Cunliffe, HE

Abstract Introduction: Triple negative breast carcinoma (TNBC) is a subtype of breast cancer with a paucity of therapeutic targets and a poor prognostic phenotype. In a retrospective cohort, we sought to determine the prevalence of the amplicon targeting the 9p24.1 locus, resulting in over-expression of PD-L1, PD-L2, and JAK2 (PDJ), and potentially actionable therapeutic targets. We then probed the genomic landscape of these PDJ positive tumors and identified co-occurring copy number aberrations, including focal amplifications and homozygous deletions. The presence of the PDJ amplicon and selected co-occurring aberrations provide a unique description of a clinically relevant subtype of TNBC. Methods: We evaluated fresh frozen and formalin-fixed paraffin embedded tumor samples from 64 patients with triple negative breast cancer whom underwent definitive surgical resection. Clinical annotation was available in 60 of the samples. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted from each biopsy using DNA content flow cytometry. Each sorted sample was interrogated with oligonucleotide array comparative genomic hybridization (aCGH). All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm. Results: We detected a high level (log2ratio greater than or equal to 2) amplicon targeting 9p24.1 in 18 of 64 patients (28%) genomic profiles with triple negative breast carcinoma. In the PDJ positive population, we detected 8 of 18 patients (44%) with co-amplification targeting myc at 8q24, 3 of 18 patients (17%) with co-amplifications targeting EGFR at 7p11, 3 of 18 patients (17%) with co-amplifications targeting PIK3CA. These co-occurring genomic events in PDJ positive tumors may provide clinically actionable targets. Other selected amplifications detected included NOTCH3, KRAS, RUNX1, TUBAL3, FGFR2, AKT1, AKT2, YPEL2, PBXL7, KIT. We detected PTEN homozygous deletion in 2 of 18 patients (11%) PDJ positive tumors. Other homozygous deletions identified in the genomic landscape included FAT1, SOX3, Park2, TNFAIP3, GPC3, RB1, and CREBBP. Conclusions: In our retrospective analysis, the amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in approximately 28% of triple negative breast cancer patients. The genomic landscape of these PDJ positive TNBCs include recurring high-level focal amplifications and targeted homozygous deletions of clinically relevant genes. The clinical implications of these data are under current investigation using model systems and are in early phase clinical trials. The efficacy of immune checkpoint inhibitors including nivolumab, pembrolizumab, and JAK2 inhibitors including ruxolitinib, in PDJ positive triple negative breast carcinoma is intriguing and remains to be elucidated. Citation Format: Gawryletz CD, Anderson KS, Cunliffe HE, Northfelt DW, McCullough AE, Lenkiewicz E, Malasi S, Pockaj BA, Barrett MT. The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma. [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 P6-03-13.

Abstract Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. Informatic analysis using the newly available “Gene Expression Based Outcome for Breast Cancer Online” (GOBO) tool indicated high levels of QSOX1 expression in Estrogen Receptor positive (ER+) subtypes of breast cancer. We confirmed this finding by evaluation of QSOX1 protein expression in breast tumors and in a panel of breast cancer cell lines. In addition, Kaplan Meyer analyses revealed QSOX1 as a significant predictive marker for both relapse and poor overall survival in Luminal B tumors, but not in other intrinsic subtypes. To investigate malignant cell mechanisms in which QSOX1 might play a key role, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in ER+ MCF7 and ER− BT549 breast cancer cell lines. Suppression of QSOX1 protein dramatically slowed cell proliferation but did not significantly effect apoptosis or cell cycle regulation. Inhibition of QSOX1 did however dramatically inhibit MCF7 and BT549 breast tumor cells from invading through Matrigel in a modified Boyden chamber assay. Inhibition of invasion could be rescued by the exogenous addition of recombinant QSOX1. Gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-9 a key mediator of breast cancer invasive behavior. Taken together, our results suggest that QSOX1 is a novel biomarker for risk of relapse and poor survival in Luminal B breast cancer, and has a pro-invasive role in malignant progression through post-translational activation of MMP-9. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-04.

