The authors have declared that no competing interests exist.
Conceived and designed the experiments: LKG JWKC WYC SHC KHL. Performed the experiments: XL ST SHC KHL SKLT IB JW. Analyzed the data: WSO LKG WYC XL ST. Contributed reagents/materials/analysis tools: LKG KHL SHC SKLT LN SLL YKL YNC EK CP LTS THH SKT. Wrote the paper: WYC LKG JWKC.
Mucinous epithelial ovarian cancer has a poor prognosis in the advanced stages and responds poorly to conventional chemotherapy. We aim to elucidate the clinicopathological factors and incidence of HER2 expression of this cancer in a large Asian retrospective cohort from Singapore. Of a total of 133 cases, the median age at diagnosis was 48.3 years (range, 15.8–89.0 years), comparatively younger than western cohorts. Most were Chinese (71%), followed by Malays (16%), others (9.0%), and Indians (5%). 24% were noted to have a significant family history of malignancy of which breast and gastrointestinal cancers the most prominent. Majority of the patients (80%) had stage I disease at diagnosis. Information on HER2 status was available in 113 cases (85%). Of these, 31 cases (27.4%) were HER2+, higher than 18.8% reported in western population. HER2 positivity appeared to be lower among Chinese and higher among Malays patients (p = 0.052). With the current standard of care, there was no discernible impact of HER2 status on overall survival. (HR = 1.79; 95% CI, 0.66–4.85; p = 0.249). On the other hand, positive family history of cancer, presence of lymphovascular invasion, and ovarian surface involvements were significantly associated with inferior overall survival on univariate and continued to be statistically significant after adjustment for stage. While these clinical factors identify high risk patients, it is promising that the finding of a high incidence of HER2 in our Asian population may allow development of a HER2 targeted therapy to improve the management of mucinous ovarian cancers.
Mucinous epithelial ovarian cancer (mEOC) accounts for 2% to 5% of all primary epithelial ovarian cancers (EOC). It is chemo- resistant
HER2 has been found to be amplified in a significant number of mEOC, varying from 18.8% in a large western study (n = 154)
(A) Previous genome-wide copy number alteration study on a small cohort of mEOC (n = 17) showed significant amplification of HER2. x-axis shows chromosomes 1-X, with alternating gray blocks. y-axis is the −log(q) where q is the false discovery rate. Positive values indicate amplification and negative values are deletion. (B) Age distribution was of normal distribution overall and for both HER2+ and HER2− cases. The median age was 48.3 (range: 15 to 89 years). (C) Frequency of reported cancers in family history. Majority of cancers were of breast and gastrointestinal (colon/stomach) origin. Note: some patients reported more than 1 case of cancer in family history.
Given the rarity of mEOC, clinicopathological factors associated with clinical outcome of mEOC have been difficult to elucidate. In previous studies on Asian patients, the cohorts have been relatively small and clinicopathological factors were not well investigated. Hence we were motivated to: (i) ascertain HER2 status and its clinical relevance in a large cohort of mEOC samples of Asian descent; and (ii) investigate and identify novel clinicopathological factors that can improve identification of high risk patients.
This study was approved by the institutional review boards (IRB) of the National Cancer Centre Singapore, KK Women's and Children's Hospital Singapore and Singapore General Hospital Singapore. IRB waiver of informed consent was approved as analyses were performed retrospectively on non-identifiable data (CIRB 2010/425/B).
The prospectively maintained gynaecologic oncology tumor databases were used to identify all patients with mEOCs. 199 cases of mucinous ovarian cancers were identified from 1963 to 2012. Tumour slides were retrieved and reviewed by 2 independent institutional gynaecologic pathologists and 133 cases dated between 1990 and 2012 which fulfilled the criteria of primary invasive mucinous ovarian cancer were selected. For the analysis, individual patient case notes were retrieved and data manually culled for age at diagnosis, ethnicity, Eastern Cooperative Oncology Group performance status, comorbidities (diabetes mellitus, hyperlipidemia, ischemic heart disease), tumor size, grade, serosal involvement, lymph node metastasis, type of surgery (hysterectomy, bilateral salpingooophorectomy, pelvic lymph node dissection, including fertility preservation), adjuvant chemotherapy, the date of initial diagnosis, recurrence and death. Death outcomes were supplemented by vital data obtained from the National Death Registry and tumor staging was determined using the 1998 FIGO criteria.
