Human Pathology (2015) 46, 850–857
www.elsevier.com/locate/humpath
Original contribution
Clinical significance of assessing Her2/neu expression in gastric cancer with dual tumor tissue paraffin blocks☆,☆☆ Xiaowen Ge MD, PhD a,1 , Haixing Wang MD a,1 , Haiying Zeng BS a , Xuejuan Jin MD, PhD b , Akesu Sujie BS a , Chen Xu MD a , Yalan Liu MD, PhD a , Jie Huang MS a , Yuan Ji MD, PhD a , Yunshan Tan MD, PhD a , Tianshu Liu MD, PhD c , Yingyong Hou MD, PhD a,⁎, Jing Qin MD, PhD d,⁎, Yihong Sun MD, PhD d , Xinyu Qin MD, PhD d a
Department of Pathology, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China c Department of Oncology, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China d Department of General Surgery, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China b
Received 11 December 2014; revised 16 February 2015; accepted 19 February 2015
Keywords: Gastric cancer; Her2/neu; Dual paraffin blocks; Trastuzumab; Immunohistochemistry
Summary One paraffin block is routinely used for human epidermal growth factor receptor 2 (Her2/neu) immunohistochemistry (IHC) assessment. Here, we investigated if picking 2 paraffin blocks for Her2/neu evaluation on 1 slide is an economical, efficient, and practical method, which may reduce false negativity of Her2/neu IHC assessment due to intratumoral heterogeneity. A total of 251 gastric cancer (GC) patients were divided into a cohort using 1 tumor tissue paraffin block (single-block group, n = 132) and a cohort using dual tumor tissue paraffin blocks (dual-block group, n = 119) when evaluating Her2/neu expression status by IHC. In dual-block group, we combined the results from 2 different paraffin blocks and used the higher one as the final score. The number of IHC 1+, 2+, and 3+ specimens in the single-block group was 31 (23.5%), 40 (30.3%), and 19 (14.4%), respectively. The combined final IHC score in the dual-block group of 1+, 2+, and 3+ was 26 (21.8%), 34 (28.6%), and 23 (19.3%), respectively. Inconsistent Her2/neu expression between blocks was found in 36 (30.3%) cases in the dual-block group. The pooled data in the single-block group and the dual-block group indicated that, when using dual blocks, the Her2/neu-positive (3+) rate of GC was higher compared to that in the single-block group. Our results implied that using dual paraffin blocks to assess Her2/neu expression of GC may help identify more patients with Her2/neu-positive GC who could benefit from targeted therapy, by reducing false-negative rate of Her2 status assessment. This is an efficient, economical, and practical method for Her2/neu evaluation of GC. © 2015 Elsevier Inc. All rights reserved.
☆ This work was supported by the Research Fund of Zhongshan Hospital for Young Scientists (grant 2013ZSQN25), Shanghai, China and Research Fund from Shanghai Technology and Science Commission (grant 13140901700), Shanghai, China. ☆☆ Disclosure: The authors have declared no conflicts of interest. ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Hou), [email protected] (J. Qin). 1 Xiaowen Ge and Haixing Wang contributed equally to this work.
http://dx.doi.org/10.1016/j.humpath.2015.02.011 0046-8177/© 2015 Elsevier Inc. All rights reserved.
Assessing Her2/neu with dual paraffin blocks
1. Introduction Gastric cancer (GC) is major global health concern and the second leading cause of cancer deaths worldwide [1]. Incidence of GC is particularly high in Japan, China, South Korea, and Chile. Approximately 65% of GC patients were diagnosed as locally advanced or metastatic stages of the disease [2]. Patients with GC have poor prognosis and few efficacious therapeutic options, particularly in advanced stages; and the mortality rate of GC is decreasing [3]. Five-year survival rate of patients with advanced or metastatic GC is around 5% to 20%, with median overall survival being less than 1 year. Most patients with stage I to III GC can be cured with curative resection. However, the recurrence rate of these patients is high. The importance of adjuvant treatment is emphasized recently, especially for metastatic and recurrent cases. Human epidermal growth factor receptor 2 (Her2), also known as Her2/neu, cerbB2, or ErbB2, is a 185-kd protein [4]. Her2/neu has an extracellular ligand binding domain, a short transmembrane domain, and an intracellular tyrosine kinase domain. Amplification of HER2/NEU gene and/or overexpression of the Her2/neu protein has been observed in many human carcinomas, including GC [5–7]. The first description of Her2/ neu overexpression in GC was reported in 1986 [8]. Adenocarcinoma is the predominant histologic type of GC (95% of tumors), which is classified into 3 subtypes: intestinal, diffuse, and mixed, based on the Laurén classification [9]. GC originating from the gastroesophageal junction is usually the intestinal type, which possesses higher prevalence of HER2/NEU gene amplification (2%-45%) compared to other Laurén subtypes (8.2%-53.4%) [7,10,11]. Overall, the overexpression prevalence of Her2/neu of GC remains controversial. Trastuzumab (Herceptin, Roche, Basel, Switzerland), an anti-Her2/neu antibody, which is recommended for treatment of Her2/ neu-positive breast cancer (BC) [12], has been proven effective for GC. GC is the second type of cancer in which trastuzumab has been proven effective [13]. According to the Trastuzumab for Gastric Cancer clinical trial reported in 2010, trastuzumabin in combination with chemotherapy significantly prolonged the median overall survival time to 13.8 months compared to 11 months in patients treated with chemotherapy alone [14,15]; in Her2/neu 3+ patients, overall survival time could be prolonged to 16 months [14,15]. There are several methods available for the detection of Her2/ neu protein overexpression and gene amplification, including immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), chromogenic in situ hybridization [16], and silver in situ hybridization [17]. In 2008, Hofmann et al [18] established the assessment criteria of Her2/neu overexpression and gene amplification for GC. These criteria then have been validated by other researchers [19]. Intratumoral heterogeneity of Her2/ neu overexpression and amplification has been observed by many pathologists for BC [20,21] and GC [18]. Notably, intratumoral heterogeneity in GC is much more significant than that in BC. In addition to tumor site, this heterogeneity of Her2/ neu overexpression and amplification may be due to sample
851
A
B
Fig. 1 The entire view of 1 slide using 2 paraffin blocks detecting Her2/neu expression by IHC. The consistent Her2/neu expression of 2 paraffin blocks of 2 representative cases in the dual-block group (A-B). A, Her2/neu expression was negative (IHC 0). B, Her2/neu expression was positive (IHC 3+). The red arrow: positive tissue controls (IHC 3+). The blue arrow: negative tissue controls (IHC 0).
size, population diversity, interobserver variability, and inconsistent specimen processing and tumor sampling among different laboratories. When using FISH for HER2/NEU status assessment, the intratumoral heterogeneity could be more common because of the thinner field of view for diagnosis. Hence, it is difficult for a pathologist to report the exact status of Her2/neu overexpression and amplification [20]. Usually, pathologists classify Her2/neu expression of GC tissue with 1 paraffin block for economical and practical reasons. Currently, the number of paraffin blocks that should be used in Her2/neu evaluation is not mentioned in the National Comprehensive Cancer Network (NCCN) guidelines [22]. Coping with intratumoral heterogeneity, assessing specimens from 2 paraffin blocks of the same patient could reduce false-negative rate of Her2/neu assessment of GC. In our laboratory, 2 specimens are fit onto a single slide for Her2/neu IHC (Fig. 1); thus, we sought to see if it is a relatively cost-effective method to identify Her2/neu-positive patients and reduce false-negative rate of Her2 IHC. In the present study, Her2/neu expression status of 251 patients was analyzed together with clinicopathological features. We compared the positive rate of Her2/neu expression between a cohort using 1 paraffin block and a cohort using dual tumor tissue paraffin blocks. The aim of the current study is to identify the clinical significance of using dual tumor tissue paraffin blocks in detecting Her2/neu expression status of GC.