Abstract Background: Estrogen receptor beta (ERβ), unlike ERα, classically functions as a tumor suppressor in vitro. However, ERβ's biological functions in vivo and predictive/prognostic value in breast cancer are controversial. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in the following 3 cohorts: 1) a cohort with all breast cancer subtypes (n = 182), 2) a prospective NCCTG adjuvant tamoxifen trial for postmenopausal women with ERα positive breast cancer with long-term follow-up (n = 198) and 3) a cohort of 80 triple negative breast cancers (TNBCs). To elucidate the biological functions of ERβ in breast cancer, novel ERβ expressing MCF7 and MDA-MB-231 cell lines were developed and characterized using multiple techniques and were examined for responsiveness to various ERβ targeted therapies. Results: About one-third of all breast tumors, regardless of sub-type, were shown to express nuclear ERβ and this expression was independent of ERα or HER2. In the NCCTG 89-30-52 cohort, breast cancer recurrence rates were significantly associated with ERβ protein expression with 10 year recurrence rates of 25%, 15% and 4% for zero, low or high levels of ERβ expression respectively. Interestingly, in TNBCs, nuclear ERβ was expressed at intermediate or high levels in 24% of tumors. In the triple negative MDA-MB-231 cell line, expression of ERβ led to inhibition of proliferation and induction of apoptosis in response to estrogen and multiple ERβ specific agonists. Conversely, these same treatments induced proliferation of ERβ-expressing MCF7 cells which endogenously express ERα. However, ERβ expression sensitized MCF7 cells to the anti-proliferative effects of anti-estrogens. Microarray analysis and RT-PCR profiling of MDA-MB-231-ERβ cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors which function as tumor suppressors, and potently inhibited canonical TGFβ signaling. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells suppressed the proliferation rates and inhibited TGFβ signaling in other TNBC cell lines, effects that were completely reversed following the depletion of cystatins from the conditioned media. Conclusions: These data demonstrate that ERβ is expressed in a substantial proportion of breast cancers and may have value as a predictive and/or prognostic biomarker. Therapeutic targeting of ERβ may have clinical benefit in multiple breast cancer sub-types; however, the specific drug of choice may vary based on ERα status. Specifically, we have demonstrated that ERβ expression in ERα+ MCF7 cells sensitizes them to the effectiveness of anti-estrogens, an observation that was confirmed in women enrolled in a prospective adjuvant tamoxifen trial. In TNBCs, where targeted therapies are lacking, our data suggest that targeting ERβ with either estrogen or ERβ specific agonists will elicit anti-tumor effects through the induction of cystatins and inhibition of TGFβ signaling resulting in tumor regression and improved patient outcomes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-03.

Abstract Background: The role of fusion genes and associated fusion transcripts has long been recognized in hematopoietic malignancies. Until quite recently it has been difficult to detect such events on a genomic scale in solid tumors. Consequently, little is known about the potential role of fusion genes, transcripts, and proteins as driver mutations, biomarkers, or therapeutic targets in breast cancer. Methods: We have developed a novel analytical pipeline, Snowshoes-FTD, for detection of fusion transcripts in breast cancer cell lines and tumor samples (Asmann, et al. NAR 2011; May 27 ePub ahead of print). Preliminary analyses have been carried out with a panel of 8 each ER+, HER2+, and triple negative (TN) primary breast tumors, 8 primary human mammary epithelial cell (HMEC) lines from biopsy samples, plus 16 normal tissues from the Illumina Body Map dataset. Results: We have identified 120 redundant, tumor-specific fusion transcripts, expressed in two or more tumors and in no non-transformed samples. Sixteen of these represent intrachromosomal fusions and 104 arise from fusion of transcripts that map to two different chromosomes. Every breast tumor expressed one or more fusion transcripts. Twenty-nine fusion transcripts appeared to be tumor subtype specific. Among these, we have identified 2 HER2+, 10 ER+, and 17 triple negative specific redundant transcripts. In general, HER2+ tumors expressed fewer fusion transcripts (range 4 to 28/tumor) compared to TN (range11 to 44/tumor). Chromosomal distribution patterns were also markedly different among the tumor subtypes. For example, ER+ tumors expressed a preponderance of redundant fusion transcripts that involve chr1 and 2, whereas TN tumors had no fusion transcripts that map to either chromosome. Conversely, the predominant locus for TN fusion transcripts was chr19, which contains only one HER2+ fusion and no ER+ fusion transcripts. Conclusions: Primary breast tumors express many chimaeric transcripts, which we presume to arise primarily from genomic rearrangements. The majority of these transcripts are redundant, and a subset are tumor subtype specific. These transcripts may mark regions of chromosomal instability. HER2+ tumors, in general, appear to evidence less chromosomal instability, as inferred from fusion transcripts; although some HER2+ tumors appear to be quite unstable. TN tumors contain many more redundant fusion transcripts, implying increased genomic instability, particularly in chr19. We conclude that these fusion transcripts represent a class of heretofore unrecognized biomarkers that may be used for sub-classification of breast tumors. Some of these transcripts appear to encode proteins that may function as tumor-subtype-specific driver mutations and may have potential as therapeutic targets in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-06-02.