Abstract R-Spondin (RSPO) proteins bind to LGR receptors and potentiate Wnt/β-catenin signaling. We have identified a therapeutic anti-RSPO3 antibody targeting the RSPO-LGR pathway. In preclinical studies, RSPO3 gene expression has shown correlation with anti-RSPO3 antibody efficacy in multiple solid tumor types. A qPCR-based RSPO3 assay has been developed as a predictive biomarker for response to the anti-RSPO3 antibody. In addition, RSPO gene fusions may play a role in the activation of Wnt signaling. A gene fusion detection workflow consisting of a RSPO3 CLIA assay, a RSPO3 RUO assay and next generation sequencing (NGS) has also been developed. We designed 6 qPCR-based assays for the RSPO3 CLIA assay development and 2 assays for the RUO assay. These assays were designed to span exon-exon junctions or target microarray probe set sequences. Amplification sensitivity and specificity were assessed for assay selection. The analytic performance of the candidate RSPO3 CLIA assay and quality control measures were established in a validation study. The validation study included: 1) performance specifications of the RSPO3 assay including analytical sensitivity, linearity, and precision, 2) determination of a reportable range, 3) establishment of a cut-off for the RSPO3 CLIA assay for patient selection, and 4) establishment of quality control procedures. 104 human cancer tissues and 24 independent patient-derived tumor xenografts (PDX) were used in these studies. To evaluate the fusion detection workflow, the RUO assay was performed on samples that tested above the CLIA assay cut-off. The delta Ct difference between the CLIA and RUO assays was calculated to identify potential fusions. The limit of quantification was established for the RSPO3 CLIA assay. The 95% reference interval was estimated to be (-2.44, 16.02) with 90% confidence interval for the lower bound (-3.45, -2.12) and upper bound (15.26, 16.57). The delta Ct cut-off for the RSPO3 CLIA assay was set based on sensitivity, specificity and prevalence. No statistically significant difference in the total variance across the tested samples was observed. A549 and OV56 were identified to be cell line controls with established acceptable delta Ct limits. Using NGS, RSPO3 fusions were identified in 6 PDX tumors with delta Ct RUO - delta Ct CLIA>7, including a novel fusion. This cut-off was further refined with NGS of 9 clinical samples. Prevalence of the RSPO3 expression and fusions will be presented. A qPCR based RSPO3 assay was developed and CLIA-validated for use as a potential predictive biomarker for response to anti-RSPO3 therapy. This RSPO3 CLIA assay, together with the fusion detection workflow, will be evaluated in a Phase 1a/b dose escalation study of anti-RSPO3 (OMP-131R10) in advanced solid tumors and in combination with FOLFIRI in metastatic colorectal cancer (NCT02482441). Citation Format: Chun Zhang, Yuwang Liu, Min Wang, Gilbert OYoung, Joy Kavanagh, Cheryl McFarlane, Fiore Cattaruzza, Pete Yeung, Jennifer Cain, Wan-Ching Yen, Marcus Fischer, Belinda Cancilla, Edwina Dobbin, Michelle McCarthy, Austin Gurney, Leonardo Faoro, John Lewicki, Tim Hoey, Ann M. Kapoun. Development of a RSPO3 CLIA-validated assay as a predictive biomarker for response to anti-RSPO3 antibody treatment in patients with solid tumors. [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 404.
BackgroundCells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects.ResultsHere, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased "in situ transcriptomics" analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma.ConclusionsIn addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy.