DISH of HER2 and chromosome 17 centromere probes were performed in an automated BenchMark ULTRA (Ventana Medical Systems, USA) slide stainer, using the INFORM HER2 Dual ISH DNA Probe Cocktail Assay (Ventana Medical Systems, USA) that allows detection of HER2 gene amplification by light microscopy. Tissue sections were deparaffinized and pretreated with Cell Conditioning 2 (CC2) at pH 6 at 86°C, and enzymatic digestion of proteins was performed with ISH protease 2 or 3 for variable lengths of time. Double-stranded DNA was denatured to allow hybridization of dinitrophenyl (DNP)-labeled HER2 DNA probes and digoxigenin (DIG)-labeled Chromosome 17 centromere probes. A stringency wash was performed at 72°C using sodium citrate, sodium chloride (SSC 10X) to wash off unbound or weakly bound probes. Detection of bound probes occurred separately, using the ultraView Silver ISH DNP and ultraView Red ISH DIG detection kits (Ventana Medical Systems, USA). Goat anti-mouse secondary antibodies conjugated to alkaline phosphatase (AP) were directed against primary DIG antibodies for detection of the centromere probes, whilst goat anti-rabbit secondary antibodies conjugated to horse radish peroxidase (HRP), were used against primary DNP antibodies.
Immunohistocytochemistry (IHC) staining of HER2 protein was performed in a BenchMark ULTRA slide stainer utilizing the ultraView Universal DAB Detection kit (Ventana Medical Systems, USA). Tissue sections were deparaffinized and rehydrated with EZ Prep concentrate (10X) solution (Ventana Medical Systems, USA) and heat-mediated antigen retrieval was performed with cell conditioning 1 (CC1) (Ventana Medical Systems, USA) at 95°C. Slides were then treated with ultraView Di-aminobenzidene (DAB) inhibitor (Ventana Medical Systems, USA) and incubated with 100 µL of rabbit anti-HER2 monoclonal antibodies, immunoglobulin G (clone SP3) (ThermoScientific, USA) at a 1∶200 antibody diluent ratio for 24 minutes. Bound antibody was detected using ultraView DAB Detection Kit, where ultraView Horse Radish Peroxidase Multimer (anti-rabbit secondary antibody) (Ventana Medical Systems, USA) were added, followed by ultraView DAB H2O2 and chromogen (Ventana Medical Systems, USA).
The region of invasive carcinoma on tissue section was first marked out by a gynecological pathologist. Twenty non-overlapping nuclei were then enumerated on first count and the HER2/Chr17 ratio was then calculated. HER2 was considered amplified if the ratio was ≥2.2 and non-amplified if the ratio was <1.8 at this count. If the ratio fell between 1.8 and 2.2, an additional 20 nuclei were enumerated and the new ratio was calculated based on 40 nuclei. HER2 was amplified if the ratio on second count was ≥2.0, and non-amplified if <2.0.
The HER2 DAKO scoring system for gastric cancer
HER2 positivity was defined as having IHC 3+, or IHC 2+ with DISH amplification. IHC 0, IHC 1+, or IHC 2+ with DISH non-amplification were considered HER2 negative.
