Diseases of the Esophagus (2015) 28, 262–268 DOI: 10.1111/dote.12190
Original article
Analysis of lymphatic and blood vessel invasion biomarkers in T1 esophagogastric adenocarcinomas for improved patient prognostication C. Perry,1 I. Soomro,2 P. Kaye,2 E. Hardy,3 S. L. Parsons,3 K. Ragunath,4 D. N. Lobo,5 S. G. Martin,1 S. Madhusudan1 Division of Oncology, School of Medicine, University of Nottingham, Departments of 2Pathology and Surgery, Divisions of 4Gastroenterology and 5Gastrointestinal Surgery, Nottingham Digestive Diseases Centre, NIHR Biomedical Research Unit, Nottingham University Hospitals, Nottingham, UK 1
3
SUMMARY. Lymphovascular invasion (LVI) in T1 esophagogastric adenocarcinoma may predict risk of recurrence despite definitive treatment with surgery or endoscopic resection. Podoplanin and CD34 are emerging biomarkers of lymphatic and blood vessel invasion, respectively, and could be adopted to refine LVI assessment. A consecutive series of 65 patients with T1 adenocarcinomas diagnosed at Nottingham University Hospitals were investigated. T1 tumors from 43/65 patients who received primary surgery only were suitable for LVI evaluation by hematoxylin and eosin (H&E) staining as well as by CD34 and Podoplanin immunohistochemistry. LVI was correlated to clinicopathological features and recurrence free survival. H&E staining detected LVI in 11.6% (5/43) of T1 tumors. CD34 and Podoplanin immunohistochemistry significantly improved LVI detection to 25.6% (11/43). Compared with LVI by H&E, immunohistochemical evaluation of blood vessel invasion (CD34) or lymphatic vessel invasion (Podoplanin) was significantly associated with higher grade (P = 0.005), submucosal invasion (T1b) (P = 0.018), lymph node positivity (N1) (P = 0.029) and poor recurrence free survival (P = 0.0003). Our study provides evidence that CD34 and Podoplanin immunohistochemistry could improve LVI detection and allow better prognostication of patients and optimum selection of definitive treatment. Larger multicenter studies are required for further validation that could have significant clinical implications. KEY WORDS: CD34, D2–40, esophagogastric adenocarcinoma, lymphovascular invasion, T1 tumor.
INTRODUCTION The incidence of esophageal and gastroesophageal junction (GEJ) adenocarcinoma is rising.1–3 Advances in endoscopic surveillance have recently increased the proportion of esophageal and GEJ adenocarcinomas diagnosed at T1 stage.4 In T1a tumors, the risk of lymph node involvement and recurrence is low, and many patients are cured by endomucosal resection (EMR).5–7 The risk of lymph node involvement in T1b tumors can be high (20–30%), and surgical resection provides the best opportunity for a cure.5,8 However, the clinical problem of which T1b tumors are at risk of lymph node metastasis remains less clearly defined. Address correspondence to: Dr Srinivasan Madhusudan, FRCP, PhD, Division of Oncology, School of Medicine, University of Nottingham, Nottingham University Hospitals, Nottingham NG5 1PB, UK. Email: srinivasan.madhusudan@nottingham .ac.uk 262
The presence of lymphovascular invasion (LVI) as assessed by standard hematoxylin and eosin (H&E) staining has been shown to predict recurrence and poor survival in patients with T1 disease.9 However, LVI detected by H&E technique has several limitations in solid tumors. H&E does not allow distinction between blood vessel invasion and lymph vessel invasion in tumors. Difficulties in determining the vessel endothelium surrounding tumor emboli or confounding fixation-related artifacts in H&E-stained tissues10 could result in false-negative or false-positive results, respectively. Moreover, whether blood vessel invasion and/or lymphatic vessel invasion contributes to poor outcome in T1 disease is also currently unknown. Hence, there is a need to refine LVI assessment to improve prognostication of patients with T1 esophagogastric adenocarcinomas. CD34 is a single-pass transmembrane sialomucin protein that is predominantly expressed in small blood vessel endothelium.11–15 Podoplanin is a type-I, © 2014 International Society for Diseases of the Esophagus
LVI in T1 esophagogastric adenocarcinoma
integral membrane, heavily O-glycosylated glycoprotein that is selectively expressed in lymphatic endothelium.16–19 Podoplanin and CD34 are emerging biomarkers for evaluation of lymphatic and blood vessel invasion in tumors, respectively.10–21 In the current study, we conducted LVI assessments in T1 tumors by H&E staining, CD34, and Podoplanin immunohistochemistry (IHC). We provide the first evidence that CD34 and Podoplanin evaluation not only improves the LVI detection rate compared with H&E but also allows better prognostication of patients.
