Research
Original Investigation
Expression of HLA Class I Antigen, Aspirin Use, and Survival After a Diagnosis of Colon Cancer Marlies S. Reimers, MD; Esther Bastiaannet, PhD; Ruth E. Langley, MD; Ronald van Eijk, PhD; Ronald L. P. van Vlierberghe, BSc; Valery E. P. Lemmens, PhD; Myrthe P. P. van Herk-Sukel, PhD; Tom van Wezel, PhD; Riccardo Fodde, PhD; Peter J. K. Kuppen, PhD; Hans Morreau, MD; Cornelis J. H. van de Velde, MD; Gerrit Jan Liefers, MD Invited Commentary page 739 IMPORTANCE Use of aspirin (which inhibits platelet function) after a colon cancer diagnosis is
associated with improved overall survival. Identifying predictive biomarkers of this effect could individualize therapy and decrease toxic effects.
Supplemental content at jamainternalmedicine.com
OBJECTIVE To demonstrate that survival benefit associated with low-dose aspirin use after a diagnosis of colorectal cancer might depend on HLA class I antigen expression. DESIGN, SETTING, AND PARTICIPANTS A cohort study with tumor blocks from 999 patients with colon cancer (surgically resected between 2002 and 2008), analyzed for HLA class I antigen and prostaglandin endoperoxide synthase 2 (PTGS2) expression using a tissue microarray. Mutation analysis of PIK3CA was also performed. Data on aspirin use after diagnosis were obtained from a prescription database. Parametric survival models with exponential (Poisson) distribution were used to model the survival. MAIN OUTCOMES AND MEASURES Overall survival. RESULTS The overall survival benefit associated with aspirin use after a diagnosis of colon cancer had an adjusted rate ratio (RR) of 0.53 (95% CI, 0.38-0.74; P < .001) when tumors expressed HLA class I antigen compared with an RR of 1.03 (0.66-1.61; P = .91) when HLA antigen expression was lost. The benefit of aspirin was similar for tumors with strong PTGS2 expression (0.68; 0.48-0.97; P = .03), weak PTGS2 expression (0.59; 0.38-0.97; P = .02), and wild-type PIK3CA tumors (0.55; 0.40-0.75; P < .001). No association was observed with mutated PIK3CA tumors (0.73; 0.33-1.63; P = .44). CONCLUSIONS AND RELEVANCE Contrary to the original hypothesis, aspirin use after colon cancer diagnosis was associated with improved survival if tumors expressed HLA class I antigen. Increased PTGS2 expression or the presence of mutated PIK3CA did not predict benefit from aspirin. HLA class I antigen might serve as a predictive biomarker for adjuvant aspirin therapy in colon cancer.
Author Affiliations: Author affiliations are listed at the end of this article.
JAMA Intern Med. 2014;174(5):732-739. doi:10.1001/jamainternmed.2014.511 Published online March 31, 2014. 732
Corresponding Author: Gerrit Jan Liefers, MD, Department of Surgery, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands ([email protected]). jamainternalmedicine.com
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archinte.jamanetwork.com/ by a University of Toledo Libraries User on 11/09/2014
Aspirin Use and Survival After Colon Cancer
T
here is a significant body of preclinical, epidemiologic, and randomized data demonstrating that aspirin has anticancer effects.1-7 Several studies2,4,8-10 have shown that aspirin use after a diagnosis of colorectal cancer improves colorectal cancer–specific and overall survival. Randomized trials7 designed to assess the cardiovascular benefits of aspirin demonstrate that allocation to aspirin reduces the risk of distant metastasis when cancer is diagnosed (hazard ratio [HR] 0.69; 95% CI, 0.5-0.95; P = .02) and on subsequent follow-up in patients without metastasis at diagnosis (0.45; 0.28-0.72; P < .001), with the largest effects seen for colorectal cancer (HR at diagnosis, 0.36; 0.18-0.74; P = .005; and HR at follow-up, 0.26; 0.11-0.57; P < .001). Although questions