2. Materials and methods 2.1. Patients and clinicopathological information collection Between March 2013 and August 2013, a total of 251 patients with primary gastric adenocarcinoma who received curative surgery without any preoperative treatment at the
852 Zhongshan Hospital were included in this study. Those patients were divided into a cohort using 1 tumor tissue paraffin block (single-block group, n = 132) and a cohort using dual tumor tissue paraffin blocks (dual-block group, n = 119) when evaluating Her2/neu expression status by IHC. Prior written informed consent was collected from all patients. The study protocol was approved by the ethics board at the Zhongshan Hospital, Fudan University, Shanghai, China. The diagnosis of GC was confirmed by histology in all cases. The following patient characteristics were collected: age, sex, histologic type (adenocarcinoma only based on World Health Organization criteria) [23], Laurén classification [9], tumor site (proximal and distal), histologic grade, microscopic tumor extension, lymphatic or venous invasion, lymph node metastasis, and pTNM stage (according to seventh edition of the Union for International Cancer Control (UICC) guidelines) [24].
2.2. Hematoxylin and eosin and IHC staining
X. Ge et al. criteria defined by Hofman et al [18]: no staining or less than 10% tumor cell positive staining as 0/negative; faintly or barely perceptible staining on at least 10% tumor cell membrane as 1+; weak to moderate positive staining on at least 10% tumor cell membrane as 2+; and cohesive moderate to strong staining on at least 10% tumor cell membrane as 3+. In this study, we classified IHC3+ as Her2/neu positive. For cohort 2 (dual blocks), we combined the results from 2 different paraffin blocks and used the higher score as the final Her2/neu IHC score.
2.4. Statistical analysis A χ2 test was used for univariate analysis; crosstabulations with qualitative variables were analyzed with the Pearson χ2 test. P value b .05 was defined as statistically significant. No adjustments were made. All analyses were performed using the statistical package SPSS version 19.0 (SPSS, Inc, an IBM Company, Chicago, IL).
GC tissues from surgical specimens were fixed in 10% buffered formalin within 30 minutes after resection. Specimens were processed following routine procedures after 24 hours of fixation. Sections were stained with hematoxylin and eosin and reviewed by 2 pathologists to confirm the diagnosis of GC. Paraffin blocks of each patient were selected by trained pathologists for subsequent IHC assessment. IHC assay using Her2/neu rabbit monoclonal antibody (clone 4B5; Ventana, Tucson, AZ) was performed with iView DAB Detection Kit (Ventana, Tucson, AZ) on a BenchMark XT automated staining system (Ventana, Tucson, AZ). In brief, the tissue sections were deparaffinized with EZ Prep (Ventana, Tucson, AZ) at 75°C and heat pretreated in Cell Conditioning 1 (Ventana, Tucson, AZ) using “standard cell conditioning” for antigen retrieval at 95°C. Tissue sections were then incubated with anti-Her2/neu primary antibody for 24 minutes at 37°C after inactivation of the endogenous peroxidase by hydrogen peroxide for 4 minutes. The tissue sections were then blocked using Endogenous Biotin Blocking Kit (Ventana, Tucson, AZ) and incubated with a biotinylated secondary antibody for 8 minutes and then with a streptavidin–horseradish peroxidase conjugate for 8 minutes at 37°C. The slides were then counterstained with Hematoxylin II (Ventana, Tucson, AZ) for 8 minutes and Bluing Reagent (Ventana, Tucson, AZ) for 8 minutes. Normal immunoglobulin G from the same species of primary antibody diluted to matching concentration of the primary antibody was used as the negative control. In the same slide, small pieces of BC tissue that Her2/neu IHC scored as 3+ and 0 were used as positive and negative controls, respectively (Fig. 1).
Surgical samples were obtained from primary tumors of 251 patients with GC. Clinicopathological characteristics of the GC patients are presented in Table 1. One hundred thirty-two patients were enrolled in cohort 1 (single-block group) and 119 patients in cohort 2 (dual-block group). In the single-block group, the age of the patients ranged from 27 to 82 years with a median of 59 years. Ninety (68.2%) patients were male, and 42 (31.8%) were female. According to the Laurén classification, 41 (31.1%) patients were diagnosed as intestinal type of GC, 53 (40.2%) were diffuse type, and 38 (28.8%) were mixed type. Based on cell cohesive status, 53 (40.2%) were graded as poorly cohesive and 79 (59.8%) were non–poorly cohesive. Lymphatic or venous invasion was found in 51 (38.6%) patients (Table 1). In the dual-block group, the age of the patients ranged from 28 to 84 years with a median of 62 years. Eighty-six (72.3%) patients were male, and 33 (27.7%) were female. According to the Laurén classification, 44 (37.0%) patients were intestinal type GC, 37 (31.1%) patients were diffuse type, and 38 (31.9%) patients were mixed type. Of these patients, 37 (31.1%) were poorly cohesive and 82 (68.9%) were non–poorly cohesive. Lymphatic or venous invasion was found in 48 (40.3%) patients (Table 1).
2.3. Definitions of Her2/neu alteration
3.2. Her2/neu protein expression status by IHC
The Her2/neu status was assessed by 2 independent observers. If there was any discrepancy, the Her2/neu status was verified by a discussion panel consisting of 3 observers. All observers were blinded with regard to the clinicopathological patient characteristics. Specimens were assessed according to the IHC scoring
In the single-block group, 42 (31.8%) cases were scored 0 for Her2/neu IHC, whereas the number of cases scored as 1+, 2+, and 3+ was 31 (23.5%), 40 (30.3%), and 19 (14.4%), respectively (Table 2). For the dual-block group, first paraffin blocks were randomly selected for IHC assessment. The number
3. Results 3.1. Characteristics of patients
Assessing Her2/neu with dual paraffin blocks
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Table 1 Clinicopathological gastric cancer patient characteristics and correlations of Her2/neu-positive (3+) rates with clinicopathologic variables Cohort 1 Clinical features Patients (n) Age (y) Mean ± SD Median Sex, n (%) Male Female Laurén phenotype, n (%) Intestinal Diffuse Mixed Combined phenotype, n (%) Poorly cohesive Non–poorly cohesive Localization, n (%) Proximal Distal pT category, n (%) pT1a pT1b pT2 pT3 pT4a pT4b pN category, n (%) pN0 pN1 pN2 pN3a pN3b Lymphatic or venous invasion, n (%) pL/V0 pL/V1 UICC stage (7th edition), n (%) IA IB IIA IIB IIIA IIIB IIIC IV Resected lymph nodes Mean ± SD Median, n Positive lymph nodes Mean ±SD Median, n Positive lymph node ratio Mean ± SD Median ⁎ P b .05.