METHODS Patients and tissue samples A consecutive series of 65 patients with T1 cancers diagnosed at Nottingham University Hospitals was evaluated in the current study. Twenty-two of 65 patients had received prior neoadjuvant chemotherapy and at definite surgery were found to be T1 tumors. Whether the tumor was downstaged by neoadjuvant chemotherapy was not clear, and therefore, these tumors (22/65) were excluded from this study. A cohort of 43 patients who received surgery only was suitable for LVI evaluation by H&E staining and IHC for CD34 and Podoplanin. No patient received EMR as the first treatment. Preoperative evaluation of patients included upper gastrointestinal (GI) endoscopy, biopsy, spiral computed tomography of the chest and abdomen, endoscopic ultrasound, and laparoscopy (where appropriate). Basic demographic, recurrence, and survival data were collected. Recurrence free survival was calculated from the date of initial surgery to the date of first relapse and was censored on December 1, 2012 for patients who did not recur. The study was approved by the Nottingham Research Ethics committee (REC/05/Q2402/72). Histopathological and immunohistochemical analysis described later were performed in surgical specimens only. IHC Paraffin-embedded tumor tissue was initially stained with H&E. For identification of blood vessels and lymph vessels, representative paraffin-embedded sections from each specimen were stained with CD34 (MCAP547, 1:500 dilution; Serotec, Oxford, UK) and with podoplanin (D2–40 antibody, 1:100 dilution, AngioBio, 11-003, Del Mar, CA, USA), respectively, as described previously.20,21 Briefly, antigen retrieval for CD34 was achieved by incubating sections in 0.01 mol/L sodium citrate buffer (pH 6.0) in an 800-W microwave for 20 minutes. Podoplanin did not require antigen retrieval. After blocking of endogenous hydrogen peroxidase and non-specific reactions, sec© 2014 International Society for Diseases of the Esophagus
263
tions were incubated for an hour at room temperature with primary antibodies. Visualization of staining was conducted using streptABC kit (StreptABComplex/ HRP Duet, Mouse/Rabbit kit, Dako Corporation, K0492, Glostrup, Denmark) according to the manufacturer’s instructions. Immunohistochemical reactions were developed with 3,3’-diaminobenzidine as the chromogenic peroxidase substrate (Dako Corporation, K3468), counterstained with Myer’s hematoxylin, and mounted. Assessment of LVI CD34-stained sections, Podoplanin-stained sections, and H&E-stained sections were examined simultaneously by two expert GI histopathologists (IS and PK). Blood vessel invasion was detected as the presence of tumor cell clusters in vessels stained positive for CD34. Lymphatic vascular invasion was detected as the presence of tumor cell clusters in vessels stained positive for Podoplanin regardless of the expression of CD34. With conventional H&E staining, invasion was considered only if the tumor cells were within an endothelium-lined, vessel-like structure. In addition, all tumors underwent a histopathological re-evaluation for tumor type, intramucosal invasion, submucosal invasion, grade, and nodal status. Statistical analysis All statistical analyses were performed in SPSS version 20 (Chicago, IL, USA). Pearson’s chisquared test was used to test for associations between categorical variables. For 2 × 2 tables, Yates’ continuity correction was applied and Fisher’s exact test used if the expected cell count was
Original article
Analysis of lymphatic and blood vessel invasion biomarkers in T1 esophagogastric adenocarcinomas for improved patient prognostication C. Perry,1 I. Soomro,2 P. Kaye,2 E. Hardy,3 S. L. Parsons,3 K. Ragunath,4 D. N. Lobo,5 S. G. Martin,1 S. Madhusudan1 Division of Oncology, School of Medicine, University of Nottingham, Departments of 2Pathology and Surgery, Divisions of 4Gastroenterology and 5Gastrointestinal Surgery, Nottingham Digestive Diseases Centre, NIHR Biomedical Research Unit, Nottingham University Hospitals, Nottingham, UK 1
3
