Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma

Background:

Trastuzumab has recently shown efficacy in the treatment of HER2-positive advanced gastric adenocarcinoma. Although antibody-based therapies target the metastatic disease, HER2 status is usually evaluated in the primary tumour because metastatic sites are rarely biopsied. The aim of this study was to compare HER2 status in primary and paired metastatic sites of gastric adenocarcinoma.

Methods:

The HER2 status was assessed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in 72 secondary lesions of gastric adenocarcinoma and in the corresponding primary tumours.

Results:

Concordance of FISH results, evaluable in 68 primary and matched metastatic sites, was 98.5%. Concordance of IHC results, available in 39 of the 72 paired cases, was 94.9%. Only one case showed discordance between primary tumour and metastasis, being negative by both IHC and FISH in the primary and showing HER2 overexpression and amplification in the corresponding pancreatic lymph node metastasis.

Conclusion:

The high concordance observed between HER2 results obtained by both IHC and FISH on primary tumours and corresponding metastases suggests that in gastric cancer HER2 status is maintained in most cases unchanged during the metastatic process.     

Droplet digital PCR measurement of HER2 in patients with gastric cancer

Background:

Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator.

Methods:

We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer.

Results:

The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients.

Conclusions:

Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.     

Frequency of HER2 expression of circulating tumour cells in patients with metastatic or recurrent gastrointestinal cancer

Background:

The clinical significance of circulating tumour cell (CTC) detection in gastrointestinal (GI) cancer remains controversial and the molecular biological characteristics of CTCs are poorly understood.

Methods:

A total of 87 patients with metastatic or recurrent GI cancer were prospectively enrolled. Circulating tumour cells and their HER2 status were assessed using the CellSearch System.

Results:

Among the 62 CTC-positive cases, we found 22 discordant cases (35.5%). Among the HER2-negative primary tumours, 17 of 54 developed HER2-positive CTCs. Five of eight had HER2-negative CTCs among the HER2-positive primary tumours.

Conclusion:

The findings in the current study suggest that it is critical to evaluate the HER2 status of not only the primary tumour but also the CTCs because the metastasising tumour cells are the primary target of systemic therapy.     

Do type 1 receptor tyrosine kinases inform treatment choice? A prospectively planned analysis of the TEAM trial

Background:

Epidermal growth factor receptors contribute to breast cancer relapse during endocrine therapy. Substitution of aromatase inhibitors (AIs) may improve outcomes in HER-positive cancers.

Methods:

Tissue microarrays were constructed. Quantitative analysis of HER1, HER2, and HER3 was performed. Data were analysed relative to disease-free survival and treatment using outcomes at 2.75 and 6.5 years.

Results:

Among 4541 eligible samples, 4225 (93%) had complete HER1–3 data. Overall, 5% were HER1-positive, 13% HER2-positive, and 21% HER3-positive; 32% (n=1351) overexpressed at least one HER receptor. In the HER1–3-negative subgroup, the hazard ratio (HR) for upfront exemestane vs tamoxifen at 2.75 years was 0.67 (95% confidence interval (CI), 0.52–0.87), in the HER1–3-positive subgroup, the HR was 1.15 (95% CI, 0.85–1.56). A prospectively planned treatment-by-marker analysis demonstrated a significant interaction between HER1–3 and treatment at 2.75 years (HR=0.58; 95% CI, 0.39–0.87; P=0.008), as confirmed by multivariate regression analysis adjusting for prognostic factors (HR=0.55; 95% CI, 0.36–0.85; P=0.005). This effect was time dependent.

Conclusion:

In the 2.75 years prior to switching patients initially treated with tamoxifen to exemestane, a significant treatment-by-marker effect exists between AI/tamoxifen treatment and HER1-3 expression, suggesting HER expression could be used to select appropriate endocrine treatment at diagnosis to prevent or delay early relapses.     

PREDICT Plus: development and validation of a prognostic model for early breast cancer that includes HER2

Background:

Predict (www.predict.nhs.uk) is an online, breast cancer prognostication and treatment benefit tool. The aim of this study was to incorporate the prognostic effect of HER2 status in a new version (Predict+), and to compare its performance with the original Predict and Adjuvant!.

Methods:

The prognostic effect of HER2 status was based on an analysis of data from 10 179 breast cancer patients from 14 studies in the Breast Cancer Association Consortium. The hazard ratio estimates were incorporated into Predict. The validation study was based on 1653 patients with early-stage invasive breast cancer identified from the British Columbia Breast Cancer Outcomes Unit. Predicted overall survival (OS) and breast cancer-specific survival (BCSS) for Predict+, Predict and Adjuvant! were compared with observed outcomes.

Results:

All three models performed well for both OS and BCSS. Both Predict models provided better BCSS estimates than Adjuvant!. In the subset of patients with HER2-positive tumours, Predict+ performed substantially better than the other two models for both OS and BCSS.