To detect significant differences in the demographic and clinical characteristics between HER2+ and HER2− patients, categorical characteristics were compared using the Chi-square test or Fisher's exact test as appropriate. Mann-Whitney U test was used to compare continuous characteristics between the 2 groups. Overall survival (OS) duration was calculated from the date of diagnosis to the date of death. Progression-free survival (PFS) duration was calculated from the date of diagnosis to the date of first progression, relapse or death, whichever occurred first. Patients who did not develop any of these time-to-event endpoints were censored at their last follow-up date. The Kaplan-Meier method was used to estimate all survival distributions, the log-rank test was used to test the differences between survival curves and Cox proportional hazard models were fitted to estimate hazard ratios to assess the association of factors with each survival endpoint. The proportional hazards assumption underlying the Cox model was verified using Schoenfeld residuals, and a 2-sided p-value<0.05 was considered statistically significant. All analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC).
133 cases qualified as primary invasive mEOC and were included in this analysis. Clinical characteristics of the patients are summarized in
Characteristics | Categories | Patients (n = 133) |
Median age at diagnosis, years (range) | - | 48.3 (15.8–89.0) |
Ethnic group | Chinese | 94 (71%) |
Malays | 21 (16%) | |
Indians | 6 (5%) | |
Others | 12 (9%) | |
Smoking history (n = 52) | Non-smoker/Ever-smoker | 38 (73%)/14 (27%) |
Family history of cancer (n = 59) | Negative/Positive | 45 (76%)/14 (24%) |
Presence of comorbidities (n = 126) | No/Yes | 48 (38%)/78 (62%) |
Stage at diagnosis (n = 125) | I | 100 (80%) |
II | 7 (6%) | |
III | 15 (12%) | |
IV | 3 (2%) | |
Tumour differentiation/grade (n = 117) | well | 71 (61%) |
moderate | 34 (29%) | |
poor | 12 (10%) | |
Tumour type (n = 130) | Mixed borderline/Non-borderline | 50 (38%)/80 (62%) |
Ovarian surface involvement (n = 122) | No/Yes | 75 (61%)/47 (39%) |
Lymphovascular invasion (n = 126) | No/Yes | 118 (94%)/8 (6%) |
Median CA125, |
- | 71.3 (3.0–8812.5) |
Received OGD (n = 126) | No/Yes | 107 (85%)/19 (15%) |
Received colonoscopy (n = 126) | No/Yes | 102 (81%)/24 (19%) |
Received appendectomy (n = 127) | No/Yes | 62 (49%)/65 (51%) |
Received omentectomy (n = 128) | No/Yes | 10 (8%)/118 (92%) |
Received chemotherapy (n = 124) | No/Yes | 68 (55%)/56 (45%) |
Received adjuvant chemotherapy (n = 123) | No/Yes | 83 (67%)/40 (33%) |
Abbreviation: OGD, oesophagogastroduodenoscopy.
Overall, the majority of patients (80.0%) with mucinous cancers were found to have stage I disease at diagnosis, with 6% of patient presenting in stage II and only 14% presented with advanced stage 3 and 4 diseases. Of the entire study cohort, 61% of mucinous tumors in our cohort were well differentiated tumors, 29% moderately differentiated and 10% poorly differentiated. 39% of cases had ovarian surface involvement (OSI) and lymphovascular invasion (LVI) was present in 6%.
HER2 status was successfully ascertained in 113 cases. Of the 133 cases, 9 cases could not be assayed for IHC and 11 cases were IHC 2+ but unsuccessful in DISH. These 20 cases were filtered out in the HER2 status analyses. In summary, 31 samples or 27.4% (95% confidence interval 20.1% to 36.3%) were HER2+ and 82 were HER2−. Excellent concordance was observed between IHC and DISH assay for IHC 0, 1+, and 3+. Of the 16 cases that were IHC 2+, 4 had amplification ratio ≥2.0.
