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Year : 2019 | Volume : 7 | Issue : 2 | Page : 53 - 58  


Original Articles
Human Epidermal Growth Factor Receptor 2 (HER2) as Surrogate Prognostic Marker in Gastric and Gastro Esophageal Junction Cancer

Shareefa Akhter1, Nassima Chanda2, Rumana Hamid3, Malik Tariq Rasool4

1Senior Resident, Department of Hematopathology, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, India

2Professor, Department of Pathology, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, India

3Professor, Department of Pathology, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, India

4Associate Professor, Radiation Oncology, Regional Cancer Centre, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar 

*Corresponding Author

Dr. Shareefa Akhter                                                                                                        

Email: malikshareefa@gmail.com                                                                                  

Abstract:

Background: HER2 is an important biomarker in gastric and gastroesophageal junction tumors. Expression of HER2 status by immunohistochemistry is heterogeneous in various patient populations. This overexpression appears to be an important prognostic factor in gastric cancer and its association with other poor prognostic factors may therefore act as a surrogate marker for poor prognosis in gastric carcinoma. We studied our patient population to look for any such association between HER2 status and various clinicopathological parameters.

Objective: To study the association between HER2 status and various clinicopathological parameters.

Methods: Patients were prospectively enrolled from August 2013 to March 2015. All relevant patient parameters were recorded. After curative resection, surgical specimens of gastric and gastroesophageal junction adenocarcinoma were analyzed for immunohistochemistry for HER2 by using standard methods. IHC score followed Hofmanns criteria. Statistical analysis of relation between HER2 status and relevant patient and disease characteristics such as gender, age at diagnosis, tumor location, histological type of tumor, stage, and size of tumor, was performed.

Results: 106 patients were enrolled into this study. Only 18 (17%) cases had HER2 score of 3+, whereas 5 (4.7%) had equivocal score (2+) and the remaining 84 (78%) tumors tested negative. HER2 status was significantly associated with location of primary tumor, gender, histopathological type of cancer, and size of primary tumor. There was no significant relation between HER2 status and age of patient, depth of invasion, nodal metastasis, or overall stage.

Conclusion: Further studies are necessary to determine the significance of HER2 status in upper gastrointestinal cancers especially that the patients need follow up where we can predict the HER2 status as an independent prognostic factor for overall survival.

Keywords: Gastric carcinoma, HER2, Pathological characteristics, Immunohistochemistry, surrogate marker

INTRODUCTION:

Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of death from cancer worldwide. 1

In Kashmir, gastric cancer comprises 9% of all cancers and is third most common cancer in men and sixth most common cancer in women. 2

Age at the time of diagnosis, tumour size, grade of primary tumor, depth of gastric wall penetration and nodal involvement, 3, 4 histological type of tumor and the presence of distant metastasis have been associated prognostic factors for survival in patients with gastric cancer. The molecular biology of gastric cancer reflects the heterogeneity of its causes and its histologic subtypes. Identification of the genetic and phenotypic variables existing among gastric cancers may lead to more directed therapeutic approaches and a more accurate prediction of clinical outcome. Epidermal growth factor receptors and epidermal growth factor levels have also been studied in gastric carcinoma. The pathophysiologic relation between these peptide receptors and their association with prognosis is not well understood.

The role of human epidermal factor receptor (HER2) is being studied extensively for its role as a prognostic marker and potential target for monoclonal antibodies. The HER2 gene, located on chromosome 17, is constitutively expressed in many types of normal epithelium. It is a 185-kDa transmembrane tyrosine kinase receptor, 5 and its gene amplification and protein overexpression play an important role in the proliferation, apoptosis, adhesion, angiogenesis and aggressiveness of many solid tumors. 6

There is increasing evidence that HER2 is an important biomarker in gastric and gastroesophageal junction tumors. 7

Analysis of HER2 status by immunohistochemistry and in-situ hybridization techniques using different scoring methods or assays suggests that HER2 is over expressed in approximately 7-34 percent of gastric tumors. 8 

HER2 appears  to be an  important prognostic  factor in  gastric  cancer;  however,  the  literature  is conflicting  in  this  respect, and not all studies have  shown  a  clear  association  between HER2  over-expression  and  poor  prognosis. So far, HER2 status in upper gastrointestinal adenocarcinomas has not been studied in Kashmiri population, where gastric carcinoma is endemic. 