remain about the optimal dose and duration of aspirin use, its efficacy in prediagnostic users, and the localization of tumors most likely to benefit, the data suggest that aspirin is a potential adjuvant therapy to prevent distant metastasis in colorectal cancer, and possibly other tumors. The precise biological mechanisms underlying the anticancer effects are unknown. Prostaglandin endoperoxide synthase 2 (PTGS2; HGNC 9605), also known as cyclooxygenase 2, overexpression has been associated 11,12 with a poor prognosis in colorectal cancer. Aspirin inhibits PTGS; at low doses (75-300 mg once daily), and given the short half-life of aspirin (approximately 30 minutes), this effect is manifested as permanent inhibition of PTGS1 in the anucleate platelet, which is unable to resynthesize the enzyme. Higher and more frequent dosing, for example, 600 mg once daily, would be required to constantly inhibit PTGS2 in systemic tissues.13-16 Despite this dose-dependent observation, data from 2 observational cohorts (the Nurses’ Health Study and Health Professionals Follow-up Study) 17 have indicated that the survival benefits of regular use of lowdose aspirin after a diagnosis of colorectal cancer are associated with the molecular characteristics of the tumor, particularly mutations in the gene PIK3CA (HGNC 8975) (a component of the PTGS2 pathway), with a multivariable HR for aspirin users compared with nonusers in tumors with mutated PIK3CA of 0.18 (95% CI, 0.06-0.61; P < .001) for cancer death and 0.54 (0.31-0.94; P = .01) for death from any cause. The metastatic potential of cancer cells that are shed into the bloodstream can be modified by environmental conditions, including platelets and bone marrow–derived cells in the vasculature.18 Platelets are thought to protect disseminating tumor cells from natural killer cells, which preferentially recognize and eliminate cells with low or absent expression of HLA class I antigen.19 We hypothesized that the survival benefit associated with low-dose aspirin use after a cancer diagnosis would be associated with tumors that have low or absent HLA class I antigen expression. We analyzed tumors from a cohort in which an association was shown 8,10 between overall survival and lowdose aspirin use after diagnosis (adjusted rate ratio [RR], 0.65; 95% CI, 0.50-0.84; P = .001) with an even larger effect in older (>70 years) patients with colon cancer (0.59; 0.440.81; P = .001) for HLA class I antigen and PTGS2 expression as well as PIK3CA mutations. jamainternalmedicine.com
Original Investigation Research
Methods Study Cohort The Eindhoven Cancer Registry was initially used to identify patients with colorectal cancer and linked to data on aspirin use from the PHARMO database network (PHARMO, the Netherlands). This research was performed according to the code of conduct for responsible use. Patient records information was made anonymous and deidentified prior to analysis according to national ethics guidelines (Code for Proper Secondary Use of Human Tissue, Dutch Federation of Medical Scientific Societies). As previously reported,8 compared with survival in aspirin nonusers, aspirin initiated or continued after diagnosis was associated with improved survival for patients with colon cancer, but not for those with rectal cancer. Paraffin-embedded tissue blocks were retrieved from 1026 patients with colon cancer who had a surgical resection between 2002 and 2008. For this study, 27 patients with more than 1 colon tumor at the time of diagnosis were excluded; thus, 999 patients were included in the analysis. There were no significant demographic differences between the patients included in the present study and the whole colon cancer cohort in the registry (N = 3586) (Supplement [eTable 1]).