132
Cohort 2 HER2 3+, n (%)
P
19
Clinical features 119
59.6 ± 10.9 59
HER2 3+, n (%)
P
23
60.7 ± 10.7 62
90 (68.2) 42 (31.8)
13 (68.4) 6 (31.6)
.981
86 (72.3) 33 (27.7)
18 (78.3) 5 (21.7)
.475
41+ (31.1) 53 (40.2) 38 (28.8)
8 (42.1) 5 (26.3) 6 (31.6)
.516
44 (37.0) 37 (31.1) 38 (31.9)
10 (43.5) 10 (43.5) 3 (13.0)
.105
53 (40.2) 79 (59.8)
13 (68.4) 6 (31.6)
.41
37 (31.1) 82 (68.9)
3 (13.0) 20 (87.0)
.037 ⁎
40 (30.3) 92 (69.7)
7 (36.8) 12 (63.2)
.503
41 (34.5) 78 (65.5)
10 (43.5) 13 (56.5)
.311
16 (12.1) 16 (12.1) 8 (6.1) 25 (18.9) 65 (49.2) 2 (1.5)
2 (10.5) 3 (15.8) 3 (15.8) 1 (5.3) 8 (42.1) 2 (10.5)
.003 ⁎
7 (5.9) 14 (11.8) 25 (21.0) 43 (36.1) 30 (25.2) 0 (0.00)
0 (0.00) 3 (13.0) 5 (21.7) 9 (39.1) 6 (26.1) 0 (0.00)
.772
45 (34.1) 19 (14.4) 18 (13.6) 34 (25.8) 16 (12.1)
5 (26.3) 2 (10.5) 5 (26.3) 2 (10.5) 5 (26.3)
.063
40 (33.6) 16 (13.4) 24 (20.2) 24 (20.2) 15 (12.6)
5 (21.7) 3 (13.0) 6 (26.1) 5 (21.7) 4 (17.4)
.69
81 (61.4) 51 (38.6)
9 (47.4) 10 (52.6)
.176
71 (59.7) 48 (40.3)
12 (52.2) 11 (47.8)
.415
.499
14 (11.8) 16 (13.4) 15 (12.6) 20 (16.8) 13 (10.9) 22 (18.5) 19 (16.0) 0 (0.00)
25 (18.9) 6 (4.5) 14 (10.6) 14 (10.6) 16 (12.1) 17 (12.9) 39 (29.5) 1 (0.8)
3 (15.8) 2 (10.5) 3 (15.8) 0 (0.00) 1 (5.3) 3 (15.8) 7 (36.8) 0 (0.00)
29.6 ± 12.2 29
37.8 ± 15.9 34
6.4 ± 9.3 3
6.7 ± 10.4 3
0.22 ± 0.27 0.11
0.18 ± 0.24 0.08
2 (8.7) 1 (4.3) 3 (13.0) 4 (17.4) 4 (17.4) 6 (26.1) 3 (13.0) 0 (0.00)
.659
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X. Ge et al.
Table 2
Comparison of Her2/neu expression status between the single-block group and the dual-block group HER2/neuimmunostaining, n (%) Single-block cohort
0 1+ 2+ 3+ Total
42 (31.8) 31 (23.5) 40 (30.3) 19 (14.4) 132
P value
Dual-block cohort The 1st paraffin block
P
43 (36.1) 27 (22.7) 31 (26.1) 18 (15.1) 119
.296
.391
of GC tumor tissues scored as 0, 1+, 2+, and 3+ for Her2/neu IHC assessment was 43 (36.1%), 27 (22.7%), 31 (26.1%), and 18 (15.1%), respectively (Table 2). For the second paraffin blocks of the dual-block group, the number of Her2/neu IHC 0, 1+, 2+, and 3+ was 53 (44.5%), 18 (15.1%), 27 (22.7%), and 21 (17.6%), respectively (Table 2).
The 2nd paraffin block 53 (44.5) 18 (15.1) 27 (22.7) 21 (17.6) 119
P
.738
Combination 36 (30.3) 26 (21.8) 34 (28.6) 23 (19.3) 119
Remarkably, the Her2/neu expression status varied from one paraffin block to another in some GC specimens in the dual-block group (Fig. 2). Her2/neu status of 83 (69.7%) cases was consistent between the 2 paraffin blocks, whereas 36 (30.3%) cases possess inconsistent Her2/neu status results (Table 3).
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Fig. 2 The intratumoral heterogeneity of Her2/neu overexpression in the dual-block group detected by IHC. The discrepancies of Her2/neu expression pattern between different paraffin blocks of 4 representative different cases (A-P). A-D, Case 1: Her2/neu expression was completely negative (B, IHC 0) in one paraffin block (A, hematoxylin-eosin staining); the other paraffin block was positive for Her2/neu (C, hematoxylin-eosin staining; D, IHC 3+). E-H, Case 2: Her2/neu immunostaining was 1+ (F) in one paraffin block (E, hematoxylin-eosin staining); the other paraffin block was positive for Her2/neu (G, hematoxylin-eosin staining; H, IHC 3+). I-L, Case 3: Her2/neu immunostaining was 2+ (J) in one paraffin block (I, hematoxylin-eosin staining); the other paraffin block was positive for Her2/neu (K, hematoxylin-eosin staining; L, IHC 3+). M-P, Case 4: Her2/neu expression was completely negative (N, IHC 0) in one paraffin block (M, hematoxylin-eosin staining); the other paraffin block was 2 + (P) (O, hematoxylin-eosin staining). Original magnifications ×1.25 (A-P).
Assessing Her2/neu with dual paraffin blocks Table 3 Consistency and inconsistency of Her2/neu expression pattern in the dual-block group Consistency, n (%) Consistent Inconsistent 0 vs 1+ 0 vs 2+ 0 vs 3+ 1+ vs 2+ 1+ vs 3+ 2+ vs 3+ Total
83 (69.7) 36 (30.3) 14 (38.9) 9 (25.0) 1 (2.8) 6 (16.7) 1 (2.8) 5 (13.9) 119
3.3. Increased Her2/neu-positive (3+) rate by using dual paraffin blocks We compared the results of the 2 cohorts. In the dual-block group, the number of Her2/neu IHC 1+, 2+, and 3+ cases was 26 (21.8%), 34 (28.6%), and 23 (19.3%), respectively (Table 2). The pooled data from 132 cases in the single-block group and 119 cases in the dual-block group showed that the Her2/neu-positive (3+) rate in the dual-block group was higher (Table 2). The Her2/neu-positive (3+) rate of the dual-block group was 19.3% compared to 14.4% in single-block group (P N .05) (Table 2). In the dual-block group, the Her2/neu-positive (3+) rate was increased from 15.1% and 17.6%, when evaluating only one paraffin block, to 19.3% (P N .05). Furthermore, 28% of patients in the dual-block group were IHC 2+ compared to 26.1% and 22.7% if only one paraffin block was used for Her2/neu IHC, implying a potentially higher Her2/neu positivity with further FISH evaluation (Table 2).
3.4. Correlation between Her2/neu expression and clinicopathological features A total of 19 GCs were classified as Her2/neu-positive (3+) in the single-block group and 23 GCs in the dual-block group (Table 1). The relationship of Her2/neu expression status with clinicopathological parameters is shown in Table 1. In the single-block group, Her2/neu-positivity (3+) was highly associated with higher pT stage of tumor according to the seventh edition of the UICC guidelines (P = .003). In the dual-block group, Her2/neu-positive (3+) GC was significantly associated with non–poorly cohesive differentiated histology (P = .037). No statistically significant differences in the Her2/neu-positive (3+) rates by sex, lymphatic invasion, or venous invasion were found for both cohorts (P N .05) (Table 1). The relative Her2/neu status is variable among the different patterns and the site of the neoplasm in the stomach (Supplementary Table 1).