METHODS:

After ethical clearance from the Institute Ethical Committee (IEC), patients were prospectively enrolled from August 2013 to March 2015. All surgical specimens of patients with gastric and gastroesophageal junction adenocarcinoma, irrespective of age, gender, or stage of disease and patients not having received any form of neo-adjuvant chemotherapy, radiotherapy or targeted therapy, operated at our hospital were included in the study. Baseline data and patient characteristics were collected from Hospital Based Cancer Registry (HBCR) of Regional Cancer Centre (RCC). Formalin-fixed, paraffin-embedded samples of tumors and corresponding normal stomach or gastroesophageal junction tissue from same specimens were evaluated for HER2 protein overexpression using immunohistochemistry (IHC). A representative block of the cancer and normal adjacent tissue was prepared from surgical specimens. Specimens were inked and fixed.  The date and time of specimen acquisition was recorded.

Immuno-Histochemical staining

HER2 IHC analysis was performed on 4 µm thick tissue sections. Briefly, after deparaffinization and rehydration steps, the tissue samples were incubated in antigen retrieval solution at 99 ℃ for 40 minutes. Endogenous peroxidase activity was quenched by 5 minute incubation with hydrogen peroxide. Sections were then incubated with HER2 antibody for 30 minutes at room temperature and then the immune-complexes were visualized with diaminobenzidine for 10 minutes and placed under a cover slip. Finally, the slides were viewed using light microscopy.

Positive and negative controls were used in all of the cases. Wherever there was a discrepancy, fresh slides were prepared and IHC was repeated with both positive and negative controls.

Scoring of Results and Statistical Analysis

IHC score criteria followed Hofmanns criteria. 8 Analysis of p-value was done with Pearson Chi-Square, Fishers Exact Test, Likelihood Ratio, and Linear-by-Linear Association. The statistical methods were performed to study the HER2 positivity rate amongst the subgroups (gender, age at diagnosis, tumor location, Laurens classification, TNM staging, lymphovascular invasion, and perineural invasion). Age was recorded as on date of diagnosis, staging was reported as per American Joint Committee on Cancer (AJCC) 7th edition 2010. A p-value less than 0.05 was considered significant. Data was analyzed using the SPSS statistical software program for Microsoft Windows.

RESULTS:

During the study period, 106 patients fulfilled the criteria for enrolment into this study. Only 18 (17%) cases had HER2 score of 3+, whereas 5 (4.7%) had equivocal score (2+) and the remaining 84 (78%) tumors had a score of 0 or 1+ (negative) (Table 1).

Most of the patients were in the sixth and seventh decade of life (Table 2) with an age range between 25 years to 85 years. Both median and mean age was 60 years. There was no statistical difference between positive and negative cases as far as age at presentation is concerned (Table 3)

Most patients in the study were men (80.2%) with a sex ratio of male to female as 4:1. There was male sex preponderance for positive cases. Out of positive cases, 16 (89%) patients were men and only 2 (11%) were women with a sex ratio of 8:1. This difference was statistically significant (p=.029). Although this study was not designed to study the risk factors of carcinoma stomach, or relation between smoking and HER2 status; but an analysis revealed that 58 (54.7%) patients were active smokers. (Table 2)

There was no association between smoking history and the status of HER2. Gastric body was the most common location of primary tumor (Table 2) followed by gastroesophageal junction. HER2 positive cases were mostly seen in gastroesophageal junction tumors (23.3%) followed by body (17%) (Table No. 3).Most common histological type was intestinal (Table No. 2). In intestinal type HER2 positivity was seen in 13 (20%) cases and in diffuse morphology it was seen in 5 (12.2%) cases (Table 3). The difference was statistically significant (p=0.044).

