Thyroid cancer is the second most common malignancy among females in Saudi Arabia accounting for 7.4% of all cancers and 10.6% of all female malignant cancers (1) . This is a comparatively higher frequency than in Western countries. For example, thyroid cancer accounts for only 3% of all cancers in the United States (2). Papillary thyroid carcinoma (PTC) is the most common thyroid cancer subtype representing 80–90% of all thyroid malignancies (3).
PTC has an excellent prognosis and is usually cured by current therapy regimens consisting of surgery followed by radio-iodine therapy. However, there is a minor fraction of patients with a dismal course of disease. Tumor recurrence occurs in approximately 5% of PTC and the mortality rate is 1–2% (4,5). Aggressive malignant behavior is strongly related to various clinicopathological variables, including tall cell variant, advanced stage, vascular invasion and nodal or distant metastasis. These parameters are statistically powerful but still not sufficient to predict unfavorable disease course in all patients. A certain prediction of aggressive tumor behavior could potentially be exploited to complement radio-iodine therapy by other cytotoxic treatments.
As our knowledge on the molecular mechanisms involved in thyroid cancer development and progression continuously increase, it can be hoped, that molecular information will eventually contribute to a better initial assessment of tumor’s aggressiveness. Human epidermal growth factor receptor 2 (HER2) is of particular interest in tumor biology as it represents a strong prognostic feature in several tumor types and simultaneously serves as a highly utile therapeutic target in breast and stomach cancer (6,7) along with possibly other cancers (8,9). HER2 encodes a 185-kDa transmembrane kinase glycoprotein (10). HER2 was first reported as overexpressed in human breast and ovarian cancers (11). HER2 overexpression has later been observed in a large variety of other human malignancies originating in different organs, such as stomach, colon, lung and pancreas (12-15).
Data on the role of HER2 in papillary thyroid cancer is not conclusive. Reported incidences of HER2 overexpression vary, from 0 to 79.5%, in studies involving thyroid patients (16-22). Given the generally excellent prognosis of papillary thyroid cancers, and the need for very large patient cohorts to find associations with rare clinical events, robust data on the prognostic role of HER2 expression in papillary thyroid cancer is lacking. Due to the high incidence of papillary thyroid cancer in Saudi Arabia, we were able to collect a cohort of 1,040 papillary thyroid cancer with follow up information. In this project we utilized this patient collection to investigate prevalence and clinical significance of HER2 overexpression in papillary thyroid cancer.
Patient selection and tissue microarray (TMA) construction
One thousand and forty patients with PTC, diagnosed between 1988 and 2011, were selected from files of the King Faisal Specialist Hospital and Research Centre. All PTC were analyzed in a TMA format. Clinical and histopathological data were available for all the patients. TMAs were constructed with 2-fold redundancy from formalin-fixed, paraffin-embedded PTC specimens as described previously (23). Tumor regions were mapped by a pathologist for coring. The TMA was constructed with 0.6 mm diameter cores spaced 0.8 mm apart using a tissue microarrayer (Semi-automated Arrayer, CM1 Mirlacher, Neuenburg, Germany). The TMA block was cut into 5 µm sections, adhered to a slide by an adhesive tape-transfer method (Instrumedics, Hackensack, NJ) and UV crosslinked. The Institutional Review Board of the King Faisal Specialist Hospital and Research Centre approved the study under Project RAC# 2080-031 on PTC archival clinical samples.
Standard protocol was followed for IHC staining. For antigen retrieval, Dako Target Retrieval Solution pH 9.0 (Catalog number S2368) was used, and the slides were microwaved at 750 W for 5 min and then at 250 W for 20 min. TMA sections were stained using FDA approved HercepTest kit from DAKO using manufacturers instruction. All slides were counterstained with hematoxylin, dehydrated, cleared and mounted. Negative controls included omission of the primary antibody. Normal tissues of different organ system were also included in the TMA to serve as control. Only fresh cut slides were stained simultaneously to minimize the influence of slide aging and maximize reproducibility of the experiment. For HER2 immunoscoring, DAKO scoring guidelines for gastric cancer were followed as no standard guidelines for HER2 scoring in thyroid tumors exist. Briefly, tumors were categorized into four groups based on intensity score (0, 1+, 2+, 3+). Intensity score 2+ and 3+ was taken as positive, as described previously (19). IHC scoring was done by two pathologists (SB & SP), blinded to the clinicopathological characteristics. Discordant scores were reviewed together to achieve agreement.