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4. Discussion Intratumoral heterogeneity of GC has been an issue in Her2/neu status diagnosis in routine clinical practice to identify patients who can benefit from trastuzumab treatment. Heterogeneous Her2/neu overexpression adversely affects the accuracy of Her2/neu status assessment [25]. Our results showed that Her2/neu overexpression observed in 1 paraffin block may not be representative of the entire tumor. Because Her2/neu IHC is usually the first assay for the assessment of Her2/neu status of GC, it is of significance to get a more precise result as an important reference to identify patients who can benefit from targeted therapy. In this study, we collected 251 cases of primary GC (132 patients in the single-block group and 119 patients in the dual-block group) and performed Her2/neu analysis by IHC. Our results indicated that, when using dual paraffin blocks, the Her2/ neu-positive (3+) rate of GC could be increased (Table 2). To our knowledge, it is the first study comparing IHC 3+ rate using dual tumor tissue paraffin blocks to detect Her2/neu expression of GC with that using single block. The evidences of intratumoral heterogeneity of Her2/neu overexpression in GC are accumulating [26–30]. In fact, it would be very interesting to evaluate whether patients with focal 3+ staining can benefit from trastuzumab therapy. Recently, trastuzumab has been found to have beneficial effects for patients with heterogenetic Her2/neu-overexpressing tumor [31,32]. In agreement with Albarracin et al [20], we think that a precise report of Her2/neu status with critical evaluation of the results about Her2/neu heterogeneity should be promoted. The NCCN guidelines recommend that multiple biopsies (8-10 spots) should be carried out to provide adequate-sized material for histologic interpretation [22]. Therefore, for patients with unresectable GC, more accurate results of Her2/neu assessment could be obtained by assessing multiple biopsies. For patients with resectable GC, according to what has been recently established in an Italian consensus conference, even less than 10% of cells overexpressing Her2/neu in GC specimen should be added to the pathology report to clinicians [33]. However, this is not a pragmatic method in routine pathologic diagnosis, because there is not a standard criterion for clinicians to follow. Oncologists may not know how to interpret diagnostic reports and treat patients accordingly without guidance of the NCCN guidelines. Therefore, we need a more pragmatic and cost-effective method to minimize false-negative rate of Her2/neu assessment due to intratumoral heterogeneity of Her2/neu expression. Following the guidelines for handling of most common and important surgical specimens in Rosai and Ackerman's Surgical Pathology (10th Edition), 4 sections through wall and including tumor border and adjacent mucosa of GC were recommended. It is not cost-effective and practical to evaluate Her2/neu IHC status of all the 4 sampled paraffin blocks in routine clinicopathological diagnosis. However, picking 2 paraffin blocks for Her2/neu evaluation on 1 slide
856 is an economical, efficient, and practical method, without additional effort and expenses while giving a more accurate Her2/neu evaluation result. The 2 picked tissue paraffin blocks can be put on 1 slide to save extra reagents of IHC. In this study, our results showed that the Her2/ neu-positive (3+) rate could be increased from 14.4% to 17.6% by assessing 1 paraffin block to 19.3% by assessing 2 paraffin blocks from the same patient (Table 2). Although this result did not reach significance, it is still meaningful because many patients have benefited since this new protocol was executed. Notably, looking at the Her2/neu IHC inconsistent cases in the dual-block group, 5.6% (2/36) of these patients would be diagnosed as Her2/neu positive (IHC3+) and 41.7% (15/36) as Her2/neu equivocal cases (IHC2+), instead of Her2/neu negative (IHC 0 and 1+), if 2 paraffin blocks were used for Her2/neu IHC assessment (Table 3). This means that another 1.7% (2/119) of the whole population in the dual-block group would be diagnosed as IHC3+ and 12.6% (15/119) as IHC2+. Every year, more than 900 patients with resectable GC undergo curative surgery at Zhongshan Hospital; our results would imply an addition of more than 15 patients with Her2/neu IHC 3+ and more than 113 with Her2/neu IHC2+ who could be subjected to FISH assessment for Her2/neu amplification. Provided that the Her2/neu FISH positive rate is about 29% in our department, there would be 48 (15 IHC3+ plus 33 IHC2+/FISH+) additional patients with GC identified who could potentially benefit from trastuzumab treatment in our hospital. The method we used in this study may provide an alternative cost-effective way to handle the intratumoral heterogeneity issue. In this respect, we recommend that, whenever possible, the Her2/neu status could be assessed on dual paraffin blocks of gastrectomy specimens. The clinicopathological characteristics of the patients in both cohorts are generally balanced (Table 1). Previously, it has been reported that Her2/neu overexpression was more common in intestinal-type GCs compared with diffuse-type GCs [25,34]. Consistently, in current study, we showed that tumors of GCs that were Her2/neu positive (3+) were significantly associated with non–poorly cohesive differentiated histology in the dual-block group (P b .05) (Table 1). Previous reports indicated that Her2/neu overexpression status in GC is associated with poor outcomes and aggressive disease progression [5,34]. In this study, we found that Her2/ neu positivity (3+) was highly associated with higher pT stage of tumor in the single-block group. These findings are in line with previous observations [7,10,11,15].
5. Conclusions Using dual tumor tissue paraffin blocks in detecting Her2/neu expression status of GC is an efficient, economical, and practical method for Her2/neu evaluation in daily clinicopathological diagnosis. It can be executed without great additional effort, while providing a more accurate Her2/neu evaluation result. Minimizing false-negative rate of Her2/neu status assessment can help
X. Ge et al. identify more patients with Her2/neu-positive GC who may potentially benefit from Her2/neu-targeted therapy.
Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.humpath.2015.03.006.
References [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90. [2] Sugano K. Gastric cancer: pathogenesis, screening, and treatment. Gastrointest Endosc Clin N Am 2008;18:513-22. [3] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10-29. [4] Tai W, Mahato R, Cheng K. The role of HER2 in cancer therapy and targeted drug delivery. J Control Release 2010;146:264-75. [5] Gravalos C, Jimeno A. HER2 in gastric cancer: a new prognostic factor and a novel therapeutic target. Ann Oncol 2008;19:1523-9. [6] Marx AH, Tharun L, Muth J, et al. HER-2 amplification is highly homogenous in gastric cancer. HUM PATHOL 2009;40:769-77. [7] Jorgensen JT. Targeted HER2 treatment in advanced gastric cancer. Oncology 2010;78:26-33. [8] Sakai K, Mori S, Kawamoto T, et al. Expression of epidermal growth factor receptors on normal human gastric epithelia and gastric carcinomas. J Natl Cancer Inst 1986;77:1047-52. [9] Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histoclinical classification. Acta Pathol Microbiol Scand 1965;64:31-49. [10] Allgayer H, Babic R, Gruetzner KU, Tarabichi A, Schildberg FW, Heiss MM. c-erbB-2 is of independent prognostic relevance in gastric cancer and is associated with the expression of tumor-associated protease systems. J Clin Oncol 2000;18:2201-9. [11] Takehana T, Kunitomo K, Kono K, et al. Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked immuno-sorbent assay. Int J Cancer 2002;98:833-7. [12] Tan AR, Swain SM. Ongoing adjuvant trials with trastuzumab in breast cancer. Semin Oncol 2003;30:54-64. [13] Roukos DH. Targeting gastric cancer with trastuzumab: new clinical practice and innovative developments to overcome resistance. Ann Surg Oncol 2010;17:14-7. [14] Petrelli NJ, Winer EP, Brahmer J, et al. Clinical cancer advances 2009: major research advances in cancer treatment, prevention, and screening—a report from the American Society of Clinical Oncology. J Clin Oncol 2009;27:6052-69. [15] Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010;376:687-97. [16] Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN. The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist 2009;14:320-68. [17] Francis GD, Jones MA, Beadle GF, Stein SR. Bright-field in situ hybridization for HER2 gene amplification in breast cancer using tissue microarrays: correlation between chromogenic (CISH) and automated silver-enhanced (SISH) methods with patient outcome. Diagn Mol Pathol 2009;18:88-95. [18] Hofmann M, Stoss O, Shi D, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology 2008;52:797-805.