Bormans type III was the most common lesion (Table No. 2), and was seen in 66 (62.3%) followed by Bormans type I which was found in 23 (21.7%). There was no association between HER2 status and Bormans tumor type (Table 3). Most of the patients in this study were of higher T-stage (Table No. 2). 80 (75.5%) patients had a T4 stage. In patients having HER2 score of 3+, a total of 13 (72%) patients had a T4 lesion (Table 3). An average lymph node yield of 10.24, was found which was considered optimum. Out of total 106 cases, 66 (62.3%) patients had nodal metastasis (Table 2), and HER2 positive rate was 17.5% and 16.7% in node negative and node positive respectively. This did not reach statistical significance.

An analysis was done for overall stage of disease also. There was 1 (0.9%) case of carcinoma in situ (stage 0), 5 (4.7%) cases of stage I, 37 (34.9%) cases of stage II and the remaining 63 (59.4%) were stage III. Subgroup analysis of stage III revealed that, 21 cases were IIIA, 19 cases were IIIB and 23 cases were IIIC.  An analysis of HER2 was done making two stage groups (≤II and >II). HER2 positive rate in stage 0, I and II was 18.6% and in stage III it was seen in 15.9% and did not reach statistical significance

Grade 1 or 2 cancer was found in 64 (60.4%) patients whereas Grade 3 tumors were found in 42 (39.6%) patients (Table 2). HER2 was positive in 13 (20%) cases in grade 1 or grade 2 cancers. In grade 3 cases, the percentage of HER2 positivity was 11.9% (Table 3). Lymphatic invasion was observed in 67 (63.2%) cases. There was no statistical significance between HER2 status and lymphatic invasion (P=0.065). Vascular invasion was observed in 68 (64.2%) of case and no relation was found between HER2 status and vascular invasion (P=0.081). Perineural invasion was observed in 43 (40.6%) cases and no relation was found between HER2 status and perineural invasion (P=043).

Size of the primary tumor was also analyzed for its significance with HER2 status. When the size of primary tumor was more than 4 cm, the incidence of HER2 positive score was 20% and it was only 13% when the size of the tumor was less than equal to 4 cm (Table 3). 

DISCUSSION: 

Gastrointestinal cancer is a major health issue in Kashmir valley and comprises more than one-third of all cancers in adult men and women. 2 This is the first study from this region, where gastric carcinoma is endemic. Study of HER2 status in our patients will add to the understanding of the biology of this tumor and may have an impact on the prognosis in our patient population. HER2 status was done only in the resected specimens and not on tissue obtained on endoscopic biopsy.

On immunohistochemistry, HER2 status was positive in 18 (17%) cases in this study. The HER2 positivity rate has not been uniform across the countries and has varied from as low as 4.4% 9 to as high as 53.4%. 10  A clear  trend  towards  a  potential  role  for  HER2  as  a negative  prognostic  factor  in  gastric  cancer has been shown, although  the  data  is  not  as  consistent  compared to what has been reported for breast cancer. 11 The explanation for large variation is likely to be multifactorial. Differences in the populations studied, use of non-standardized assays using different antibodies and the application of different scoring criteria for the stained slides may have a significant impact on the incidence on HER2 status.

Despite advances in histology, molecular biology and multimodality treatment approach to gastric cancer, the outcome remains poor, mainly due to late presentation. We observed that 59% presented with stage III disease. We did not observe any association between the depth of tumor and nodal metastasis with HER2 overexpression. This correlates with the findings of Chao He, et al 12 The median age at diagnosis of gastric cancer patients in United States is 70 years. 13 In our patient population, both median and mean age was 60 years. We did not find any relation of HER2 status with the age at presentation. Other studies have also not found any correlation with age at presentation. 12

There was male sex preponderance for positive cases with a sex ratio of 8:1. This difference was statistically significant (p=.029). This is in concordance with a study done by Kataoka Y, et al 14 but other study by Chao He, et al 12 did not find any correlation of HER2 status with gender. Whether there is any real correlation between gender and HER2 status is unknown.

