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Supplementary Table 1. Details of the secretome array, listed by each probset.

Supplementary Table 4. Pathway associated probesets up-regulated in cancer as compared to both normal fallopian and ovarian surface epithelium.

Supplementary Table 3. List of pathways identified for the statistically significant up-regulated probesets in cancer as compared to normal ovarian surface epithelium.

Supplementary Table 5. Non-pathway associated probesets up-regulated in cancer as compared to both normal fallopian and ovarian surface epithelium.

Supplementary Table 2. List of pathways identified for the statistically significant up-regulated probesets in cancer as compared to normal fallopian tube.

Supplementary Methods

supplementary table legend

Purpose: To generate a comprehensive “Secretome” of proteins potentially found in the blood and derive a virtual Affymetrix array. To validate the utility of this database for the discovery of novel serum-based biomarkers using ovarian cancer transcriptomic data.Experimental Design: The secretome was constructed by aggregating the data from databases of known secreted proteins, transmembrane or membrane proteins, signal peptides, G-protein coupled receptors, or proteins existing in the extracellular region, and the virtual array was generated by mapping them to Affymetrix probeset identifiers. Whole-genome microarray data from ovarian cancer, normal ovarian surface epithelium, and fallopian tube epithelium were used to identify transcripts upregulated in ovarian cancer.Results: We established the secretome from eight public databases and a virtual array consisting of 16,521 Affymetrix U133 Plus 2.0 probesets. Using ovarian cancer transcriptomic data, we identified candidate blood-based biomarkers for ovarian cancer and performed bioinformatic validation by demonstrating rediscovery of known biomarkers including CA125 and HE4. Two novel top biomarkers (FGF18 and GPR172A) were validated in serum samples from an independent patient cohort.Conclusions: We present the secretome, comprising the most comprehensive resource available for protein products that are potentially found in the blood. The associated virtual array can be used to translate gene-expression data into cancer biomarker discovery. A list of blood-based biomarkers for ovarian cancer detection is reported and includes CA125 and HE4. FGF18 and GPR172A were identified and validated by ELISA as being differentially expressed in the serum of ovarian cancer patients compared with controls. Clin Cancer Res; 21(21); 4960–9. ©2015 AACR.