PCR detects clonal rearrangements of the Ig gene in lymphoproliferative disorders. False negativity occurs in germinal centre/post-germinal centre lymphomas (GC/PGCLs) as they display a high rate of somatic hypermutation (SHM), which causes primer mismatching when detecting Ig rearrangements by PCR.To investigate the degree of SHM in a group of GC/PGCLs and assess the rate of false negativity when using BIOMED-2 PCR when compared with previously published strategies.DNA was isolated from snap-frozen tissue from 49 patients with GC/PGCL (23 diffuse large B cell lymphomas (DLBCLs), 26 follicular lymphomas (FLs)) and PCR-amplified for complete (VDJH), incomplete (DJH) and Ig kappa/lambda rearrangements using the BIOMED-2 protocols, and compared with previously published methods using consensus primers. Germinal centre phenotype was defined by immunohistochemistry based on CD10, Bcl-6 and MUM-1.Clonality detection by amplifying Ig rearrangements using BIOMED-2 family-specific primers was considerably higher than that found using consensus primers (74% DLBCL and 96% FL vs 69% DLBCL and 73% FL). Addition of BIOMED-2 DJH rearrangements increased detection of clonality by 22% in DLBCL. SHM was present in VDJH rearrangements from all patients with DLBCL (median (range) 5.7% (2.5-13.5)) and FL (median (range) 5.3% (2.3-11.9)) with a clonal rearrangement.Use of BIOMED-2 primers has significantly reduced the false negative rate associated with GC/PGCL when compared with consensus primers, and the inclusion of DJH rearrangements represents a potential complementary target for clonality assessment, as SHM is thought not to occur in these types of rearrangements.
Abstract: Objective : The mutational status of the immunoglobulin (Ig) V H gene in B‐cell chronic lymphocytic leukaemia (B‐CLL) identifies two subgroups of patients with significantly different outcomes. We investigated the association of ZAP‐70 expression with IgV H mutational status in B‐CLL by quantifying ZAP‐70 mRNA, to evaluate its use as a surrogate marker for mutational status. The aim of this study was to develop a quantitative reverse transcriptase‐polymerase chain reaction (RQ‐PCR) assay for the detection of ZAP‐70 expression in a group of patients whose mutational status and cytogenetics had been determined previously. Methods : RQ‐PCR was used to analyse ZAP‐70 expression from 42 B‐CLL patients. B cells were purified using CD19 magnetic bead system and total RNA was isolated. RQ‐PCR was performed using Taqman PCR. Results : Twenty‐five patients (60%) had mutated and 17 (40%) had unmutated IgV H genes; 94% (16/17) of patients with unmutated IgV H gene were ZAP‐70 positive as assessed by RQ‐PCR and 92% (23/25) of patients with mutated IgV H gene were ZAP‐70 negative. In three patients, ZAP‐70 expression and IgV H mutational status were discordant. Conclusion : This paper describes an RQ‐PCR assay for the detection of ZAP‐70 expression and confirms that IgV H unmutated CLL cells have a high expression of ZAP‐70 in comparison with IgV H mutated CLL. This robust method acts as a surrogate marker for IgV H mutational status albeit with <100% concordance. However, it does provide better concordance with mutational status than that reported using flow cytometry.
Wnt signaling plays several roles in hematopoiesis, promoting hemopoietic stem cell (HSC) self-renewal, providing proliferative signals for immature progenitors and regulating lineage commitment. To ascertain which Wnt proteins and receptors are important during hematopoietic development, we used two systems; in vitro hematopoietic differentiation of embryonic stem (ES) cells and tissues isolated from sites specific for hematopoiesis during mouse embryogenesis. Initially genes involved in hematopoiesis were profiled and indicate differentiating ES cells undergo a wave of primitive hematopoiesis (Day 3.75) similar to the mouse yolk sac, followed by a wave of more definitive hematopoiesis (Day 7.75) comparable to the aorta-gonad-mesonephros (AGM) and E15.5 liver with lineage commitment by Day 15. A similar biphasic expression pattern occurred for Wnt/Fzd/LRP genes with Wnt 3, 5a, 8a, Fzd4, and LRP5 becoming upregulated during primitive hematopoiesis, followed by Wnt3a, 6, 7b, 10b, and 16 during more definitive hematopoiesis. High expression of Wnt5a, Fzd4, and LRP5 during the first phase of hematopoiesis suggests these genes are involved in early hematopoietic regulation. Wnt3a and 16 were also expressed at specific stages, with Wnt16 detected when the earliest lymphoid progenitors are formed (AGM and 2°BC of ES differentiation). Wnt3a expression corresponded with the induction of definitive hematopoiesis a period, which involves rapid expansion of HSC (Day 7.75 of ES differentiation, AGM and E15.5 liver). Supplementation with Wnt3a during ES hematopoietic differentiation increased proliferation and appeared to promote stem cell expansion. Overall this study provides valuable information on the Wnt/Fzd/LRP involved in supporting embryonic hematopoiesis.
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