Tissue Microarray Production and Immunohistochemistry Three 1.0-mm-diameter cores were obtained from formalinfixed, paraffin-embedded tumor blocks using hematoxylineosin–stained sections for tumor identification (with a qualified pathologist [H.M.] confirming the identification of the tumor) and transferred into a receiver paraffin block using tissue microarray (TMA Master; 3DHistech Ltd). Immunohistochemical staining was performed on 4-μm sections cut from each receiver block and mounted on glass. For each primary antibody, all slides were stained simultaneously to avoid interassay variation. Immunohistochemical analyses to detect HLA class I antigen expression with mouse monoclonal antibodies HCA2 and HC10 (heavy chains of HLA-A), using diaminobenzidine solution (Dako) for visualization of the antibodies, were performed by 2 independent observers (M.S.R. and R.L.P.v.V.), as previously described,20 with good interobserver agreement (κ = 0.5-0.7). The mouse monoclonal antibodies HCA2 and HC10 that were used recognize the heavy chains of HLA class I antigen. Their reactivity spectrum has been described in detail.18 The HLA class I antigen expression status was determined according to the International HLA and Immunogenetics Workshop,21 with tumor cell HLA class I antigen status defined as follows: loss of HLA class I antigen: less than 5% expressing both HCA2 and HC10 or less than 5% expressing either of the markers, and expression of HLA class I antigen: 5% or more expressing both markers. Normal epithelial, stromal, or lymphoid cells served as positive internal controls. Expression of PTGS2 was analyzed automatically with a double staining to separately visualize stromal cells (using diaminobenzidine solution for visualization of anticollagen I, anticollagen VI, and elastin [all polyclonal rabbit antibodies obJAMA Internal Medicine May 2014 Volume 174, Number 5
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archinte.jamanetwork.com/ by a University of Toledo Libraries User on 11/09/2014
733
Research Original Investigation
Aspirin Use and Survival After Colon Cancer
Figure 1. Representative Images of Immunohistochemical Staining for HLA Class I Antigen Expression (HCA2 and HC10) and Prostaglandin Endoperoxide Synthase 2 (PTGS2) A
E
B
C
F
G
Staining performed according to standard protocols, as detailed in the Methods section. A, HC10-negative tumor. B, HC10-positive tumor. C, HCA2-negative tumor. D, HCA2-positive tumor. E, Tumor with weak PTGS2 expression. F, Tumor with strong PTGS2 expression. G, Enlarged view of sample in F.
See the Tissue Microarray Production and Immunohistochemistry subsection of the Methods section for details of the immunohistochemical staining methods used. Original magnifications ×20 (A-F) and ×40 (G).
tained from Abcam]) and positive tumor cells (with the monoclonal mouse antibody anti-PTGS2 [Cayman Chemical Co]), using Vector Blue (Vector Laboratories) for visualization of the PTGS2 antibody. Slides were scanned (Pannoramic Midi; 3DHistech) and PTGS2 expression was assessed (AxioVision, 4.6; Zeiss) using the criteria proposed by Buskens et al22 and comparable to the scoring method used by Chan et al.2 Representative images of the immunohistochemical staining are shown in Figure 1. Microsatellite stability status was determined by immunohistochemical analyses as previously described.23 In short, 4 antibodies directed against mutL homologue 21 (MLH1, clone ES05; DakoCytomation), mutS homologue 2 (MSH2, clone g2191129; BD Biosciences), mutS homologue 6 (MSH6, clone EPR3945; Epitomics), and postmeiotic segregation of Saccharomyces cerevisiae 2 (PMS2, clone A16-4; BD Biosciences) were used. The criteria used to confirm microsatellite instability in the tissues are described elsewhere.23,24 All slides were stained simultaneously to avoid interassay variation. Slides that underwent the entire immunohistochemical staining procedure without primary antibodies served as negative controls. The quality of the staining, the scoring method, and discrepancies between the 2 observers were checked by a pathologist (H.M).
domly chosen, with a 1:2 ratio for aspirin user to nonuser, using a fully automated system (Tissue Preparation System with VERSANT Tissue Preparation Reagents; Siemens Healthcare Diagnostics) as described previously.25 Hydrolysis probe assays were performed for the major known mutations (hotspots) in exon 9, c.1624G>A; p.E542K, c.1633G>A; p.E545K as well as in exon 20, c.3140A>G; and p.H1047R as previously described. 26 Hydrolysis probe assays were analyzed using quantitative polymerase chain reaction analysis software (CFX Manager, version 3/0; BioRad). To identify additional nonhotspot mutations, Sanger sequencing was performed on exon 9 and exon 20 of all samples. Mutation detection was performed by 2 observers independently (M.S.R. and R.v.E.) using DNA variant analysis software (Mutation Surveyor, version 4.0.9; Softgenetics). All primers and probes used for the assays are listed in the Supplement (eTable 2).