SUMMARY. Lymphovascular invasion (LVI) in T1 esophagogastric adenocarcinoma may predict risk of recurrence despite definitive treatment with surgery or endoscopic resection. Podoplanin and CD34 are emerging biomarkers of lymphatic and blood vessel invasion, respectively, and could be adopted to refine LVI assessment. A consecutive series of 65 patients with T1 adenocarcinomas diagnosed at Nottingham University Hospitals were investigated. T1 tumors from 43/65 patients who received primary surgery only were suitable for LVI evaluation by hematoxylin and eosin (H&E) staining as well as by CD34 and Podoplanin immunohistochemistry. LVI was correlated to clinicopathological features and recurrence free survival. H&E staining detected LVI in 11.6% (5/43) of T1 tumors. CD34 and Podoplanin immunohistochemistry significantly improved LVI detection to 25.6% (11/43). Compared with LVI by H&E, immunohistochemical evaluation of blood vessel invasion (CD34) or lymphatic vessel invasion (Podoplanin) was significantly associated with higher grade (P = 0.005), submucosal invasion (T1b) (P = 0.018), lymph node positivity (N1) (P = 0.029) and poor recurrence free survival (P = 0.0003). Our study provides evidence that CD34 and Podoplanin immunohistochemistry could improve LVI detection and allow better prognostication of patients and optimum selection of definitive treatment. Larger multicenter studies are required for further validation that could have significant clinical implications. KEY WORDS: CD34, D2–40, esophagogastric adenocarcinoma, lymphovascular invasion, T1 tumor.
INTRODUCTION The incidence of esophageal and gastroesophageal junction (GEJ) adenocarcinoma is rising.1–3 Advances in endoscopic surveillance have recently increased the proportion of esophageal and GEJ adenocarcinomas diagnosed at T1 stage.4 In T1a tumors, the risk of lymph node involvement and recurrence is low, and many patients are cured by endomucosal resection (EMR).5–7 The risk of lymph node involvement in T1b tumors can be high (20–30%), and surgical resection provides the best opportunity for a cure.5,8 However, the clinical problem of which T1b tumors are at risk of lymph node metastasis remains less clearly defined. Address correspondence to: Dr Srinivasan Madhusudan, FRCP, PhD, Division of Oncology, School of Medicine, University of Nottingham, Nottingham University Hospitals, Nottingham NG5 1PB, UK. Email: srinivasan.madhusudan@nottingham .ac.uk 262
The presence of lymphovascular invasion (LVI) as assessed by standard hematoxylin and eosin (H&E) staining has been shown to predict recurrence and poor survival in patients with T1 disease.9 However, LVI detected by H&E technique has several limitations in solid tumors. H&E does not allow distinction between blood vessel invasion and lymph vessel invasion in tumors. Difficulties in determining the vessel endothelium surrounding tumor emboli or confounding fixation-related artifacts in H&E-stained tissues10 could result in false-negative or false-positive results, respectively. Moreover, whether blood vessel invasion and/or lymphatic vessel invasion contributes to poor outcome in T1 disease is also currently unknown. Hence, there is a need to refine LVI assessment to improve prognostication of patients with T1 esophagogastric adenocarcinomas. CD34 is a single-pass transmembrane sialomucin protein that is predominantly expressed in small blood vessel endothelium.11–15 Podoplanin is a type-I, © 2014 International Society for Diseases of the Esophagus
LVI in T1 esophagogastric adenocarcinoma
integral membrane, heavily O-glycosylated glycoprotein that is selectively expressed in lymphatic endothelium.16–19 Podoplanin and CD34 are emerging biomarkers for evaluation of lymphatic and blood vessel invasion in tumors, respectively.10–21 In the current study, we conducted LVI assessments in T1 tumors by H&E staining, CD34, and Podoplanin immunohistochemistry (IHC). We provide the first evidence that CD34 and Podoplanin evaluation not only improves the LVI detection rate compared with H&E but also allows better prognostication of patients.