Fluorescence in situ hybridization (FISH)
For HER2 dual-color FISH on paraffin-embedded TMA was performed using commercially available DNA probes LSI HER2/CEP 17; Vysis Inc. BX51 Olympus fluorescence microscope (Olympus, Richardson, TX, USA) was used for screening the FISH slides. The HER2 locus specific probe located on chromosome 17, was labeled with Spectrum Orange whilst the centromere was labelled with Spectrum Green (LSI HER2/CEP 17; Vysis Inc.). Histologic TMA tissue sections, 5 µm thick, were deparaffinized with a series of xylene prior to immersion in 100% ethanol. FISH was carried according to the manufacturer’s instructions. FISH scoring was performed independent of the IHC result. The number of HER2 (red) and CEP17 (green) signals were scored for each sample in 20 nuclei. The HER2/CEP17 ratio was calculated according to ASCO/CAP guidelines. A HER2/CEP17 ratio of 1 was considered normal, less than 1.8 as non-amplified and more than 2.2 as amplified (24).
Data analysis and statistics
The JMP 10.0 (SAS Institute Inc., Cary, NC, USA) software package was used for data analyses. We examined the association of HER2 expression with clinicopathological parameters, biomarker expression using chi-square tests and also performed survival analysis by using the Mantel-Cox log-rank test. Survival curves were generated using Kaplan-Meier method with significance evaluated using the Mantel-Cox log-rank test. Values of P<0.05 were considered statistically significant.
The details of 1,040 patients selected for analyses are as follows. The mean age of the patients at initial surgery was 40.4 years (range, 6–92 years), and 261 were (25.1%) males and 779 (74.9%) were females. The mean duration of follow-up was 76.5 months (range, 0–280 months). Seven hundred and ninety one (78.3%) of the tumours were classical papillary carcinomas; 153 (15.1%) were the follicular variant of PTC; and 66 (6.5%) were the tall cell variant. Extrathyroidal extension was seen in 462 (52.9%) cases and AJCC staging was as follows: 693 (68.6%) stage I; 51 (5.1%) stage II; 84 (8.3%) stage III; and 182 (18.0%) stage IV. Details of surgical margin status were available in only 490 cases and involved surgical margins were noted in 266 (54.2%) cases.
Immunohistochemical analysis of HER2 expression was interpretable in 991 PTC spots. While 796 cancers (80.3%) showed negative HER2 staining (score 0 and 1), there were 363 with score 0, 433 (43.7%) with 1+, 194 (19.6%) with 2+, and 1 (0.1%) with 3+ immunostaining. Total number of cases which demonstrated overexpression of 2+/3+ were 195 (19.7%). Representative cases are shown in Figure 1. HER2 positivity (2+/3+) in PTC was significantly associated with early stage tumors (Stage I) (P=0.0076). However, HER2 expression was not associated with age, gender, lymphovascular invasion or extrathyroidal extention (Table 1). There was no difference in survival between patients showing variable HER2 protein expression (P=0.7442) (Figure 2).
HER2 by FISH was interpretable in 913 PTC spots. Amplification was not seen in any of our cases and the incidence of HER2 gain in our cohort was only 3% (26 of 913) of cases. Tumors with representative FISH findings are shown in Figure 3. There was a tendency towards higher protein expression levels in tumors with a HER2 gene copy gain as compared to cancers with normal HER2 gene copy status, but this association did not reach statistical significance (P=0.0816) (Figure 4). No association was seen between HER2 FISH gain and tumor phenotype or patient survival.
Our analyses revealed a 2+ HER2 staining in 19.6% and a 3+ HER2 staining in only 1 (0.1%) of our 991 interpretable tumors. This is in the range of earlier studies that were also done using FDA approved HER2 detection kits. Mdah et al. found 8.6% 2+ positive and 6.9% 3+ positive cases in a series of 58 papillary thyroid cancers (20). Sugishita et al. found 46% 2+ positive and 38% 3+ positive cases in a series of 37 papillary thyroid cancers (17). Mondi et al. found 33% 1+ positive however, no 2+ or 3+ positive cases, in a set of six papillary thyroid cancers (19). The reported data on HER2 expression were more variable in earlier studies using other non-FDA approved reagents. Here, Haugen et al. found 78% HER2 positive cases in 14 papillary thyroid cancers (22), Balta et al. observed 14.9% HER2 positive cases in 47 papillary thyroid cancers (16), Wu et al. identified 79.5% HER2 positive cases in 331 papillary thyroid cancers (25) and, Utrilla et al. described 52% HER2 positive cases in 25 papillary thyroid cancers (26).