PIK3CA Mutation Analysis Samples of DNA were extracted from 1 to 2 variable-length, 2.0mm-diameter cores taken from 663 of the 999 blocks ran734
D
Statistical Analysis The vital status of patients (alive vs dead) was established from medical records or through linkage of cancer registry data with the municipal population registries. Follow-up started 30 days from diagnosis of colorectal cancer (T0), because information on hospital prescriptions was not available, and was continued until the last contact date (January 1, 2012) or the date of death. Patients who died within 30 days of diagnosis were excluded from the survival analyses (2.4% for colon cancer). Nonusers were classified as
JAMA Internal Medicine May 2014 Volume 174, Number 5
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archinte.jamanetwork.com/ by a University of Toledo Libraries User on 11/09/2014
jamainternalmedicine.com
Aspirin Use and Survival After Colon Cancer
Original Investigation Research
Table 1. Baseline Characteristics of Patients With Colon Cancer According to Tumor HLA Class I Antigen Expression and Use of Aspirin After Diagnosisa HLA Antigen Loss (n = 320)
HLA Antigen Expression (n = 643)
All Patients (N = 999)b
No Aspirin
Aspirin
Female
494 (49.4)
142 (54.0)
22 (38.6)
Male
505 (50.6)
121 (46.0)
35 (61.4)
≤65
342 (34.2)
110 (41.8)
8 (14.0)
66-74
304 (30.4)
66 (25.1)
26 (45.6)
≥75
353 (35.4)
87 (33.1)
23 (40.4)
2002-2004
451 (45.1)
102 (38.8)
27 (47.4)
2005-2007
548 (54.8)
161 (61.2)
30 (52.6)
Characteristic
P Valuec
No Aspirin
Aspirin
261 (50.1)
44 (36.1)
260 (49.9)
78 (63.9)
188 (36.1)
23 (18.8)
P Valuec
Sex, No. (%) .04
.005
Age, y, No. (%)
Original Investigation
Expression of HLA Class I Antigen, Aspirin Use, and Survival After a Diagnosis of Colon Cancer Marlies S. Reimers, MD; Esther Bastiaannet, PhD; Ruth E. Langley, MD; Ronald van Eijk, PhD; Ronald L. P. van Vlierberghe, BSc; Valery E. P. Lemmens, PhD; Myrthe P. P. van Herk-Sukel, PhD; Tom van Wezel, PhD; Riccardo Fodde, PhD; Peter J. K. Kuppen, PhD; Hans Morreau, MD; Cornelis J. H. van de Velde, MD; Gerrit Jan Liefers, MD Invited Commentary page 739 IMPORTANCE Use of aspirin (which inhibits platelet function) after a colon cancer diagnosis is
associated with improved overall survival. Identifying predictive biomarkers of this effect could individualize therapy and decrease toxic effects.
Supplemental content at jamainternalmedicine.com
OBJECTIVE To demonstrate that survival benefit associated with low-dose aspirin use after a diagnosis of colorectal cancer might depend on HLA class I antigen expression. DESIGN, SETTING, AND PARTICIPANTS A cohort study with tumor blocks from 999 patients with colon cancer (surgically resected between 2002 and 2008), analyzed for HLA class I antigen and prostaglandin endoperoxide synthase 2 (PTGS2) expression using a tissue microarray. Mutation analysis of PIK3CA was also performed. Data on aspirin use after diagnosis were obtained from a prescription database. Parametric survival models with exponential (Poisson) distribution were used to model the survival. MAIN OUTCOMES AND MEASURES Overall survival. RESULTS The overall survival benefit associated with aspirin use after a diagnosis of colon cancer had an adjusted rate ratio (RR) of 0.53 (95% CI, 0.38-0.74; P < .001) when tumors expressed HLA class I antigen compared with an RR of 1.03 (0.66-1.61; P = .91) when HLA antigen expression was lost. The benefit of aspirin was similar for tumors with strong PTGS2 expression (0.68; 0.48-0.97; P = .03), weak PTGS2 expression (0.59; 0.38-0.97; P = .02), and wild-type PIK3CA tumors (0.55; 0.40-0.75; P < .001). No association was observed with mutated PIK3CA tumors (0.73; 0.33-1.63; P = .44). CONCLUSIONS AND RELEVANCE Contrary to the original hypothesis, aspirin use after colon cancer diagnosis was associated with improved survival if tumors expressed HLA class I antigen. Increased PTGS2 expression or the presence of mutated PIK3CA did not predict benefit from aspirin. HLA class I antigen might serve as a predictive biomarker for adjuvant aspirin therapy in colon cancer.