Assessing Her2/neu with dual paraffin blocks [19] Ruschoff J, Dietel M, Baretton G, et al. HER2 diagnostics in gastric cancer—guideline validation and development of standardized immunohistochemical testing. Virchows Arch 2010;457:299-307. [20] Albarracin C, Edgerton ME, Gilcrease MZ, et al. Is it too soon to start reporting HER2 genetic heterogeneity? Arch Pathol Lab Med 2010; 134:162-3 [author reply 163]. [21] Seol H, Lee HJ, Choi Y, et al. Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance. Mod Pathol 2012;25:938-48. [22] Ajani JA, Barthel JS, Bekaii-Saab T, et al. Gastric cancer. J Natl Compr Canc Netw 2010;8:378-409. [23] Bosman FT, Carneiro F, Hruban RH, Theise ND. WHO classification of tumours of the digestive system. Lyon, France: International Agency for Research on Cancer (IARC); 2010. [24] Sobin LH, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumours. 7th ed. Oxford: Wiley-Blackwell; 2009. [25] Warneke VS, Behrens HM, Boger C, et al. Her2/neu testing in gastric cancer: evaluating the risk of sampling errors. Ann Oncol 2013;24:725-33. [26] Bozzetti C, Negri FV, Lagrasta CA, et al. Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma. Br J Cancer 2011;104:1372-6. [27] Abraham SC, Park SJ, Lee JH, Mugartegui L, Wu TT. Genetic alterations in gastric adenomas of intestinal and foveolar phenotypes. Mod Pathol 2003;16:786-95.
857 [28] Kim MA, Lee HJ, Yang HK, Bang YJ, Kim WH. Heterogeneous amplification of ERBB2 in primary lesions is responsible for the discordant ERBB2 status of primary and metastatic lesions in gastric carcinoma. Histopathology 2011;59:822-31. [29] Tafe LJ, Janjigian YY, Zaidinski M, et al. Human epidermal growth factor receptor 2 testing in gastroesophageal cancer: correlation between immunohistochemistry and fluorescence in situ hybridization. Arch Pathol Lab Med 2011;135:1460-5. [30] Yan B, Yau EX, Bte Omar SS, et al. A study of HER2 gene amplification and protein expression in gastric cancer. J Clin Pathol 2010;63:839-42. [31] Sukov WR, Duek AC, Tenner KS, et al. Benefit of adjuvant trastuzumab in breast cancer patients with focal HER-2 amplified colonies: data from N9831 Intergroup Adjuvant Trial [abstract]. J Clin Oncol 2009;15s:520 [Suppl.]. [32] Arena V, Pennacchia I, Vecchio FM, Carbone A. HER-2 intratumoral heterogeneity. Mod Pathol 2013;26:607-9. [33] Consensus workshop e Raccomandazioni sull’impiego delle diverse metodiche per la determinazione dello stato di HER2 nel carcinoma mammario e nel carcinoma gastrico. Catania 2010. [34] Tanner M, Hollmen M, Junttila TT, et al. Amplification of HER-2 in gastric carcinoma: association with topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab. Ann Oncol 2005;16:273-8.
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Original contribution
Clinical significance of assessing Her2/neu expression in gastric cancer with dual tumor tissue paraffin blocks☆,☆☆ Xiaowen Ge MD, PhD a,1 , Haixing Wang MD a,1 , Haiying Zeng BS a , Xuejuan Jin MD, PhD b , Akesu Sujie BS a , Chen Xu MD a , Yalan Liu MD, PhD a , Jie Huang MS a , Yuan Ji MD, PhD a , Yunshan Tan MD, PhD a , Tianshu Liu MD, PhD c , Yingyong Hou MD, PhD a,⁎, Jing Qin MD, PhD d,⁎, Yihong Sun MD, PhD d , Xinyu Qin MD, PhD d a
Department of Pathology, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China c Department of Oncology, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China d Department of General Surgery, Zhongshan Hospital, Fudan University, Fenglin Rd #180, Shanghai, 200032, PR China b
Received 11 December 2014; revised 16 February 2015; accepted 19 February 2015
Keywords: Gastric cancer; Her2/neu; Dual paraffin blocks; Trastuzumab; Immunohistochemistry
Summary One paraffin block is routinely used for human epidermal growth factor receptor 2 (Her2/neu) immunohistochemistry (IHC) assessment. Here, we investigated if picking 2 paraffin blocks for Her2/neu evaluation on 1 slide is an economical, efficient, and practical method, which may reduce false negativity of Her2/neu IHC assessment due to intratumoral heterogeneity. A total of 251 gastric cancer (GC) patients were divided into a cohort using 1 tumor tissue paraffin block (single-block group, n = 132) and a cohort using dual tumor tissue paraffin blocks (dual-block group, n = 119) when evaluating Her2/neu expression status by IHC. In dual-block group, we combined the results from 2 different paraffin blocks and used the higher one as the final score. The number of IHC 1+, 2+, and 3+ specimens in the single-block group was 31 (23.5%), 40 (30.3%), and 19 (14.4%), respectively. The combined final IHC score in the dual-block group of 1+, 2+, and 3+ was 26 (21.8%), 34 (28.6%), and 23 (19.3%), respectively. Inconsistent Her2/neu expression between blocks was found in 36 (30.3%) cases in the dual-block group. The pooled data in the single-block group and the dual-block group indicated that, when using dual blocks, the Her2/neu-positive (3+) rate of GC was higher compared to that in the single-block group. Our results implied that using dual paraffin blocks to assess Her2/neu expression of GC may help identify more patients with Her2/neu-positive GC who could benefit from targeted therapy, by reducing false-negative rate of Her2 status assessment. This is an efficient, economical, and practical method for Her2/neu evaluation of GC. © 2015 Elsevier Inc. All rights reserved.
☆ This work was supported by the Research Fund of Zhongshan Hospital for Young Scientists (grant 2013ZSQN25), Shanghai, China and Research Fund from Shanghai Technology and Science Commission (grant 13140901700), Shanghai, China. ☆☆ Disclosure: The authors have declared no conflicts of interest. ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Hou), [email protected] (J. Qin). 1 Xiaowen Ge and Haixing Wang contributed equally to this work.
http://dx.doi.org/10.1016/j.humpath.2015.02.011 0046-8177/© 2015 Elsevier Inc. All rights reserved.
Assessing Her2/neu with dual paraffin blocks
1. Introduction Gastric cancer (GC) is major global health concern and the second leading cause of cancer deaths worldwide [1]. Incidence of GC is particularly high in Japan, China, South Korea, and Chile. Approximately 65% of GC patients were diagnosed as locally advanced or metastatic stages of the disease [2]. Patients with GC have poor prognosis and few efficacious therapeutic options, particularly in advanced stages; and the mortality rate of GC is decreasing [3]. Five-year survival rate of patients with advanced or metastatic GC is around 5% to 20%, with median overall survival being less than 1 year. Most patients with stage I to III GC can be cured with curative resection. However, the recurrence rate of these patients is high. The importance of adjuvant treatment is emphasized recently, especially for metastatic and recurrent cases. Human epidermal growth factor receptor 2 (Her2), also known as Her2/neu, cerbB2, or ErbB2, is a 185-kd protein [4]. Her2/neu has an extracellular ligand binding domain, a short transmembrane domain, and an intracellular tyrosine kinase domain. Amplification of HER2/NEU gene and/or overexpression of the Her2/neu protein has been observed in many human carcinomas, including GC [5–7]. The first description of Her2/ neu overexpression in GC was reported in 1986 [8]. Adenocarcinoma is the predominant histologic type of GC (95% of tumors), which is classified into 3 subtypes: intestinal, diffuse, and mixed, based on the Laurén classification [9]. GC originating from the gastroesophageal junction is usually the intestinal type, which possesses higher prevalence of HER2/NEU gene amplification (2%-45%) compared to other Laurén subtypes (8.2%-53.4%) [7,10,11]. Overall, the overexpression prevalence of Her2/neu of GC remains controversial. Trastuzumab (Herceptin, Roche, Basel, Switzerland), an anti-Her2/neu antibody, which is recommended for treatment of Her2/ neu-positive breast cancer (BC) [12], has been proven effective for GC. GC is the second type of cancer in which trastuzumab has been proven effective [13]. According to the Trastuzumab for Gastric Cancer clinical trial reported in 2010, trastuzumabin in combination with chemotherapy significantly prolonged the median overall survival time to 13.8 months compared to 11 months in patients treated with chemotherapy alone [14,15]; in Her2/neu 3+ patients, overall survival time could be prolonged to 16 months [14,15]. There are several methods available for the detection of Her2/ neu protein overexpression and gene amplification, including immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), chromogenic in situ hybridization [16], and silver in situ hybridization [17]. In 2008, Hofmann et al [18] established the assessment criteria of Her2/neu overexpression and gene amplification for GC. These criteria then have been validated by other researchers [19]. Intratumoral heterogeneity of Her2/ neu overexpression and amplification has been observed by many pathologists for BC [20,21] and GC [18]. Notably, intratumoral heterogeneity in GC is much more significant than that in BC. In addition to tumor site, this heterogeneity of Her2/ neu overexpression and amplification may be due to sample
851
A
B
Fig. 1 The entire view of 1 slide using 2 paraffin blocks detecting Her2/neu expression by IHC. The consistent Her2/neu expression of 2 paraffin blocks of 2 representative cases in the dual-block group (A-B). A, Her2/neu expression was negative (IHC 0). B, Her2/neu expression was positive (IHC 3+). The red arrow: positive tissue controls (IHC 3+). The blue arrow: negative tissue controls (IHC 0).
