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A novel multiplex assay combining autoantibodies plus PSA has potential implications for classification of prostate cancer from non-malignant cases.

Xie C, Kim HJ, Haw JG, Kalbasi A, Gardner BK, Li G, Rao J, Chia D, Liong M, Punzalan RR, Marks LS, Pantuck AJ, de la Taille A, Wang G, Mukouyama H, Zeng G - J Transl Med (2011)

Bottom Line: One major obstacle is the lack of a sensitive and multiplex approach for quantifying autoAb against a large panel of clinically relevant tumor-associated antigens (TAA).Peptide epitopes from the above 6 PCAA were identified and confirmed that autoAb against these peptide epitopes reacted specifically with the full-length protein.The A+PSA index also reduced false positive rate and improved the area under a receiver operating characteristic curve.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095-1738, USA.

ABSTRACT

Background: The lack of sufficient specificity and sensitivity among conventional cancer biomarkers, such as prostate specific antigen (PSA) for prostate cancer has been widely recognized after several decades of clinical implications. Autoantibodies (autoAb) among others are being extensively investigated as potential substitute markers, but remain elusive. One major obstacle is the lack of a sensitive and multiplex approach for quantifying autoAb against a large panel of clinically relevant tumor-associated antigens (TAA).

Methods: To circumvent preparation of phage lysates and purification of recombinant proteins, we identified B cell epitopes from a number of previously defined prostate cancer-associated antigens (PCAA). Peptide epitopes from cancer/testis antigen NY-ESO-1, XAGE-1b, SSX-2,4, as well as prostate cancer overexpressed antigen AMACR, p90 autoantigen, and LEDGF were then conjugated with seroMAP microspheres to allow multiplex measurement of autoAb present in serum samples. Moreover, simultaneous quantification of autoAb plus total PSA was achieved in one reaction, and termed the "A+PSA" assay.

Results: Peptide epitopes from the above 6 PCAA were identified and confirmed that autoAb against these peptide epitopes reacted specifically with the full-length protein. A pilot study was conducted with the A+PSA assay using pre-surgery sera from 131 biopsy-confirmed prostate cancer patients and 121 benign prostatic hyperplasia and/or prostatitis patients. A logistic regression-based A+PSA index was found to enhance sensitivities and specificities over PSA alone in distinguishing prostate cancer from nonmalignant cases. The A+PSA index also reduced false positive rate and improved the area under a receiver operating characteristic curve.

Conclusions: The A+PSA assay represents a novel platform that integrates autoAb signatures with a conventional cancer biomarker, which may aid in the diagnosis and prognosis of prostate cancer and others.

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Related in: MedlinePlus

Identification and validation of B cell epitopes from cancer/testis antigen XAGE-1b. (A). ELISA was used to screen candidate peptides from XAGE-1b for recognition by patients' sera. Three patients (#1-3) were positive for either XAGE-1b:1-25 or 57-81. Sera were diluted at 1:25, 1:125, and 1:625 with BSA serving as a control target. The mean OD of 8 HD and the OD of one seronegative patient (#4) are also shown. The use of sera from NSCLC patients for screening is due to higher frequency of Ab against these shared antigens in NSCLC patients. Previous work has shown that peptide epitopes identified using one type of sera are equally recognized by sera from other cancer patients. (B). Western blots confirmed recognition of the full-length XAGE-1b protein. Lane 1, 2, and 3 contained, respectively, lysate from 293 cells transfected with a control plasmid, a plasmid encoding XAGE-1b (denoted with an arrow), and lysate from LNCaP-CL1 cells (expressing XAGE-1b but at a much lower level based on real-time PCR, data not shown).
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Figure 1: Identification and validation of B cell epitopes from cancer/testis antigen XAGE-1b. (A). ELISA was used to screen candidate peptides from XAGE-1b for recognition by patients' sera. Three patients (#1-3) were positive for either XAGE-1b:1-25 or 57-81. Sera were diluted at 1:25, 1:125, and 1:625 with BSA serving as a control target. The mean OD of 8 HD and the OD of one seronegative patient (#4) are also shown. The use of sera from NSCLC patients for screening is due to higher frequency of Ab against these shared antigens in NSCLC patients. Previous work has shown that peptide epitopes identified using one type of sera are equally recognized by sera from other cancer patients. (B). Western blots confirmed recognition of the full-length XAGE-1b protein. Lane 1, 2, and 3 contained, respectively, lysate from 293 cells transfected with a control plasmid, a plasmid encoding XAGE-1b (denoted with an arrow), and lysate from LNCaP-CL1 cells (expressing XAGE-1b but at a much lower level based on real-time PCR, data not shown).

Mentions: Similar to NY-ESO-1, XAGE-1b and SSX-2,4 are cancer/testis antigens shared among cancers of the prostate, lung, breast and others [20,21]. To identify dominant B cell epitopes from XAGE-1b, computer-aided algorithms were applied to predict the peptide epitopes [18]. Two candidate peptides were screened by ELISA (Figure 1A) with serum samples from cancer patients. Three of 48 cancer patients were tested positive reacting with XAGE-1b peptides based on previously described criterion [18]. Two of the 3 seropositive patients reacted only with XAGE:1-25 peptide; while the other reacted with both XAGE:1-25 and XAGE:57-81 peptides. Western blot confirmed that sera recognizing the XAGE:1-25 peptide reacted with the full-length XAGE-1b protein from a transfected 293 cell line (Figure 1B).