Author Affiliations: Author affiliations are listed at the end of this article.
JAMA Intern Med. 2014;174(5):732-739. doi:10.1001/jamainternmed.2014.511 Published online March 31, 2014. 732
Corresponding Author: Gerrit Jan Liefers, MD, Department of Surgery, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands ([email protected]). jamainternalmedicine.com
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archinte.jamanetwork.com/ by a University of Toledo Libraries User on 11/09/2014
Aspirin Use and Survival After Colon Cancer
T
here is a significant body of preclinical, epidemiologic, and randomized data demonstrating that aspirin has anticancer effects.1-7 Several studies2,4,8-10 have shown that aspirin use after a diagnosis of colorectal cancer improves colorectal cancer–specific and overall survival. Randomized trials7 designed to assess the cardiovascular benefits of aspirin demonstrate that allocation to aspirin reduces the risk of distant metastasis when cancer is diagnosed (hazard ratio [HR] 0.69; 95% CI, 0.5-0.95; P = .02) and on subsequent follow-up in patients without metastasis at diagnosis (0.45; 0.28-0.72; P < .001), with the largest effects seen for colorectal cancer (HR at diagnosis, 0.36; 0.18-0.74; P = .005; and HR at follow-up, 0.26; 0.11-0.57; P < .001). Although questions remain about the optimal dose and duration of aspirin use, its efficacy in prediagnostic users, and the localization of tumors most likely to benefit, the data suggest that aspirin is a potential adjuvant therapy to prevent distant metastasis in colorectal cancer, and possibly other tumors. The precise biological mechanisms underlying the anticancer effects are unknown. Prostaglandin endoperoxide synthase 2 (PTGS2; HGNC 9605), also known as cyclooxygenase 2, overexpression has been associated 11,12 with a poor prognosis in colorectal cancer. Aspirin inhibits PTGS; at low doses (75-300 mg once daily), and given the short half-life of aspirin (approximately 30 minutes), this effect is manifested as permanent inhibition of PTGS1 in the anucleate platelet, which is unable to resynthesize the enzyme. Higher and more frequent dosing, for example, 600 mg once daily, would be required to constantly inhibit PTGS2 in systemic tissues.13-16 Despite this dose-dependent observation, data from 2 observational cohorts (the Nurses’ Health Study and Health Professionals Follow-up Study) 17 have indicated that the survival benefits of regular use of lowdose aspirin after a diagnosis of colorectal cancer are associated with the molecular characteristics of the tumor, particularly mutations in the gene PIK3CA (HGNC 8975) (a component of the PTGS2 pathway), with a multivariable HR for aspirin users compared with nonusers in tumors with mutated PIK3CA of 0.18 (95% CI, 0.06-0.61; P < .001) for cancer death and 0.54 (0.31-0.94; P = .01) for death from any cause. The metastatic potential of cancer cells that are shed into the bloodstream can be modified by environmental conditions, including platelets and bone marrow–derived cells in the vasculature.18 Platelets are thought to protect disseminating tumor cells from natural killer cells, which preferentially recognize and eliminate cells with low or absent expression of HLA class I antigen.19 We hypothesized that the survival benefit associated with low-dose aspirin use after a cancer diagnosis would be associated with tumors that have low or absent HLA class I antigen expression. We analyzed tumors from a cohort in which an association was shown 8,10 between overall survival and lowdose aspirin use after diagnosis (adjusted rate ratio [RR], 0.65; 95% CI, 0.50-0.84; P = .001) with an even larger effect in older (>70 years) patients with colon cancer (0.59; 0.440.81; P = .001) for HLA class I antigen and PTGS2 expression as well as PIK3CA mutations. jamainternalmedicine.com