Due to the high prevalence of papillary thyroid cancer in Saudi Arabia we were able to collect a cohort of patients that is substantially larger than available for comparable studies evaluating potential prognostic biomarkers. TMA studies on PTCs have so far included 331 tumors (25). High number of cases are imperatively needed for analyzing the possible role of biomarkers in the context of tumor aggressiveness, as only few papillary thyroid cancers show a dismal clinical course with distant metastases and/or recurrent disease after radio-iodine therapy. Tumor specific life expectancy was 89.9% after 5 years and 86.2% after 10 years in a register based study on 2,729 patients with papillary thyroid cancer from Sweden (27). This is comparable to our cohort with a survival rate of over 90% after 5 years (28).
The lack of unequivocal associations of HER2 expression with parameters of malignancy such as extrathyroidal tumor expansion, pT category, UICC stage, nodal (pN) or distant metastasis (pM), and tumor recurrence, demonstrates that HER2 expression is not a parameter of poor prognosis in papillary thyroid cancer. This is different from breast cancer, where HER2 expression is strongly linked to high tumor aggressiveness in patients that are not treated with anti-HER2 drugs (29-33).
The difference in the impact of HER2 expression may be caused by variations in the molecular environment of thyroid and breast cancer cells or just be due to different HER2 expression levels. Papillary thyroid cancers mostly exhibit a 2+ HER2 expression while breast cancers have a 3+ expression caused by high level gene amplification in most cases (34-39). The absence of a prognostic impact of HER2 expression in our study is in line with several earlier studies evaluating papillary thyroid cancers and also finding no association with tumor stage (16-19), metastasis (16,17,19,20), tumor subtype (16-20) and extrathyroidal extension (17,19).
The utility of HER2 as a therapeutic target has particularly been demonstrated for breast cancers showing unequivocal HER2 amplification with a HER2/centromere 17 ratio that usually markedly exceeds the threshold value of 2.0. Although the discussion continues on whether high level HER2 expression can occur in the absence of amplification and whether 2+ overexpression, caused by high polysomy can also result in some response to anti-HER2 therapy. After more than 15 years of clinical experience with anti-HER2 therapy, it appears that such events are not frequent or evident (40). Based on the experience in breast cancer, it thus seems unlikely that papillary thyroid cancer will be a good candidate for anti-HER2 therapy.
In our study, most HER2 positive cancers only showed 2+ positivity, exclusive of one 3+ case, with no cases even borderlining HER2 gene amplification. This is in line with data from Mondi et al. (19) and Mdah et al. who showed 6.9% HER2 overexpression in his patient cohort of 58 PTC and also failed to find amplification using chromogenic in situ hybridization (20). However, the study of Sugishita et al. suggested a HER2 amplification in 22% of cases (17). In this study, the cut-off level for amplification was selected at 1.3 instead of the typically used threshold of 2.0. None of the tumors in the 69 cases of Sugishita et al. had reached the cut-off level of 2.0. It is noteworthy, that a slight elevation of HER2 signals as compared to centromere 17 signals is mostly caused by allele duplication in the S- and G2 phases of the cell cycle which results in a visible duplication of the small HER2 signals but not of the much larger and confluent centromere signals (41).
Elevated HER2 copy numbers correlating with higher levels of protein expression was expected in our study. It is well known, that even a mildly increased gene copy number will often result in a mildly increased expression of the corresponding gene product (42). Even though a statistical significance was not obtained due to small number of cases, a clear tendency was indeed seen towards a higher protein expression in cancers with a HER2 gene copy number gain. Elevated protein expression measured by IHC has also been reported for EGFR family members in other cancers with increased gene copy numbers not meeting the formal criteria for gene amplification types (43,44).
Our results demonstrated that mild HER2 overexpression occurs at relevant frequency in papillary thyroid cancer and in the absence of gene amplification. In conclusion, expression of HER2 seems to hold no value as a prognostic factor in PTC. It appears possible, however, that next generation anti-HER2 drugs that may target even lower level HER2 expressing cancer cells could have an effect on a subset of papillary thyroid cancers.
We thank Sandeep Kumar Parvathareddy, Sarah Siraj, Valorie Balde, Mary Joan Galvez, Felisa De Vera, Hassan Al-Dossari, Padmanaban Annaiyappanaidu and Zeeshan Qadri, for technical assistance.
Conflicts of Interest: The authors have no conflicts of interest to declare.
Ethical Statement: The Institutional Review Board of the King Faisal Specialist Hospital and Research Centre approved the study under Project RAC# 2080-031 on PTC archival clinical samples.
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