-generation sequencing (NGS) promises to deliver clinical mutation analysis for multiple actionable biomarkers at once. However, most targeted therapies are developed with companion diagnostic assays after rigorous analytical and clinical validation. In order to compare the consistency of biomarker results under standard laboratory workflow, we measured the detection of known EGFR mutations in 17 clinical and 2 reference samples using the cobas® EGFR Mutation Test version 1(cobas test), the TruSeq and TruSight panels using the Illumina MiSeq and AmpliSeq panel using the Thermo Fisher PGM instruments. Samples were tested in triplicate at 2 sites for each platform with 2 sites performing cobas testing and sequencing. Archived clinical samples were NSCLC adenocarcinoma with average tumor cell content at 60.3% ± 21.0% and mutant allele frequency of 36.0% ± 17.0% based on pyrosequencing during initial clinical testing. Three samples had scar tissue present and the remainder had little to no necrosis. The average age was 67.6 ± 7.0 years and samples came from 10 women, 6 men and 1 gender not specified. Data were analyzed to assess the inter- and intra- platform discordant rates, the mean error rates relative to the known mutation status, platform invalid rates, average turnaround time and repeat testing rates. All platforms had low discordant and mean error rates when using the 2.5% mutation frequency cutoff and a read depth (RD) limit = 100. One PGM site had a sample invalid rate between 20%-51% for different EGFR amplicons at RD = 100; the upper limit of the invalid rate for PGM sites was >70% with RD = 500. The cobas z 480 platform had a 0-0.32% intra-platform discordant rate and a 0.65-0.93% mean error rate. The MiSeq platform had a 0-0.69% intra-platform discordant rate and a 0-0.69% mean error rate. The PGM platform had a 0.99-3.15% intra-platform discordant rate and a 1.5-4.02% mean error rate. The mean numbers of all non-silent COSMIC mutations detected by PGM and MiSeq at the 2.5% cutoff were 4.36 and 2.32, respectively. However, the mean number of EGFR mutations detected relative to the expected number was 100% for MiSeq and 92.4% and 100% for PGM at 2.5% and 10% frequency cutoffs, respectively. The 3 platforms were assessed for samples that required repeat testing, re-extraction of DNA and the turnaround time (TAT) from DNA isolation to result. In all three cases, the cobas test required the least TAT, fewer repeats and re-extractions relative to PGM and MiSeq. One MiSeq site required the longest turnaround time (14 days) but did not require repeat testing for invalid runs. Re-extraction from backup sections was common for both PGM and MiSeq but not cobas. The cobas test provides accurate, precise, and fast actionable results for NSCLC patients. NGS approaches can result in accurate and precise results when adequate RD is achieved but TAT remains a challenge relative to the cobas test. Citation Format: John F. Palma, Yu Chuan Tai, Wei-min Liu, Steve Anderson, Anup Madan, John W. Longshore, Arundhati Rao. RT-PCR and NGS comparison: detection of known EGFR mutations in non-small-cell lung cancer clinical samples with routine laboratory testing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1394.

Abstract P2-02-21: Longitudinal analysis of circulating tumor cells and cell free tumor DNA by next generation sequencing in triple negative breast cancer

As the practice of genetically profiling patient tumors is considered for making clinical treatment decisions, recent methodologies for screening of genomic aberrations in circulating tumor cells (CTCs) and cell-free plasma DNA (cfDNA) may provide non-invasive tools for such applications. Genomic analysis of DNA from CTCs and plasma can also provide useful insight into tumor heterogeneity and thus disease progression by revealing sub-populations of tumor cells that evolve during treatment, have novel drug-resistant genotypes, or carry alternate cancer driver mutations not identified by the sequencing of primary tumors. Comprehensive evaluation of DNA isolated from CTCs and cfDNA from a breast cancer patient by whole exome sequencing was performed to better understand the role of liquid biopsies in investigating the etiology of tumor progression. The patient was diagnosed with metastatic triple negative breast cancer (TNBC) six years after remission from estrogen receptor (ER-3+), progesterone receptor (PR-1+), human epidermal receptor growth factor 2 negative (Her2-), grade 3 intra-ductal carcinoma of the right breast. Metastatic lesions were found in the spine, pelvis and sacrum and bone-marrow. The patient was enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001; ClinicalTrials.gov ID NCT01957514) and initially received weekly cisplatin infusions followed by additional targeted therapy. Peripheral blood was obtained during regular clinic visits over the 272 days the patient was enrolled in the clinical trial. CTCs were identified and enumerated from each blood draw using the AccuCyte® -CyteFinder® (AC/CF) system (RareCyte, Seattle WA). Multiple CTCs along with white blood cells (WBCs) were picked from various time points throughout the treatment regimen. The selected CTCs and WBCs were whole genome amplified and whole exome sequencing was performed to identify tumor specific variants. A comparative analysis with variants present in genomic DNA isolated from the bone-marrow metastasis tissue biopsy samples and cfDNA revealed the evolution of tumor-specific variants during therapy. Each CTC had somatic alterations in genes associated with therapies in current use or those in the clinical trials setting. Sequencing analysis of cfDNA provided similar information on potential therapeutic approaches. The monitoring of disease over time through genomic analysis of CTCs and cfDNA can identify novel sub-populations related to disease progression for the tailoring of cancer treatment regimens. Further analysis is being performed to better understand the evolution of the genomic heterogeneity among CTCs at the same time point and across different time points and therefore better understand the etiology of progression of metastatic breast cancer in this patient. Citation Format: Howard K, Austin S, Ramirez AB, Ritter L, Boles D, Pruitt J, Collins H, Mahen E, Leonti A, Maassel L, Subia C, Tuuli S, Heying N, Deutsch K, Cox J, Lo FY, Stilwell JL, Kaldjian EP, Dorschner M, Blau S, Blau A, Eisenberg M, Anderson S, Madan A. Longitudinal analysis of circulating tumor cells and cell free tumor DNA by next generation sequencing in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-21.