Circulating Proteolytic Products of Carboxypeptidase N for Early Detection of Breast Cancer Y. Li, Y. Li, T. Chen, A.S. Kuklina, P. Bernard, F.J. Esteva, H. Shen, M. Ferrari, and Y. Hu January © Copyright 2014 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Background  Current circulating biomarkers for breast cancer have poor diagnostic sensitivity  Sensitivity of CA15-3 and CA27.29: 30% in early stage; % in advanced cases; also increased in benign diseases  Mammography screening for breast cancer is not recommended for young women (< 40 years) and not adopted commonly for older women  Radiation is harmful for the false positive patients in follow- up procedures  New and noninvasive ways for early diagnosis of breast cancer are in great need See Editorial by Diamandis EP. Tumor Microenvironment–Released Peptides: Could They Form the Basis for an Early-Diagnosis Breast Cancer Test? Clinical Chemistry 2014; 60: 4-6.

© Copyright 2009 by the American Association for Clinical Chemistry Background  Proteolytic activity in the tumor microenviroment (not necessarily deriving from tumor cells) may release peptides into circulation that are potential biomarkers for monitoring tumor progression See Editorial by Diamandis EP. Tumor Microenvironment–Released Peptides: Could They Form the Basis for an Early-Diagnosis Breast Cancer Test? Clinical Chemistry 2014; 60: 4-6.

© Copyright 2009 by the American Association for Clinical Chemistry Background  Carboxypeptidase N (CPN), also known as an arginine/lysine carboxypeptidase, is a member of a larger family of zinc metallopeptidases  CPN cleaves the carboxy (C)-terminal arginine or lysine of endogenous peptides  Substrates: bradykinin (BK9), anaphylatoxins (C3a, C4a, and C5a), and fibrinopeptides A and B  EDTA is an inhibitor of CPN  The relationship between breast cancer, CPN, and circulating concentrations of the proteolytic products of CPN has not been established See Editorial by Diamandis EP. Tumor Microenvironment–Released Peptides: Could They Form the Basis for an Early-Diagnosis Breast Cancer Test? Clinical Chemistry 2014; 60: 4-6.

© Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods Injection 2 weeks4 weeks6 weeks (Nude mice, n=8; breast cancer cell line: MDA-MD-231) Breast cancer mouse model : 8 weeks Serum collection Tissue collection Breast cancer (BC) patients: ControlBC-IBC-IIBC-IIIBC-IVTotal Blood plasma Tissue slices

© Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods Data Analysis MALDI-TOF MS Nanopore fractionation Circulating peptides were isolated by nanoporous silica chip and analyzed by mass spectrometry

© Copyright 2009 by the American Association for Clinical Chemistry Results Figure 1. Ex vivo peptide cleavage assay on the synthetic C3f peptide. A. C3f peptide cleavage assay in NIF (normal interstitial fluid), CM ( conditioned cell-culture medium), and TIF (tumor interstitial fluid). B. Specific cleavage sites are indicated on the C3f peptide, a substrate of the enzymes factor I, chymotrypsin and CPN. C. Detecting CPN expression in different assay conditions. C3f, synthetic peptide His 6 -C3f_S R His 6 only; NIF or TIF, normal or tumor interstitial fluid only; EDTA, an inhibitor of CPN; NIF (CPN -) and TIF (CPN -), CPN-depleted normal and tumor tissue interstitial fluid; CM (CPN -), CPN-depleted conditioned medium. A B C

© Copyright 2009 by the American Association for Clinical Chemistry Result Figure 2. CPN expression in mouse tissues and sera. A. CPN immunoblotting expression in normal breast interstitial fluid (NIF) and tumor interstitial fluid (TIF). Coomassie Brilliant Blue (CBB) stained gel was used for the evaluation of equal loading. Both immunoblot and CBB stained gel were subjected to quantitative image analysis by software Image J, and the result was presented by histogram. B. Immunohistochemistry analysis of CPN in normal breast tissue and tumor tissue collected at the 2 nd and 8 th week. C. Immunoblotting of CPN in mouse sera. Ponceau S-stained serum albumin (SA) serves as internal control for loading. (Groups: 0, 2 nd, 4 th, 6 th, 8 th week; n = 4 / group). A BC

© Copyright 2009 by the American Association for Clinical Chemistry Result (3) Figure 3. The vertical scattering plot of the normalized peak intensities in MALDI TOF MS for the potential peptide signatures in serum samples collected from tumor-bearing animals (breast cancer mouse model). The mass-to-charge ratio and sequence identification is listed in each panel. Mouse number: n = 8; Student t- test, * P < 0.05, ** P < 0.01, *** P <

© Copyright 2009 by the American Association for Clinical Chemistry Result (4) Figure 4. CPN expression in human tissues and plasma. A. IHC stains for CPN in human control cohort (cancer adjunct tissue or adenosis) and tumor tissues. B. The histogram shows the percentage of the scoring results of IHC stains for CPN (n = 10, 7, 77 and 16 for control, BC-I, BC-II and BC-III slides, respectively). Scoring method of IHC staining pattern refers to the protocol reported by Zhang et al. (30). C. CPN expression by immunoblotting in human plasma from healthy controls and breast cancer (BC) patients at different stages of disease (I, II, III and IV). Ponceau S-stained serum albumin (SA) served as internal control. n = 4 / group. A B C

© Copyright 2009 by the American Association for Clinical Chemistry Result (5) Figure 5. Profiles of the potential peptide signatures in plasma from healthy women and women with breast cancer of different pathological stages. Sample number: n = 10, healthy controls; n = 11, stage I breast cancer (BC-I); n = 12, stage II breast cancer (BC-II); n = 15, stage III breast cancer (BC-III); n = 10, stage IV breast cancer (BC-IV); Student t-test, * P < 0.05, *** P <

© Copyright 2009 by the American Association for Clinical Chemistry Discussion  CPN expression was increased in tumor as compared with normal breast tissue, but no changes in blood levels were observed  Six circulating CPN-catalyzed peptides reflect the higher CPN activity in tumors as early as the first pathologic stage of breast cancer

© Copyright 2009 by the American Association for Clinical Chemistry Questions  Is it possible to use a single biomarker for cancer diagnosis or prognosis?  What are the advantages and disadvantages of using multiple circulating peptides as cancer biomarkers?  Why did the selected CPN-catalyzed peptide concentrations correlate imperfectly with tumor burden?

© Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at Download the free Clinical Chemistry app on iTunes for additional content! Follow us