Original Investigation Research
Methods Study Cohort The Eindhoven Cancer Registry was initially used to identify patients with colorectal cancer and linked to data on aspirin use from the PHARMO database network (PHARMO, the Netherlands). This research was performed according to the code of conduct for responsible use. Patient records information was made anonymous and deidentified prior to analysis according to national ethics guidelines (Code for Proper Secondary Use of Human Tissue, Dutch Federation of Medical Scientific Societies). As previously reported,8 compared with survival in aspirin nonusers, aspirin initiated or continued after diagnosis was associated with improved survival for patients with colon cancer, but not for those with rectal cancer. Paraffin-embedded tissue blocks were retrieved from 1026 patients with colon cancer who had a surgical resection between 2002 and 2008. For this study, 27 patients with more than 1 colon tumor at the time of diagnosis were excluded; thus, 999 patients were included in the analysis. There were no significant demographic differences between the patients included in the present study and the whole colon cancer cohort in the registry (N = 3586) (Supplement [eTable 1]).
Tissue Microarray Production and Immunohistochemistry Three 1.0-mm-diameter cores were obtained from formalinfixed, paraffin-embedded tumor blocks using hematoxylineosin–stained sections for tumor identification (with a qualified pathologist [H.M.] confirming the identification of the tumor) and transferred into a receiver paraffin block using tissue microarray (TMA Master; 3DHistech Ltd). Immunohistochemical staining was performed on 4-μm sections cut from each receiver block and mounted on glass. For each primary antibody, all slides were stained simultaneously to avoid interassay variation. Immunohistochemical analyses to detect HLA class I antigen expression with mouse monoclonal antibodies HCA2 and HC10 (heavy chains of HLA-A), using diaminobenzidine solution (Dako) for visualization of the antibodies, were performed by 2 independent observers (M.S.R. and R.L.P.v.V.), as previously described,20 with good interobserver agreement (κ = 0.5-0.7). The mouse monoclonal antibodies HCA2 and HC10 that were used recognize the heavy chains of HLA class I antigen. Their reactivity spectrum has been described in detail.18 The HLA class I antigen expression status was determined according to the International HLA and Immunogenetics Workshop,21 with tumor cell HLA class I antigen status defined as follows: loss of HLA class I antigen: less than 5% expressing both HCA2 and HC10 or less than 5% expressing either of the markers, and expression of HLA class I antigen: 5% or more expressing both markers. Normal epithelial, stromal, or lymphoid cells served as positive internal controls. Expression of PTGS2 was analyzed automatically with a double staining to separately visualize stromal cells (using diaminobenzidine solution for visualization of anticollagen I, anticollagen VI, and elastin [all polyclonal rabbit antibodies obJAMA Internal Medicine May 2014 Volume 174, Number 5
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archinte.jamanetwork.com/ by a University of Toledo Libraries User on 11/09/2014
733
Research Original Investigation
Aspirin Use and Survival After Colon Cancer
Figure 1. Representative Images of Immunohistochemical Staining for HLA Class I Antigen Expression (HCA2 and HC10) and Prostaglandin Endoperoxide Synthase 2 (PTGS2) A
E
B
C
F
G
Staining performed according to standard protocols, as detailed in the Methods section. A, HC10-negative tumor. B, HC10-positive tumor. C, HCA2-negative tumor. D, HCA2-positive tumor. E, Tumor with weak PTGS2 expression. F, Tumor with strong PTGS2 expression. G, Enlarged view of sample in F.