A novel multiplex assay combining autoantibodies plus PSA has potential implications for classification of prostate cancer from non-malignant cases.

Xie C, Kim HJ, Haw JG, Kalbasi A, Gardner BK, Li G, Rao J, Chia D, Liong M, Punzalan RR, Marks LS, Pantuck AJ, de la Taille A, Wang G, Mukouyama H, Zeng G - J Transl Med (2011)

Identification and validation of B cell epitopes from cancer/testis antigen XAGE-1b. (A). ELISA was used to screen candidate peptides from XAGE-1b for recognition by patients' sera. Three patients (#1-3) were positive for either XAGE-1b:1-25 or 57-81. Sera were diluted at 1:25, 1:125, and 1:625 with BSA serving as a control target. The mean OD of 8 HD and the OD of one seronegative patient (#4) are also shown. The use of sera from NSCLC patients for screening is due to higher frequency of Ab against these shared antigens in NSCLC patients. Previous work has shown that peptide epitopes identified using one type of sera are equally recognized by sera from other cancer patients. (B). Western blots confirmed recognition of the full-length XAGE-1b protein. Lane 1, 2, and 3 contained, respectively, lysate from 293 cells transfected with a control plasmid, a plasmid encoding XAGE-1b (denoted with an arrow), and lysate from LNCaP-CL1 cells (expressing XAGE-1b but at a much lower level based on real-time PCR, data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102624&req=5

Figure 1: Identification and validation of B cell epitopes from cancer/testis antigen XAGE-1b. (A). ELISA was used to screen candidate peptides from XAGE-1b for recognition by patients' sera. Three patients (#1-3) were positive for either XAGE-1b:1-25 or 57-81. Sera were diluted at 1:25, 1:125, and 1:625 with BSA serving as a control target. The mean OD of 8 HD and the OD of one seronegative patient (#4) are also shown. The use of sera from NSCLC patients for screening is due to higher frequency of Ab against these shared antigens in NSCLC patients. Previous work has shown that peptide epitopes identified using one type of sera are equally recognized by sera from other cancer patients. (B). Western blots confirmed recognition of the full-length XAGE-1b protein. Lane 1, 2, and 3 contained, respectively, lysate from 293 cells transfected with a control plasmid, a plasmid encoding XAGE-1b (denoted with an arrow), and lysate from LNCaP-CL1 cells (expressing XAGE-1b but at a much lower level based on real-time PCR, data not shown).
Mentions: Similar to NY-ESO-1, XAGE-1b and SSX-2,4 are cancer/testis antigens shared among cancers of the prostate, lung, breast and others [20,21]. To identify dominant B cell epitopes from XAGE-1b, computer-aided algorithms were applied to predict the peptide epitopes [18]. Two candidate peptides were screened by ELISA (Figure 1A) with serum samples from cancer patients. Three of 48 cancer patients were tested positive reacting with XAGE-1b peptides based on previously described criterion [18]. Two of the 3 seropositive patients reacted only with XAGE:1-25 peptide; while the other reacted with both XAGE:1-25 and XAGE:57-81 peptides. Western blot confirmed that sera recognizing the XAGE:1-25 peptide reacted with the full-length XAGE-1b protein from a transfected 293 cell line (Figure 1B).

Bottom Line: One major obstacle is the lack of a sensitive and multiplex approach for quantifying autoAb against a large panel of clinically relevant tumor-associated antigens (TAA).Peptide epitopes from the above 6 PCAA were identified and confirmed that autoAb against these peptide epitopes reacted specifically with the full-length protein.The A+PSA index also reduced false positive rate and improved the area under a receiver operating characteristic curve.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095-1738, USA.

ABSTRACT

Background: The lack of sufficient specificity and sensitivity among conventional cancer biomarkers, such as prostate specific antigen (PSA) for prostate cancer has been widely recognized after several decades of clinical implications. Autoantibodies (autoAb) among others are being extensively investigated as potential substitute markers, but remain elusive. One major obstacle is the lack of a sensitive and multiplex approach for quantifying autoAb against a large panel of clinically relevant tumor-associated antigens (TAA).

Methods: To circumvent preparation of phage lysates and purification of recombinant proteins, we identified B cell epitopes from a number of previously defined prostate cancer-associated antigens (PCAA). Peptide epitopes from cancer/testis antigen NY-ESO-1, XAGE-1b, SSX-2,4, as well as prostate cancer overexpressed antigen AMACR, p90 autoantigen, and LEDGF were then conjugated with seroMAP microspheres to allow multiplex measurement of autoAb present in serum samples. Moreover, simultaneous quantification of autoAb plus total PSA was achieved in one reaction, and termed the "A+PSA" assay.

Results: Peptide epitopes from the above 6 PCAA were identified and confirmed that autoAb against these peptide epitopes reacted specifically with the full-length protein. A pilot study was conducted with the A+PSA assay using pre-surgery sera from 131 biopsy-confirmed prostate cancer patients and 121 benign prostatic hyperplasia and/or prostatitis patients. A logistic regression-based A+PSA index was found to enhance sensitivities and specificities over PSA alone in distinguishing prostate cancer from nonmalignant cases. The A+PSA index also reduced false positive rate and improved the area under a receiver operating characteristic curve.

Conclusions: The A+PSA assay represents a novel platform that integrates autoAb signatures with a conventional cancer biomarker, which may aid in the diagnosis and prognosis of prostate cancer and others.

Show MeSH
Related in: MedlinePlus