Chumsri, S

Abstract Background: Tumor endothelial cells (ECs) are a population of stromal cells required for tumor growth that cooperate with tumors to form angiogenic structures. In blood, circulating ECs (CECs) are normal constituents of healthy individuals, although a Cancer Associated Vascular Endothelial cell (CAVE) subtype has been observed in cancer patients. The CAVE population has been isolated and identified using their large size or multicellular clustering and a pooled mixture of classical EC markers (i.e. CD31 and CD146). However, there has been no attempt to differentiate CAVEs from the many EC subtypes. This is not surprising as in-depth phenotyping of ECs requires an array of biomarkers that until recently has not been feasible. A multi-phenotypic screening of EC markers was tested on CAVEs from 116 blood samples in 3 types of solid tumors. This data suggests that CAVEs exist as a common and diverse subtype of tumor derived CECs that may express cytokeratin (CK) and various EC biomarkers, correlating to disease stage. Methods: Peripheral blood samples from 116 cancer patients (stage I-IV) were drawn from 2012-2014 including breast (n=42), lung (n=39) and prostate (n=35), as well as blood from 34 healthy controls. Blood was processed by an established filtration approach, i.e. the CellSieveTM microfiltration technique (Creatv MicroTech), filtering blood by size exclusion and staining cells for CK 8, 18 & 19, EpCAM and CD45. After identification and imaging, the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique was used to remove fluorescence signal and restain all cells with CD31, CD146, CD144, & DAPI. After reimaging, QUAS-R was again used to remove fluorescence and restain the cells for CD14, CD105, CD34, & DAPI. Results: Out of 116 patient samples, we identified CAVEs in 63 patients (54%) based on positivity of CD31, CD144 or CD146, but none were found in healthy controls. CAVEs per 7.5mL sample in patients averaged 5.1 (breast), 5.6 (prostate) and 7.9 (lung). Presence of CAVEs appeared related to stage with 26% in stage 1, 61% in stage 2, 68% in stage 3, and 74% in stage 4 patients. No CAVEs were positive for CD14 or CD45. CD31 was the most present marker, found on 93% of CAVEs, followed by CD144 (85%), CD34 (64%), CD146 (45%), & CD105 (4%). In contrast with the previous study on this topic, CK was found in 67% of CAVEs, but was not a universal marker. Conclusions: It has been reported that CK+ and CD45- CECs are isolated from the blood of cancer patients in colon and lung cancers, prompting some to classify them as circulating tumor cells. However, subtyping these CECs is incomplete when characterized with only 3-4 biomarkers. A multi-phenotypic subtyping technique was used to properly identify and subtype these CECs in cancer patients. This data suggest that a subset of CECs, e.g. CAVEs, are found in circulation as CK+/CD45-, but exist as a heterogeneous population of cancer specific circulating cells that require further study. Citation Format: Daniel L. Adams, R. Katherine Alpaugh, Steven H. Lin, Jeffrey R. Marks, Raymond Bergan, Stuart S. Martin, Sarany Chumsri, Massimo Cristofanilli, Cha-Mei Tang, Steingrimur Stefansson. Identifying, subtyping and classifying tumor associated circulating endothelial cells in patients with solid tumors [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 778. doi:10.1158/1538-7445.AM2017-778

Abstract Background: Non-amplification ERBB2 alterations (ERBB2 mut) in advanced/metastatic breast cancer (mBC) are not detected by IHC or FISH, but when detected by DNA sequencing assays can lead to clinical responses to anti-HER2 targeted therapy. We queried a database of more than 43,000 clinical cases to uncover the frequency, type and associated genomic alterations (GA) in mBC driven by ERBB2 mut and highlight clinical responses to small molecule drug and antibody-based anti-HER2 therapeutics. Methods: DNA was extracted from 40 microns of FFPE sections from 5,605 mBC. Comprehensive genomic profiling (CGP) was performed using a hybrid-capture, adaptor ligation based next generation sequencing assay of up to 315 genes to a mean coverage depth of >600X. The results were analyzed for base substitutions, short insertions and deletions, selected rearrangements, and copy number changes. Results: 698 (12.5%) of 5,605 mBC featured ERBB2 alterations. 596 (10.6%) featured ERBB2 amplifications and 137 (2.4%) featured ERBB2mut. 35 (0.6%) of total mBC had both ERBB2amp and ERBB2mut, which accounted for 5.0% of all ERBB2 altered mBC. The 137 ERBB2mut mBC cases had a median age of 61 years (range 29 to 93 years) and were sequenced to a mean depth of 600X. Samples utilized for CGP included 52 (38%) from the patient's primary BC and 85 (62%) from metastatic sites including bone/soft tissue/skin (12%), liver (20%), LN (14%), serous cavities (6%), lung (4%) and miscellaneous sites (6%). 71 (52%) mBC were submitted as carcinoma NOS, 44 (32%) as IDC, 22 (16%) as ILC and 1 (1%) as mucinous mBC. Of the 137 ERBB2mut cases, 8 featured more than 1 ERBB2 mut. There were 124 (85%) ERBB2 kinase domain mutations and 15 (10%) extra-cellular domain ERBB2mut. The most common genes co-altered in ERBB2mut mBC were TP53 (49%), PIK3CA (42%), CDH1 (37%), MYC (17%), and CCND1 (16%). The enrichment of ERBB2mut in CDH1 mut mBR was significant (p=0.0006) and associated with relapsed lobular mBC. Multiple case examples of kinase domain and extra-cellular domain ERBB2mut mBC responding to a variety of anti-HER2 targeted therapies will be presented. Conclusions: In this large series of 5,605 mBC, 20% of the total ERBB2 alterations were non-amplification ERBB2mut not detectable by standard of care IHC and FISH slide-based HER2 tests. Given the demonstration of ERBB2mut driven mBC responsive to anti-HER2 targeted therapies in this study, expansion of clinical trials designed to detect these ERBB2mut cases with CGP and optimize the targeted therapies for these patients is strongly recommended. Citation Format: Ross JS, Wang K, Ali SM, Chumsri S, Elvin JA, Vergilio J-A, Suh J, Yelensky R, Lipson D, Chmielecki J, Miller VA, Stephens PJ. Non-amplification ERBB2 genomic alterations in 5,605 cases of refractory and metastatic breast cancer: An emerging opportunity for anti-HER2 targeted therapies. [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 P3-07-05.