The observed proportion of HER2 positivity was higher in Malays than in Chinese in our cohort (
Characteristics | Categories | HER2+ | HER2− | |
Median age at diagnosis, years (range) | - | 43.6 (17.6–74.9) | 49.4 (15.8–82.1) | 0.094 |
Ethnic group | Chinese (n = 80) | 18 (23%) | 62 (78%) | 0.052 |
Malays (n = 16) | 9 (56%) | 7 (44%) | ||
Indians (n = 6) | 1 (17%) | 5 (83%) | ||
Others (n = 11) | 3 (27%) | 8 (73%) | ||
Smoking history | Non-smoker (n = 29) | 9 (31%) | 20 (69%) | 0.720 |
Ever-smoker (n = 14) | 3 (21%) | 11 (79%) | ||
Family history of cancer | Negative (n = 39) | 12 (31%) | 27 (69%) | 0.728 |
Positive (n = 11) | 4 (36%) | 7 (64%) | ||
Presence of comorbidities | No (n = 40) | 14 (35%) | 26 (65%) | 0.267 |
Yes (n = 68) | 17 (25%) | 51 (75%) | ||
Stage at diagnosis | I (n = 88) | 29 (33%) | 59 (67%) | 0.285 |
II (n = 5) | 0 (-) | 5 (100%) | ||
III (n = 11) | 2 (18%) | 9 (82%) | ||
IV (n = 3) | 0 (-) | 3 (100%) | ||
Tumour differentiation/grade | well (n = 64) | 19 (30%) | 45 (70%) | 0.717 |
moderate (n = 28) | 10 (36%) | 18 (64%) | ||
poor (n = 9) | 2 (22%) | 7 (78%) | ||
Tumour type | Mixed borderline (n = 45) | 17 (38%) | 28 (62%) | 0.063 |
Non-borderline (n = 65) | 14 (22%) | 51 (78%) | ||
Ovarian surface involvement | No (n = 66) | 23 (35%) | 43 (65%) | 0.075 |
Yes (n = 38) | 7 (18%) | 31 (82%) | ||
Lymphovascular invasion | No (n = 100) | 29 (29%) | 71 (71%) | 0.186 |
Yes (n = 7) | 0 (-) | 7 (100%) | ||
Median CA125, |
- | 37.2 (6.1–415.7) | 102.5 (3.0–8812.5) | |
Received OGD | No (n = 93) | 29 (31%) | 64 (69%) | 0.148 |
Yes (n = 16) | 2 (13%) | 14 (88%) | ||
Received colonoscopy | No (n = 90) | 29 (32%) | 61 (68%) | 0.057 |
Yes (n = 19) | 2 (11%) | 17 (89%) | ||
Received appendectomy | No (n = 51) | 15 (29%) | 36 (71%) | 0.833 |
Yes (n = 58) | 16 (28%) | 42 (72%) | ||
Received omentectomy | No (n = 7) | 3 (43%) | 4 (57%) | 0.400 |
Yes (n = 103) | 28 (27%) | 75 (73%) | ||
Received chemotherapy | No (n = 57) | 18 (32%) | 39 (68%) | 0.419 |
Yes (n = 49) | 12 (24%) | 37 (76%) | ||
Received adjuvant chemotherapy | No (n = 69) | 19 (28%) | 50 (72%) | 0.745 |
Yes (n = 36) | 11 (31%) | 25 (69%) |
Abbreviation: OGD, oesophagogastroduodenoscopy.
After a median follow-up of 2.8 years (range, 0–19.99 years), 29 recurrences and 22 deaths, the median overall survival (OS) was not reached for the study population and the 5-year OS rate was 75.4%. Overall, there was no statistically significant difference in OS between HER2+ and HER2− patients (p = 0.249) (
No statistical significance was observed for HER2+ compared to HER2− cases in (A) overall survival (log-rank p = 0.249), and (B) progression free survival (log-rank p = 0.120).