size, population diversity, interobserver variability, and inconsistent specimen processing and tumor sampling among different laboratories. When using FISH for HER2/NEU status assessment, the intratumoral heterogeneity could be more common because of the thinner field of view for diagnosis. Hence, it is difficult for a pathologist to report the exact status of Her2/neu overexpression and amplification [20]. Usually, pathologists classify Her2/neu expression of GC tissue with 1 paraffin block for economical and practical reasons. Currently, the number of paraffin blocks that should be used in Her2/neu evaluation is not mentioned in the National Comprehensive Cancer Network (NCCN) guidelines [22]. Coping with intratumoral heterogeneity, assessing specimens from 2 paraffin blocks of the same patient could reduce false-negative rate of Her2/neu assessment of GC. In our laboratory, 2 specimens are fit onto a single slide for Her2/neu IHC (Fig. 1); thus, we sought to see if it is a relatively cost-effective method to identify Her2/neu-positive patients and reduce false-negative rate of Her2 IHC. In the present study, Her2/neu expression status of 251 patients was analyzed together with clinicopathological features. We compared the positive rate of Her2/neu expression between a cohort using 1 paraffin block and a cohort using dual tumor tissue paraffin blocks. The aim of the current study is to identify the clinical significance of using dual tumor tissue paraffin blocks in detecting Her2/neu expression status of GC.
2. Materials and methods 2.1. Patients and clinicopathological information collection Between March 2013 and August 2013, a total of 251 patients with primary gastric adenocarcinoma who received curative surgery without any preoperative treatment at the
852 Zhongshan Hospital were included in this study. Those patients were divided into a cohort using 1 tumor tissue paraffin block (single-block group, n = 132) and a cohort using dual tumor tissue paraffin blocks (dual-block group, n = 119) when evaluating Her2/neu expression status by IHC. Prior written informed consent was collected from all patients. The study protocol was approved by the ethics board at the Zhongshan Hospital, Fudan University, Shanghai, China. The diagnosis of GC was confirmed by histology in all cases. The following patient characteristics were collected: age, sex, histologic type (adenocarcinoma only based on World Health Organization criteria) [23], Laurén classification [9], tumor site (proximal and distal), histologic grade, microscopic tumor extension, lymphatic or venous invasion, lymph node metastasis, and pTNM stage (according to seventh edition of the Union for International Cancer Control (UICC) guidelines) [24].
2.2. Hematoxylin and eosin and IHC staining
X. Ge et al. criteria defined by Hofman et al [18]: no staining or less than 10% tumor cell positive staining as 0/negative; faintly or barely perceptible staining on at least 10% tumor cell membrane as 1+; weak to moderate positive staining on at least 10% tumor cell membrane as 2+; and cohesive moderate to strong staining on at least 10% tumor cell membrane as 3+. In this study, we classified IHC3+ as Her2/neu positive. For cohort 2 (dual blocks), we combined the results from 2 different paraffin blocks and used the higher score as the final Her2/neu IHC score.
2.4. Statistical analysis A χ2 test was used for univariate analysis; crosstabulations with qualitative variables were analyzed with the Pearson χ2 test. P value b .05 was defined as statistically significant. No adjustments were made. All analyses were performed using the statistical package SPSS version 19.0 (SPSS, Inc, an IBM Company, Chicago, IL).
GC tissues from surgical specimens were fixed in 10% buffered formalin within 30 minutes after resection. Specimens were processed following routine procedures after 24 hours of fixation. Sections were stained with hematoxylin and eosin and reviewed by 2 pathologists to confirm the diagnosis of GC. Paraffin blocks of each patient were selected by trained pathologists for subsequent IHC assessment. IHC assay using Her2/neu rabbit monoclonal antibody (clone 4B5; Ventana, Tucson, AZ) was performed with iView DAB Detection Kit (Ventana, Tucson, AZ) on a BenchMark XT automated staining system (Ventana, Tucson, AZ). In brief, the tissue sections were deparaffinized with EZ Prep (Ventana, Tucson, AZ) at 75°C and heat pretreated in Cell Conditioning 1 (Ventana, Tucson, AZ) using “standard cell conditioning” for antigen retrieval at 95°C. Tissue sections were then incubated with anti-Her2/neu primary antibody for 24 minutes at 37°C after inactivation of the endogenous peroxidase by hydrogen peroxide for 4 minutes. The tissue sections were then blocked using Endogenous Biotin Blocking Kit (Ventana, Tucson, AZ) and incubated with a biotinylated secondary antibody for 8 minutes and then with a streptavidin–horseradish peroxidase conjugate for 8 minutes at 37°C. The slides were then counterstained with Hematoxylin II (Ventana, Tucson, AZ) for 8 minutes and Bluing Reagent (Ventana, Tucson, AZ) for 8 minutes. Normal immunoglobulin G from the same species of primary antibody diluted to matching concentration of the primary antibody was used as the negative control. In the same slide, small pieces of BC tissue that Her2/neu IHC scored as 3+ and 0 were used as positive and negative controls, respectively (Fig. 1).
Surgical samples were obtained from primary tumors of 251 patients with GC. Clinicopathological characteristics of the GC patients are presented in Table 1. One hundred thirty-two patients were enrolled in cohort 1 (single-block group) and 119 patients in cohort 2 (dual-block group). In the single-block group, the age of the patients ranged from 27 to 82 years with a median of 59 years. Ninety (68.2%) patients were male, and 42 (31.8%) were female. According to the Laurén classification, 41 (31.1%) patients were diagnosed as intestinal type of GC, 53 (40.2%) were diffuse type, and 38 (28.8%) were mixed type. Based on cell cohesive status, 53 (40.2%) were graded as poorly cohesive and 79 (59.8%) were non–poorly cohesive. Lymphatic or venous invasion was found in 51 (38.6%) patients (Table 1). In the dual-block group, the age of the patients ranged from 28 to 84 years with a median of 62 years. Eighty-six (72.3%) patients were male, and 33 (27.7%) were female. According to the Laurén classification, 44 (37.0%) patients were intestinal type GC, 37 (31.1%) patients were diffuse type, and 38 (31.9%) patients were mixed type. Of these patients, 37 (31.1%) were poorly cohesive and 82 (68.9%) were non–poorly cohesive. Lymphatic or venous invasion was found in 48 (40.3%) patients (Table 1).