Xiang-Shan Fan, et al 15 found a significantly higher frequency of HER2 over-expression in proximal than in distal gastric cancer. The result is similar to those reported recently in most other studies. 16 We found that HER2 positive cases were mostly seen in gastroesophageal junction tumors (23.3%) followed by body (17%) whereas in antropyloric region the HER2 positivity rate was seen in only 3 (10.3%) patients.

A number of studies have shown that HER2 overexpression and amplification are related to the histological type of tumor, with higher levels found in the intestinal phenotype compared to the diffuse and mixed types. Most common histological type in our studied patients was intestinal (61.3%) with a HER2 positivity rate of 20%. In diffuse morphology it was seen in 5 (12.2%) cases. The difference was statistically significant (p=0.044). This is in concordance with the results of study conducted by Tanner M et al 17 but because of the wide variation in the prevalence of HER2 expression, it is difficult to evaluate the clinical importance of these findings. The present results also pinpoint the usefulness of Laurens classification, since this divides gastric adenocarcinomas into different pathobiologically relevant subgroups. 

We found that most common lesion was Bormans type III (62.3%) followed by Bormans type I (21.7%). There was no association between HER2 and Bormans type. 

Histological grade of the primary tumor has been evaluated as a prognostic factor in gastric carcinoma and the results were similar to Xiang-Shan Fan et al. 15 It has been suggested that lymphovascular invasion may be a clinically useful marker of biologic aggressiveness. 18 Although we observed high incidence of lymphovascular invasion, but there was no statistical significance between HER2 status and lymphatic or vascular invasion. Siewert et al 19 demonstrated, by analyzing the 10-year results of 1654 patients with curative gastrectomy, that tumor size is associated with prognosis in gastric carcinoma. Yonemura et al 20 found a positive correlation between the intensity of HER2 staining and tumor size. With an average tumor size of 4.89 cm, we observed that higher tumor size (> 4 cm) predicted HER2 positivity and reached statistical significance.

With a dismal prognosis in upper gastrointestinal cancers due to multiple factors such as late presentation and lack of effective chemotherapy, to study the HER2 status has opened up more options of treatment in a subset of patients. ToGA study 21 is the first randomized, prospective, multicenter, phase III trial to evaluate the efficacy and safety of trastuzumab in patients with HER2-neu-positive gastric and gastroesophageal junction adenocarcinoma in combination with cisplatin and a fluoropyrimidine. There  was  a  significant improvement  in  the  median  overall  survival  with  the  addition  of trastuzumab to chemotherapy compared to chemotherapy alone in patients with HER2 overexpression (13.8 vs.11 months, respectively; p  =  .046).  This  study  established  trastuzumab  in  combination  with chemotherapy  as  a  new  standard  of  care  for  patients with  HER2  positive advanced  or  metastatic  gastric  and  gastroesophageal junction adenocarcinoma. 

CONCLUSION:

Immunohistochemistry for HER2 has added to the armamentarium of a pathologist to further guide the clinician in managing gastric cancer. In our patient population, HER2 overexpression is found in 17% patients of gastric and gastroesophageal junction carcinoma and was related to location of primary tumor, gender, histopathological type of cancer, and size of primary tumor. No significant relation could be deduced between the HER2 status and age of patient, depth of invasion, nodal metastasis, overall stage, lymphovascular invasion, perineural invasion, smoking rates, or grade of primary tumor. Oncologists may incorporate the use of trastuzumab in HER2 positive gastric and gastroesophageal junction cancers as is currently being done in HER2 positive breast cancer. This may benefit a subgroup of patient population after carefully selecting them for this treatment. Further studies are necessary to determine the significance of HER2 status in upper gastrointestinal cancers especially that the patients need follow up where we can predict the HER2 status as an independent prognostic factor for overall survival. This study was not designed to look for disease free survival (DFS) or overall survival (OS). 