See the Tissue Microarray Production and Immunohistochemistry subsection of the Methods section for details of the immunohistochemical staining methods used. Original magnifications ×20 (A-F) and ×40 (G).
tained from Abcam]) and positive tumor cells (with the monoclonal mouse antibody anti-PTGS2 [Cayman Chemical Co]), using Vector Blue (Vector Laboratories) for visualization of the PTGS2 antibody. Slides were scanned (Pannoramic Midi; 3DHistech) and PTGS2 expression was assessed (AxioVision, 4.6; Zeiss) using the criteria proposed by Buskens et al22 and comparable to the scoring method used by Chan et al.2 Representative images of the immunohistochemical staining are shown in Figure 1. Microsatellite stability status was determined by immunohistochemical analyses as previously described.23 In short, 4 antibodies directed against mutL homologue 21 (MLH1, clone ES05; DakoCytomation), mutS homologue 2 (MSH2, clone g2191129; BD Biosciences), mutS homologue 6 (MSH6, clone EPR3945; Epitomics), and postmeiotic segregation of Saccharomyces cerevisiae 2 (PMS2, clone A16-4; BD Biosciences) were used. The criteria used to confirm microsatellite instability in the tissues are described elsewhere.23,24 All slides were stained simultaneously to avoid interassay variation. Slides that underwent the entire immunohistochemical staining procedure without primary antibodies served as negative controls. The quality of the staining, the scoring method, and discrepancies between the 2 observers were checked by a pathologist (H.M).
domly chosen, with a 1:2 ratio for aspirin user to nonuser, using a fully automated system (Tissue Preparation System with VERSANT Tissue Preparation Reagents; Siemens Healthcare Diagnostics) as described previously.25 Hydrolysis probe assays were performed for the major known mutations (hotspots) in exon 9, c.1624G>A; p.E542K, c.1633G>A; p.E545K as well as in exon 20, c.3140A>G; and p.H1047R as previously described. 26 Hydrolysis probe assays were analyzed using quantitative polymerase chain reaction analysis software (CFX Manager, version 3/0; BioRad). To identify additional nonhotspot mutations, Sanger sequencing was performed on exon 9 and exon 20 of all samples. Mutation detection was performed by 2 observers independently (M.S.R. and R.v.E.) using DNA variant analysis software (Mutation Surveyor, version 4.0.9; Softgenetics). All primers and probes used for the assays are listed in the Supplement (eTable 2).
PIK3CA Mutation Analysis Samples of DNA were extracted from 1 to 2 variable-length, 2.0mm-diameter cores taken from 663 of the 999 blocks ran734
D
Statistical Analysis The vital status of patients (alive vs dead) was established from medical records or through linkage of cancer registry data with the municipal population registries. Follow-up started 30 days from diagnosis of colorectal cancer (T0), because information on hospital prescriptions was not available, and was continued until the last contact date (January 1, 2012) or the date of death. Patients who died within 30 days of diagnosis were excluded from the survival analyses (2.4% for colon cancer). Nonusers were classified as
JAMA Internal Medicine May 2014 Volume 174, Number 5
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archinte.jamanetwork.com/ by a University of Toledo Libraries User on 11/09/2014
jamainternalmedicine.com
Aspirin Use and Survival After Colon Cancer
Original Investigation Research
Table 1. Baseline Characteristics of Patients With Colon Cancer According to Tumor HLA Class I Antigen Expression and Use of Aspirin After Diagnosisa HLA Antigen Loss (n = 320)
HLA Antigen Expression (n = 643)
All Patients (N = 999)b
No Aspirin
Aspirin
Female
494 (49.4)
142 (54.0)
22 (38.6)
Male
505 (50.6)
121 (46.0)
35 (61.4)
≤65
342 (34.2)
110 (41.8)
8 (14.0)
66-74
304 (30.4)
66 (25.1)
26 (45.6)
≥75
353 (35.4)
87 (33.1)
23 (40.4)
2002-2004
451 (45.1)
102 (38.8)
27 (47.4)
2005-2007
548 (54.8)
161 (61.2)
30 (52.6)
Characteristic
P Valuec
No Aspirin
Aspirin
261 (50.1)
44 (36.1)
260 (49.9)
78 (63.9)
188 (36.1)
23 (18.8)
P Valuec
Sex, No. (%) .04
.005
Age, y, No. (%)