2.3. Definitions of Her2/neu alteration
3.2. Her2/neu protein expression status by IHC
The Her2/neu status was assessed by 2 independent observers. If there was any discrepancy, the Her2/neu status was verified by a discussion panel consisting of 3 observers. All observers were blinded with regard to the clinicopathological patient characteristics. Specimens were assessed according to the IHC scoring
In the single-block group, 42 (31.8%) cases were scored 0 for Her2/neu IHC, whereas the number of cases scored as 1+, 2+, and 3+ was 31 (23.5%), 40 (30.3%), and 19 (14.4%), respectively (Table 2). For the dual-block group, first paraffin blocks were randomly selected for IHC assessment. The number
3. Results 3.1. Characteristics of patients
Assessing Her2/neu with dual paraffin blocks
853
Table 1 Clinicopathological gastric cancer patient characteristics and correlations of Her2/neu-positive (3+) rates with clinicopathologic variables Cohort 1 Clinical features Patients (n) Age (y) Mean ± SD Median Sex, n (%) Male Female Laurén phenotype, n (%) Intestinal Diffuse Mixed Combined phenotype, n (%) Poorly cohesive Non–poorly cohesive Localization, n (%) Proximal Distal pT category, n (%) pT1a pT1b pT2 pT3 pT4a pT4b pN category, n (%) pN0 pN1 pN2 pN3a pN3b Lymphatic or venous invasion, n (%) pL/V0 pL/V1 UICC stage (7th edition), n (%) IA IB IIA IIB IIIA IIIB IIIC IV Resected lymph nodes Mean ± SD Median, n Positive lymph nodes Mean ±SD Median, n Positive lymph node ratio Mean ± SD Median ⁎ P b .05.
132
Cohort 2 HER2 3+, n (%)
P
19
Clinical features 119
59.6 ± 10.9 59
HER2 3+, n (%)
P
23
60.7 ± 10.7 62
90 (68.2) 42 (31.8)
13 (68.4) 6 (31.6)
.981
86 (72.3) 33 (27.7)
18 (78.3) 5 (21.7)
.475
41+ (31.1) 53 (40.2) 38 (28.8)
8 (42.1) 5 (26.3) 6 (31.6)
.516
44 (37.0) 37 (31.1) 38 (31.9)
10 (43.5) 10 (43.5) 3 (13.0)
.105
53 (40.2) 79 (59.8)
13 (68.4) 6 (31.6)
.41
37 (31.1) 82 (68.9)
3 (13.0) 20 (87.0)
.037 ⁎
40 (30.3) 92 (69.7)
7 (36.8) 12 (63.2)
.503
41 (34.5) 78 (65.5)
10 (43.5) 13 (56.5)
.311
16 (12.1) 16 (12.1) 8 (6.1) 25 (18.9) 65 (49.2) 2 (1.5)
2 (10.5) 3 (15.8) 3 (15.8) 1 (5.3) 8 (42.1) 2 (10.5)
.003 ⁎
7 (5.9) 14 (11.8) 25 (21.0) 43 (36.1) 30 (25.2) 0 (0.00)
0 (0.00) 3 (13.0) 5 (21.7) 9 (39.1) 6 (26.1) 0 (0.00)
.772
45 (34.1) 19 (14.4) 18 (13.6) 34 (25.8) 16 (12.1)
5 (26.3) 2 (10.5) 5 (26.3) 2 (10.5) 5 (26.3)
.063
40 (33.6) 16 (13.4) 24 (20.2) 24 (20.2) 15 (12.6)
5 (21.7) 3 (13.0) 6 (26.1) 5 (21.7) 4 (17.4)
.69
81 (61.4) 51 (38.6)
9 (47.4) 10 (52.6)
.176
71 (59.7) 48 (40.3)
12 (52.2) 11 (47.8)
.415
.499
14 (11.8) 16 (13.4) 15 (12.6) 20 (16.8) 13 (10.9) 22 (18.5) 19 (16.0) 0 (0.00)
25 (18.9) 6 (4.5) 14 (10.6) 14 (10.6) 16 (12.1) 17 (12.9) 39 (29.5) 1 (0.8)
3 (15.8) 2 (10.5) 3 (15.8) 0 (0.00) 1 (5.3) 3 (15.8) 7 (36.8) 0 (0.00)
29.6 ± 12.2 29
37.8 ± 15.9 34
6.4 ± 9.3 3
6.7 ± 10.4 3
0.22 ± 0.27 0.11
0.18 ± 0.24 0.08
2 (8.7) 1 (4.3) 3 (13.0) 4 (17.4) 4 (17.4) 6 (26.1) 3 (13.0) 0 (0.00)
.659
854
X. Ge et al.
Table 2
Comparison of Her2/neu expression status between the single-block group and the dual-block group HER2/neuimmunostaining, n (%) Single-block cohort
0 1+ 2+ 3+ Total
42 (31.8) 31 (23.5) 40 (30.3) 19 (14.4) 132
P value
Dual-block cohort The 1st paraffin block
P
43 (36.1) 27 (22.7) 31 (26.1) 18 (15.1) 119
.296
.391
of GC tumor tissues scored as 0, 1+, 2+, and 3+ for Her2/neu IHC assessment was 43 (36.1%), 27 (22.7%), 31 (26.1%), and 18 (15.1%), respectively (Table 2). For the second paraffin blocks of the dual-block group, the number of Her2/neu IHC 0, 1+, 2+, and 3+ was 53 (44.5%), 18 (15.1%), 27 (22.7%), and 21 (17.6%), respectively (Table 2).
The 2nd paraffin block 53 (44.5) 18 (15.1) 27 (22.7) 21 (17.6) 119
P
.738
Combination 36 (30.3) 26 (21.8) 34 (28.6) 23 (19.3) 119
Remarkably, the Her2/neu expression status varied from one paraffin block to another in some GC specimens in the dual-block group (Fig. 2). Her2/neu status of 83 (69.7%) cases was consistent between the 2 paraffin blocks, whereas 36 (30.3%) cases possess inconsistent Her2/neu status results (Table 3).
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Fig. 2 The intratumoral heterogeneity of Her2/neu overexpression in the dual-block group detected by IHC. The discrepancies of Her2/neu expression pattern between different paraffin blocks of 4 representative different cases (A-P). A-D, Case 1: Her2/neu expression was completely negative (B, IHC 0) in one paraffin block (A, hematoxylin-eosin staining); the other paraffin block was positive for Her2/neu (C, hematoxylin-eosin staining; D, IHC 3+). E-H, Case 2: Her2/neu immunostaining was 1+ (F) in one paraffin block (E, hematoxylin-eosin staining); the other paraffin block was positive for Her2/neu (G, hematoxylin-eosin staining; H, IHC 3+). I-L, Case 3: Her2/neu immunostaining was 2+ (J) in one paraffin block (I, hematoxylin-eosin staining); the other paraffin block was positive for Her2/neu (K, hematoxylin-eosin staining; L, IHC 3+). M-P, Case 4: Her2/neu expression was completely negative (N, IHC 0) in one paraffin block (M, hematoxylin-eosin staining); the other paraffin block was 2 + (P) (O, hematoxylin-eosin staining). Original magnifications ×1.25 (A-P).