Limitations

We did not have facility for florescent in-situ hybridization (FISH) at our centre, and under the grant allocation, there was no provision to send samples to other centres for FISH. Although the study did not include relation of HER2 status and disease free and overall survival, as a subsidiary end point, we are following patients for relapses and survival and may come up with further analysis in near future. 

Funding support

This work was supported by a grant from the Sher-i-Kashmir Institute of Medical Sciences, Srinagar. 

REFERENCES: 

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Table 1: HER2 Scoring Results (n=106)

Result

Frequency

%

Negative

84

79.2

Equivocal

4

3.8

Positive

18

17

Total

106

100

Table 2: Clinicopathological characterises of Patients (n=106)

Parameter

Frequency

%

Age (years)

21-30

2

1.8

31-40

5

4.7

41-50

16

15

51-60

33

31

61-70

41

38.6

71-80

8

7.4

> 80

1

0.9

Gender

Male

85

80.2

Female

21

19.8

Smoking

Absent

48

45.3

Present

58

54.7

Location

Antropylorus

29

27.4

Body

47

44.3

GE junction

30

28.3

Histopathological type

Intestinal

65

61.3

Diffuse

41

38.7

Borrman type

I

23

21.7

II

17

16

III

66

62.3

T-stage

T0

1

0.9

T1

1

0.9

T2

8

7.5

T3

16

15

T4

80

75.5

N-stage

N0

40

37.7

N1

22

20.7

N2

23

21.6

N3

21

19.8

Stage

0

1

0.9

I

5

4.7

II

37

34.9

III

63

59.4

Grade

1

40

37.7

2

24

22.6

3

42

39.6

Table 3: Analysis of HER2 Scoring with Clinicopathological Characteristics (n=106)

Parameter

HER2 scoring

P value

Negative

Equivocal

Positive

Total

Age (years)

< 60

27 (77.1%)

2 (5.7%)

6 (17.1%)

35 (100%)

0.064

> 60

57 (80.3%)

2 (2.8%)

12 (16.9%)

71 (100%)

Gender

Female

17 (81%)

2 (9.5%)

2 (9.5%)

21 (100%)

0.029

Male

67 (78.8%)

2 (2.4%)

16 (18.8%)

85 (100%)

Tumor location

Antropylorus

24 (82.8%)

2 (6.9%)

3 (10.3%)

29 (100%)

0.319

Body

39 (82.6%)

0

8 (17%)

47 (100%)

GE junction

21 (70%)

2 (6.7%)

7 (23.3%)

30 (100%)

Histo-

Pathological

Type

Intestinal

49 (75.4%)

3 (4.6%)

13 (20%)

65 (100%)

0.044

Diffuse

35 (85.4%)

1 (2.4%)

5 (12.2%)

14 (100%)

Borrman type

I

18 (78.3%)

1 (4.3%)

4 (17.4%)

23 (100%)

0.505

II

14 (82.4%)

0

3 (17.6%)

17 (100%)

III

52 (78.8%)

3 (4.5%)

11 (16.7%)

66 (100%)

T stage

T1/T2/T3

20 (76.9%)

1 (3.8%)

5 (19.2%)

26 (100%)

0.093

T4

64 (80%)

3 (3.8%)

13 (16.2%)

80 (100%)

N stage

Negative

32 (80%)

1 (2.5%)

7 (17.5%)

40 (100%)

0.08

Positive

52 (78.8%)

3 (4.5%)

11 (16.7%)

66 (100%)

Grade

G1/G2

48 (75%)

3 (4.7%)

13 (20.3%)

64 (100%)

0.039

G3

36 (85.7%)

1 (2.4%)

5 (11.9%)

42 (100%)

Primary

Tumor size

< 4

38 (82.6%)

2 (4.3%)

6 (13%)

46 (100%)

0.05

> 4

46 (76.7%)

2 (3.3%)

12 (20%)

60 (100%)

 





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