METHODS Patients and tissue samples A consecutive series of 65 patients with T1 cancers diagnosed at Nottingham University Hospitals was evaluated in the current study. Twenty-two of 65 patients had received prior neoadjuvant chemotherapy and at definite surgery were found to be T1 tumors. Whether the tumor was downstaged by neoadjuvant chemotherapy was not clear, and therefore, these tumors (22/65) were excluded from this study. A cohort of 43 patients who received surgery only was suitable for LVI evaluation by H&E staining and IHC for CD34 and Podoplanin. No patient received EMR as the first treatment. Preoperative evaluation of patients included upper gastrointestinal (GI) endoscopy, biopsy, spiral computed tomography of the chest and abdomen, endoscopic ultrasound, and laparoscopy (where appropriate). Basic demographic, recurrence, and survival data were collected. Recurrence free survival was calculated from the date of initial surgery to the date of first relapse and was censored on December 1, 2012 for patients who did not recur. The study was approved by the Nottingham Research Ethics committee (REC/05/Q2402/72). Histopathological and immunohistochemical analysis described later were performed in surgical specimens only. IHC Paraffin-embedded tumor tissue was initially stained with H&E. For identification of blood vessels and lymph vessels, representative paraffin-embedded sections from each specimen were stained with CD34 (MCAP547, 1:500 dilution; Serotec, Oxford, UK) and with podoplanin (D2–40 antibody, 1:100 dilution, AngioBio, 11-003, Del Mar, CA, USA), respectively, as described previously.20,21 Briefly, antigen retrieval for CD34 was achieved by incubating sections in 0.01 mol/L sodium citrate buffer (pH 6.0) in an 800-W microwave for 20 minutes. Podoplanin did not require antigen retrieval. After blocking of endogenous hydrogen peroxidase and non-specific reactions, sec© 2014 International Society for Diseases of the Esophagus
263
tions were incubated for an hour at room temperature with primary antibodies. Visualization of staining was conducted using streptABC kit (StreptABComplex/ HRP Duet, Mouse/Rabbit kit, Dako Corporation, K0492, Glostrup, Denmark) according to the manufacturer’s instructions. Immunohistochemical reactions were developed with 3,3’-diaminobenzidine as the chromogenic peroxidase substrate (Dako Corporation, K3468), counterstained with Myer’s hematoxylin, and mounted. Assessment of LVI CD34-stained sections, Podoplanin-stained sections, and H&E-stained sections were examined simultaneously by two expert GI histopathologists (IS and PK). Blood vessel invasion was detected as the presence of tumor cell clusters in vessels stained positive for CD34. Lymphatic vascular invasion was detected as the presence of tumor cell clusters in vessels stained positive for Podoplanin regardless of the expression of CD34. With conventional H&E staining, invasion was considered only if the tumor cells were within an endothelium-lined, vessel-like structure. In addition, all tumors underwent a histopathological re-evaluation for tumor type, intramucosal invasion, submucosal invasion, grade, and nodal status. Statistical analysis All statistical analyses were performed in SPSS version 20 (Chicago, IL, USA). Pearson’s chisquared test was used to test for associations between categorical variables. For 2 × 2 tables, Yates’ continuity correction was applied and Fisher’s exact test used if the expected cell count was