Background: Targeted therapy for HER2+ breast cancer has significantly improved outcomes for this aggressive subtype. However, a subset of patients do not achieve pathological complete response (pCR). In CALGB 40601, a randomized Phase III Trial for neoadjuvant treatment of HER2+ primary breast cancer with Paclitaxel (T: taxane) combined with HER2 antibody therapy (H: Herceptin/Trastuzumab), the small molecule inhibitor Lapatinib (L), or the antibody-inhibitor combination, pCR frequency was 56% for the combination (THL arm), 46% for Trastuzumab (TH arm), and 32% for Lapatinib (TL arm, closed early because of lower efficacy) (PMID: 26527775). While a recent publication reports relapse-free survival (RFS), overall survival (OS), and RNA-based gene expression signatures that can predict pCR (PMID: 33095682), understanding the proteogenomic landscape of treatment response should facilitate identification of alternative and therapeutically tractable protein targets for treatment-resistant tumors. Methods: Microscaled proteogenomic profiling (PMID: 31988290) was performed on treatment-naïve, flash-frozen core needle biopsies from the CALGB 40601 trial obtained from the Alliance for Clinical Trials in Oncology tissue bank. Multi-omics profiling included whole-exome sequencing (WES), RNA-sequencing, and mass spectroscopy-based proteomics and phosphoproteomics from one or two cores from each patient. Results: Eighty baseline core biopsies from 54 patients, including 22 patients from the THL arm, 24 from the TH arm, and 8 from the TL arm, from the CALGB 40601 tissue archive were of sufficient quality to yield genomics, transcriptomics, and/or proteomics profiling data. The frequency of pCR for profiled samples was representative of the overall trial cohort. Linear models were employed to identify baseline determinants of pCR for each arm and to assess differences in genes associated with response between the TH and THL arms. Pathways associated with RNA processing, translation, and the proteasome were elevated in pCR tumors in TH and THL arms, while cell cycle, DNA replication and repair pathways were higher in pCR only in the THL arm. While enrichment of similar pathways was observed in pCR in the transcriptome, the proteome specifically showed enrichment of pathways associated with extracellular matrix and EMT in non-pCR in the THL but not the TH arm. In particular, “EMT”, “ECM-receptor interaction”, and “extracellular structure organization” constituted the most enriched pathways and GO terms that were higher in non-pCR than in pCR tumors from the combination arm (THL) in the proteomics data despite showing no enrichment in the transcriptomics data. Driving this pathway enrichment were several collagens and matrix metalloproteinases that were significantly elevated in non-pCR tumors at the protein but not the RNA level. Finally, kinase target enrichment of differential phosphorylation sites suggested that the activity of PAK1, a regulator of cytoskeletal remodeling, is elevated in non-pCR tumors from the THL arm (p=0.006), but not the TH arm (p=0.69). Conclusion: Proteogenomic analysis of archival HER2+ breast cancer core biopsies provides opportunities for identifying proteins and phosphorylation sites in treatment-naive tumors that are associated with pCR to neoadjuvant Paclitaxel/anti-HER2 therapy. Notably, proteomic but not transcriptomic data showed that ECM and EMT pathways were elevated in non-pCR tumors; thus, signatures encompassing these pathways may serve as biomarkers for aggressive HER2+ breast cancer that is more likely to evade treatment. Non-pCR tumors in the THL arm were also marked by elevated levels of PAK1 target phosphorylation sites, suggesting that this kinase may be a potential therapeutic target in HER2+ breast cancer that is refractory to combination anti-HER2 therapy. Citation Format: Eric J. Jaehnig, Aranzazu Fernandez-Martinez, Tanmayi Vashist, Matthew V. Holt, LaTerrica Williams, Jonathan Lei, Beom-Jun Kim, Yongchao Dou, Viktoriya Korchina, Richard Gibbs, Donna Muzny, Harshavardhan Doddapaneni, Henry Rodriguez, Ana Robles, Tara Hiltke, DR Mani, Michael Gillette, Terry Hyslop, Yujia Wen, Linda McCart, George Miles, Steven Carr, Bing Zhang, Shankha Satpathy, Matthew Ellis, Meenakshi Anurag. Proteogenomic profiling of fresh frozen core biopsies from CALGB 40601 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-36.

Introduction: Cytotoxic chemotherapy for sporadic Triple Negative Breast Cancer (TNBC) remains the standard of care and the recent approval for adjuvant PD1 therapy is not biomarker guided. Pathological complete response (pCR) is often not achieved and portends poor survival. Predictive markers for individual drugs have proven elusive. Approach: Microscaled proteogenomics (MPG) was applied to snap-frozen TNBC clinical trial core needle biopsies obtained before treatment with carboplatin and docetaxel (WashU: NCT201404107 and BCM: NCT02544987). Clinical endpoints for discovery analysis were pathological complete response (pCR) and residual cancer burden (RCB). Standard non-parametric statistical tests were employed to identify proteogenomic features associated with these endpoints. Results: Copy number aberrations (CNA) are a recurrent feature of TNBC and a potential driver of chemotherapy sensitivity. We therefore sought CNA with concordant changes at the mRNA and protein levels that also associate with pCR status. Genes located within a recurrent interstitial deletion at chromosomal location 19q13.3 were the most significantly down-regulated at mRNA and protein level in chemotherapy resistant cases. 19q13.3 encodes multiple DNA damage response (DDR) genes; however, only LIG1, a DNA ligase required for lagging strand synthesis and DNA repair, showed concordant changes at both the mRNA and protein level. In multiple independent TNBC data sets, LIG1 deletion was associated lack of pCR and poor metastasis-free survival. Additionally in the BrighTNess TNBC trial lower LIG1 mRNA levels were associated with increased chemotherapy resistance in the carboplatin containing arms (no pCR and residual cancer burden I-III; p=0.0008 and 0.003 respectively). In PDX-derived short-term cultures and PDXs treated with docetaxel or carboplatin, a specific association of carboplatin resistance with LIG1 deletion was observed. LIG1 depleted-tumors did not harbor elevated scores for homologous recombination defect signature, suggesting LIG1 loss is an orthogonal pathway for TNBC pathogenesis The high chromosomal instability index in LIG1 deletion tumors in our TNBC study was robustly reproduced in multiple datasets (including TCGA-BRCA ; Metastatic breast cancer project). LIG1 copy number deletion was also associated with poor progression free survival, and high chromosomal instability in multiple other cancers (including TCGA-​UCEC HR=2.23, TCGA-HNSC HR=1.46, TCGA-PRAD HR=2.07, TCGA- COAD HR=1.75 and TCGA-KIRP HR=4.00). Conclusion: Deletion of LIG1 is associated with chromosomal instability in TNBC and occurs in tumors without genomic evidence for defects in homologous recombination. Other clinical features of LIG1 deleted TNBC and how LIG1 loss may cause chromosomal instability and tumorigenesis will be discussed. Citation Format: Meenakshi Anurag, Eric Jaehnig, Jonathan Lei, Beom-Jun Kim, Anh Minh Tran Huynh, Yongchao Dou, Tanmayi Vashist, Erik Bergstrom, Xuxu Gou, Viktoriya Korchina, Donna Marie Muzny, Kristen Otte, Harshavardhan Doddapaneni, Lacey Dobrolecki, Gloria Vittone Echeverria, Bora Lim, Mothaffar Rimawi, Karsten Krug, Ian Hageman, Henry Rodriguez, Ana I. Robles, Tara Hiltke, Kent Osborne, Michael Gillette, George Miles, Steven Carr, Michael T Lewis, Bing Zhang, Foluso Ademuyiwa, Shankha Satpathy, Matthew J. Ellis. LIG1 deletion predicts chemotherapy resistance, chromosomal instability, and poor prognosis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1010.