Advanced age at diagnosis, positive family history of cancer, advanced disease stage at diagnosis, presence of LVI and ovarian surface involvement (OSI) were significantly associated with inferior OS on univariate analysis (
Variable | Univariate Analysis | Stage-Adjusted Analysis | ||
HR (95% CI) | HR (95% CI) | |||
Age at diagnosis (per year increase) | 1.03 (1.01–1.06) | 1.02 (0.99–1.05) | 0.191 | |
Ethnic group (Malays vs Chinese) | 0.39 (0.12–1.27) | 0.127 | 0.68 (0.20–2.33) | 0.943 |
Ethnic group (Indians vs Chinese) | 0.00 (NE) | 0.00 (NE) | ||
Ethnic group (Others vs Chinese) | 0.00 (NE) | 0.00 (NE) | ||
Smoking history (Ever-smoker vs non-smoker) | 0.98 (0.20–4.91) | 0.980 | 0.47 (0.06–3.45) |
0.458 |
Family history of cancer (Positive vs Negative) | 5.88 (1.40–24.79) | 7.95 (1.30–48.65) |
||
Presence of comorbidities (Yes vs No) | 1.16 (0.55–2.42) | 0.694 | 0.75 (0.34–1.65) | 0.475 |
Stage at diagnosis (II vs I) | 4.85 (1.57–14.97) | - | - | |
Stage at diagnosis (III vs I) | 15.42 (6.54–36.36) | - | ||
Stage at diagnosis (IV vs I) | 28.51 (7.38–110.22) | - | ||
Tumour differentiation/grade (moderate vs well) | 1.15 (0.48–2.78) | 0.093 | 1.15 (0.47–2.82) | 0.848 |
Tumour differentiation/grade (poor vs well) | 2.77 (1.05–7.29) | 1.35 (0.46–3.95) | ||
Tumour type (Non-borderline vs mixed borderline) | 1.75 (0.81–3.80) | 0.153 | 1.33 (0.58–3.07) | 0.500 |
Ovarian surface involvement (Yes vs No) | 7.80 (3.14–19.35) | 4.14 (1.45–11.80) | ||
Lymphovascular invasion (Yes vs No) | 10.25 (4.39–23.92) | 5.58 (2.05–15.21) | ||
CA125 (per |
1.00 (1.00–1.00) | 0.916 | 1.00 (1.00–1.00) | 0.461 |
Received chemotherapy (Yes vs No) | 4.26 (1.83–9.89) | 1.66 (0.60–4.60) |
0.335 | |
Received adjuvant chemotherapy (Yes vs No) | 1.18 (0.58–2.41) | 0.646 | 1.01 (0.43–2.38) | 0.974 |
HER2 status (HER2− vs HER2+) | 1.79 (0.66–4.85) | 0.249 | 1.01 (0.34–2.97) | 0.988 |
0.75 (0.49–1.15) | 0.185 | 0.72 (0.44–1.18) |
0.193 |
Abbreviation: HR, hazard ratio; CI, confidence interval; NE, not estimable.
Based on Wald test.
To interpret with caution as there were <10 deaths in the fitted multivariable model.
Departures from proportionality assumption. The time-varying effects of receipt of chemotherapy were further accounted for by including a time-by-covariate interaction term in the Cox model. Based on the extended model, there was no significant association between OS and chemotherapy.