Assessing Her2/neu with dual paraffin blocks Table 3 Consistency and inconsistency of Her2/neu expression pattern in the dual-block group Consistency, n (%) Consistent Inconsistent 0 vs 1+ 0 vs 2+ 0 vs 3+ 1+ vs 2+ 1+ vs 3+ 2+ vs 3+ Total
83 (69.7) 36 (30.3) 14 (38.9) 9 (25.0) 1 (2.8) 6 (16.7) 1 (2.8) 5 (13.9) 119
3.3. Increased Her2/neu-positive (3+) rate by using dual paraffin blocks We compared the results of the 2 cohorts. In the dual-block group, the number of Her2/neu IHC 1+, 2+, and 3+ cases was 26 (21.8%), 34 (28.6%), and 23 (19.3%), respectively (Table 2). The pooled data from 132 cases in the single-block group and 119 cases in the dual-block group showed that the Her2/neu-positive (3+) rate in the dual-block group was higher (Table 2). The Her2/neu-positive (3+) rate of the dual-block group was 19.3% compared to 14.4% in single-block group (P N .05) (Table 2). In the dual-block group, the Her2/neu-positive (3+) rate was increased from 15.1% and 17.6%, when evaluating only one paraffin block, to 19.3% (P N .05). Furthermore, 28% of patients in the dual-block group were IHC 2+ compared to 26.1% and 22.7% if only one paraffin block was used for Her2/neu IHC, implying a potentially higher Her2/neu positivity with further FISH evaluation (Table 2).
3.4. Correlation between Her2/neu expression and clinicopathological features A total of 19 GCs were classified as Her2/neu-positive (3+) in the single-block group and 23 GCs in the dual-block group (Table 1). The relationship of Her2/neu expression status with clinicopathological parameters is shown in Table 1. In the single-block group, Her2/neu-positivity (3+) was highly associated with higher pT stage of tumor according to the seventh edition of the UICC guidelines (P = .003). In the dual-block group, Her2/neu-positive (3+) GC was significantly associated with non–poorly cohesive differentiated histology (P = .037). No statistically significant differences in the Her2/neu-positive (3+) rates by sex, lymphatic invasion, or venous invasion were found for both cohorts (P N .05) (Table 1). The relative Her2/neu status is variable among the different patterns and the site of the neoplasm in the stomach (Supplementary Table 1).
855
4. Discussion Intratumoral heterogeneity of GC has been an issue in Her2/neu status diagnosis in routine clinical practice to identify patients who can benefit from trastuzumab treatment. Heterogeneous Her2/neu overexpression adversely affects the accuracy of Her2/neu status assessment [25]. Our results showed that Her2/neu overexpression observed in 1 paraffin block may not be representative of the entire tumor. Because Her2/neu IHC is usually the first assay for the assessment of Her2/neu status of GC, it is of significance to get a more precise result as an important reference to identify patients who can benefit from targeted therapy. In this study, we collected 251 cases of primary GC (132 patients in the single-block group and 119 patients in the dual-block group) and performed Her2/neu analysis by IHC. Our results indicated that, when using dual paraffin blocks, the Her2/ neu-positive (3+) rate of GC could be increased (Table 2). To our knowledge, it is the first study comparing IHC 3+ rate using dual tumor tissue paraffin blocks to detect Her2/neu expression of GC with that using single block. The evidences of intratumoral heterogeneity of Her2/neu overexpression in GC are accumulating [26–30]. In fact, it would be very interesting to evaluate whether patients with focal 3+ staining can benefit from trastuzumab therapy. Recently, trastuzumab has been found to have beneficial effects for patients with heterogenetic Her2/neu-overexpressing tumor [31,32]. In agreement with Albarracin et al [20], we think that a precise report of Her2/neu status with critical evaluation of the results about Her2/neu heterogeneity should be promoted. The NCCN guidelines recommend that multiple biopsies (8-10 spots) should be carried out to provide adequate-sized material for histologic interpretation [22]. Therefore, for patients with unresectable GC, more accurate results of Her2/neu assessment could be obtained by assessing multiple biopsies. For patients with resectable GC, according to what has been recently established in an Italian consensus conference, even less than 10% of cells overexpressing Her2/neu in GC specimen should be added to the pathology report to clinicians [33]. However, this is not a pragmatic method in routine pathologic diagnosis, because there is not a standard criterion for clinicians to follow. Oncologists may not know how to interpret diagnostic reports and treat patients accordingly without guidance of the NCCN guidelines. Therefore, we need a more pragmatic and cost-effective method to minimize false-negative rate of Her2/neu assessment due to intratumoral heterogeneity of Her2/neu expression. Following the guidelines for handling of most common and important surgical specimens in Rosai and Ackerman's Surgical Pathology (10th Edition), 4 sections through wall and including tumor border and adjacent mucosa of GC were recommended. It is not cost-effective and practical to evaluate Her2/neu IHC status of all the 4 sampled paraffin blocks in routine clinicopathological diagnosis. However, picking 2 paraffin blocks for Her2/neu evaluation on 1 slide
856 is an economical, efficient, and practical method, without additional effort and expenses while giving a more accurate Her2/neu evaluation result. The 2 picked tissue paraffin blocks can be put on 1 slide to save extra reagents of IHC. In this study, our results showed that the Her2/ neu-positive (3+) rate could be increased from 14.4% to 17.6% by assessing 1 paraffin block to 19.3% by assessing 2 paraffin blocks from the same patient (Table 2). Although this result did not reach significance, it is still meaningful because many patients have benefited since this new protocol was executed. Notably, looking at the Her2/neu IHC inconsistent cases in the dual-block group, 5.6% (2/36) of these patients would be diagnosed as Her2/neu positive (IHC3+) and 41.7% (15/36) as Her2/neu equivocal cases (IHC2+), instead of Her2/neu negative (IHC 0 and 1+), if 2 paraffin blocks were used for Her2/neu IHC assessment (Table 3). This means that another 1.7% (2/119) of the whole population in the dual-block group would be diagnosed as IHC3+ and 12.6% (15/119) as IHC2+. Every year, more than 900 patients with resectable GC undergo curative surgery at Zhongshan Hospital; our results would imply an addition of more than 15 patients with Her2/neu IHC 3+ and more than 113 with Her2/neu IHC2+ who could be subjected to FISH assessment for Her2/neu amplification. Provided that the Her2/neu FISH positive rate is about 29% in our department, there would be 48 (15 IHC3+ plus 33 IHC2+/FISH+) additional patients with GC identified who could potentially benefit from trastuzumab treatment in our hospital. The method we used in this study may provide an alternative cost-effective way to handle the intratumoral heterogeneity issue. In this respect, we recommend that, whenever possible, the Her2/neu status could be assessed on dual paraffin blocks of gastrectomy specimens. The clinicopathological characteristics of the patients in both cohorts are generally balanced (Table 1). Previously, it has been reported that Her2/neu overexpression was more common in intestinal-type GCs compared with diffuse-type GCs [25,34]. Consistently, in current study, we showed that tumors of GCs that were Her2/neu positive (3+) were significantly associated with non–poorly cohesive differentiated histology in the dual-block group (P b .05) (Table 1). Previous reports indicated that Her2/neu overexpression status in GC is associated with poor outcomes and aggressive disease progression [5,34]. In this study, we found that Her2/ neu positivity (3+) was highly associated with higher pT stage of tumor in the single-block group. These findings are in line with previous observations [7,10,11,15].
5. Conclusions Using dual tumor tissue paraffin blocks in detecting Her2/neu expression status of GC is an efficient, economical, and practical method for Her2/neu evaluation in daily clinicopathological diagnosis. It can be executed without great additional effort, while providing a more accurate Her2/neu evaluation result. Minimizing false-negative rate of Her2/neu status assessment can help
X. Ge et al. identify more patients with Her2/neu-positive GC who may potentially benefit from Her2/neu-targeted therapy.
Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.humpath.2015.03.006.
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