Studies of therapeutic resistance in cancer have conventionally focused on identification of acquired exome mutations in tissue or circulating DNA at progression. However, this strategy has generated limited insights into resistance to CDK4/6 inhibitor and endocrine therapy combinations, which are the key first-line treatment modality in ER+ breast cancer. We utilized an alternative approach of integrated proteogenomic analysis of 8 pairs of pre- and post-treatment biopsies, 67 rapid autopsy samples, and 17 plasma samples from 12 ER+ breast cancer patients treated with CDK4/6 inhibitor combinations at a large academic center. In addition to whole exome sequencing, RNA sequencing, and immunohistochemistry (IHC) profiling on all tissue samples, we performed deep-scale mass spectrometry-based proteomics and phospho-proteomics on 35 rapid autopsy samples from 5 patients that had sufficient protein yields for analysis. We describe a patient with acquired Rb IHC loss after treatment progression without a Rb1 genetic alteration detected in exome sequencing or Rb transcript loss at her post-progression biopsy. Integrated proteogenomic analysis of ten autopsy lesions from this patient revealed convergent Rb protein loss across all tumor lesions, including lesions with and without Rb1 exome alterations. ESR1 mutations were frequently acquired at post-treatment biopsies at high cancer cell fractions, but a pre-existing ESR1-mutant subclone was nevertheless lost in a patient who acquired a concurrent Rb1-mutant subclone in the same tumor lesion. This suggests ESR1 mutations confer a relative fitness advantage that can nevertheless be mitigated in the presence of more potent synchronous resistance mechanisms. To investigate whether circulating tumor DNA reflects DNA shed specifically by progressing tumor lesions, we modeled the DNA shedding of tumor lesions into the plasma for 3 patients with both rapid autopsy and serial plasma samples available. In some instances, new lesions began shedding significant amounts of DNA months prior to their initial radiographic appearance. Together, these results illustrate the value of integrated multi-omics interrogation of different sample types to investigate therapeutic resistance and advance precision oncology. Citation Format: Christopher T. Chen, Ignaty Leshchiner, Liz Martin, Harry Kane, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Michael Gillette, Shankha Satpathy, Christopher Pinto, Daniel McLoughlin, Read Allen, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Steven Carr, Laxmi Parida, Gad Getz, Dejan Juric. Proteogenomic characterization of CDK4/6 inhibitor-resistant ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P065.