Variable | Univariate Analysis | Stage-Adjusted Analysis | ||
HR (95% CI) | HR (95% CI) | |||
Age at diagnosis (per year increase) | 1.02 (0.99–1.04) | 0.057 | 1.01 (0.99–1.04) | 0.287 |
Ethnic group (Malays vs Chinese) | 0.43 (0.15–1.21) | 0.167 | 0.70 (0.24–2.06) | 0.731 |
Ethnic group (Indians vs Chinese) | 0.37 (0.05–2.70) | 0.37 (0.05–2.76) | ||
Ethnic group (Others vs Chinese) | 0.00 (NE) | 0.00 (NE) | ||
Smoking history (Ever-smoker vs non-smoker) | 2.09 (0.66–6.58) | 0.199 | 0.81 (0.16–4.04) | 0.797 |
Family history of cancer (Positive vs Negative) | 2.22 (0.72–6.86) | 0.154 | 2.91 (0.75–11.26) | 0.121 |
Presence of comorbidities (Yes vs No) | 1.19 (0.61–2.34) | 0.605 | 0.91 (0.44–1.87) | 0.799 |
Stage at diagnosis (II vs I) | 3.88 (1.30–11.52) | - | - | |
Stage at diagnosis (III vs I) | 10.45 (4.80–22.71) | - | ||
Stage at diagnosis (IV vs I) | 16.43 (4.54–59.51) | - | ||
Tumour differentiation/grade (moderate vs well) | 1.24 (0.54–2.88) | 1.20 (0.52–2.78) | 0.224 | |
Tumour differentiation/grade (poor vs well) | 4.33 (1.86–10.07) | 2.25 (0.89–5.67) | ||
Tumour type (Non-borderline vs mixed borderline) | 1.91 (0.95–3.84) | 0.065 | 1.33 (0.63–2.80) | 0.447 |
Ovarian surface involvement (Yes vs No) | 8.86 (3.82–20.53) | 5.26 (2.04–13.58) | ||
Lymphovascular invasion (Yes vs No) | 8.13 (3.59–18.42) | 4.60 (1.81–11.71) | ||
CA125 (per |
1.00 (1.00–1.00) | 0.617 | 1.00 (1.00–1.00) | 0.803 |
Received chemotherapy (Yes vs No) | 5.92 (2.60–13.50) | 3.01 (1.18–7.68) | ||
Received adjuvant chemotherapy (Yes vs No) | 1.69 (0.89–3.23) | 0.106 | 1.63 (0.76–3.49) | 0.212 |
HER2 status (HER2− vs HER2+) | 2.02 (0.82–4.96) | 0.120 | 1.30 (0.50–3.38) | 0.586 |
0.72 (0.48–1.08) | 0.109 | 0.69 (0.43–1.09) |
0.111 |
Abbreviation: HR, hazard ratio; CI, confidence interval; NE, not estimable.
P values for age at diagnosis, CA125 and HER2 amplification ratio were based on Wald test, and P values for all other variables were based on the log-rank test.
Based on Wald test.
To interpret with caution as there were <10 deaths in the fitted multivariable model.
Ovarian mucinous tumors tend to have a poor prognosis in advanced stages and response to chemotherapy is generally poor in comparison to other histology subtypes of ovarian cancer. There is mounting evidence that distinct mutations and genomic aberrations exist in each histological subtype of ovarian cancers, suggesting that treatment of ovarian cancer could be stratified according to histology subtypes.
The current study reports on HER2 status and clinicopathological factors in the largest cohort of mEOC in an Asian setting. It carries three significant implications. Firstly, compared to a recent report by Anglesio et al that the incidence of HER2 positivity was 18.8% (n = 154) in the western populations
Secondly, we found that 80.0% of our mEOC patients had stage I disease at diagnosis as compared to 55% to 60% in reports from the western populations.
Thirdly, contrary to the report from McCaughan et al who found HER2+ ovarian cancer patients to have a poor survival rate
Angelsio et al also showed that HER2+ and KRAS mutations (i.e. KRAS+) are almost mutually exclusive in mucinous ovarian cancers. Interestingly, a double negative subtype (i.e. HER2− and KRAS−) showed poorer prognosis, similar to the poorer prognosis observed for triple-negative subtype in breast cancer
Although our study is the largest Asian mucinous ovarian cancer cohort to date, there are limitations in the study. In our cohort, differences in follow up duration between HER2+ and HER2− patients may have affected the survival outcomes, although we have taken the steps to assess the sensitivity due to the difference. The sensitivity analyses did not show significance. The role of tumor intra-heterogeneity may also affect HER2 expression
This study is the largest cohort to report clinicopathological factors and the incidence of HER2 mucinous ovarian cancer in an Asian setting. The data shed light on the differences in HER2 prevalence between Asian and western cohorts, and within our Asian cohort, between ethnic subpopulations. The high prevalence of HER2 in mEOC suggests the potential for HER2 targeted treatment in this relatively chemo-resistant and rare cancer.