Conclusion:

Predict+ is the first clinical breast cancer prognostication tool that includes tumour HER2 status. Use of the model might lead to more accurate absolute treatment benefit predictions for individual patients.     

Epstein-Barr virus as a marker of biological aggressiveness in breast cancer

Purpose:

Although a potential role of the Epstein-Barr virus (EBV) in the pathogenesis of breast cancer (BC) has been underlined, results remain conflicting. Particularly, the impact of EBV infection on biological markers of BC has received little investigation.

Methods:

In this study, we established the frequency of EBV-infected BC using real-time quantitative PCR (RT–PCR) in 196 BC specimens. Biological and pathological characteristics according to EBV status were evaluated.

Results:

EBV DNA was present in 65 of the 196 (33.2%) cases studied. EBV-positive BCs tended to be tumours with a more aggressive phenotype, more frequently oestrogen receptor negative (P=0.05) and with high histological grade (P=0.01). Overexpression of thymidine kinase activity was higher in EBV-infected BC (P=0.007). The presence of EBV was weakly associated with HER2 gene amplification (P=0.08).

Conclusion:

Our study provides evidence for EBV-associated BC undergoing distinct carcinogenic processes, with more aggressive features.     

Lapatinib monotherapy in patients with relapsed, advanced, or metastatic breast cancer: efficacy, safety, and biomarker results from Japanese patients phase II studies

Background:

HER2-positive metastatic breast cancer (MBC) relapsing after trastuzumab-based therapy may require continued HER2 receptor inhibition to control the disease and preserve the patients'' quality-of-life. Efficacy and safety of lapatinib monotherapy was evaluated in Japanese breast cancer patients after trastuzumab-based therapies.

Methods:

In studies, EGF100642 and EGF104911 evaluated the efficacy and safety of oral lapatinib given 1500 mg once daily in patients with advanced or MBC. All patients progressed on anthracyclines and taxanes; HER2-positive patients had also progressed on trastuzumab.

Results:

For HER2-positive tumours (n=100), objective response rate was 19.0% (95% confidence interval (CI): 11.8–28.1) and clinical benefit rate (CBR) was 25.0% (95% CI: 16.9–34.7). One out of 22 HER2-negative tumour was documented as complete response (n=22). The median time-to-progression (TTP) in the HER2-positive and HER2-negative groups was 13.0 and 8.0 weeks (P=0.007); median overall survival was 58.3 and 40.0 weeks, respectively. The most frequent adverse event was diarrhoea. TTP and CBR were significantly associated with HER2 expression. Patients with tumours harbouring an H1047R PIK3CA mutation or low expression of PTEN derived clinical benefit from lapatinib.

Conclusion:

Lapatinib monotherapy had shown anti-tumour activity in Japanese patients with HER2-positive MBC that relapsed after trastuzumab-based therapy, including those with brain metastases. Patients benefiting from lapatinib may have biomarker profiles differing from that reported for trastuzumab.     

Role of HER3 expression and PTEN loss in patients with HER2-overexpressing metastatic breast cancer (MBC) who received taxane plus trastuzumab treatment

Background:

The aim of this study was to investigate the role of human epidermal growth factor receptor (HER3) and PTEN expression in patients with HER2-overexpressing metastatic breast cancer (MBC).

Methods:

One hundred twenty-five MBC patients who were treated with taxane plus trastuzumab chemotherapy as first-line therapy were included in this analysis. Immunohistochemical (IHC) staining with HER3 and PTEN antibodies were conducted retrospectively.

Results:

Patients who had negative HER3 staining (62.4%) had a better progression-free survival (PFS) than did those who had positive HER3 staining (P=0.001; median PFS, 21 vs 11 months). Patients who had a PTEN score >20 (78.1%) showed longer PFS than did those with a PTEN score ⩽20 (P=0.006; median PFS, 13 vs 9 months). Patients who had a PTEN score >20 exhibited a longer overall survival (OS) than did those with a PTEN score ⩽20 (P=0.005; median OS, 48 vs 25 months). HER3 negativity and PTEN loss were identified as independent risk factors for PFS. PTEN loss was identified as an independent risk factor for OS.

Conclusion:

HER3 and PTEN expressions may be predictive markers, and PTEN expression may be a predictive and prognostic biomarker for trastuzumab treatment in HER2-positive MBCs.     

Exploring the use and impact of adjuvant Trastuzumab for HER2-positive breast cancer patients in a large UK cancer network Do the results of international clinical trials translate into a similar benefit for patients in South East Wales?

Background:

Trastuzumab was approved in the United Kingdom for adjuvant treatment of human epidermal growth factor receptor 2 (HER2)+ breast cancer in 2006 at significant economic cost and with limited evidence in smaller T1N0 tumours. The South East Wales Cancer Network covers a population of 1 420 000 and maintains a database of treatments used. We examined this database to ensure the outcome of Trastuzumab use is as expected, especially in patients with T1N0 cancers.

Ethods:

M Case notes of patients with HER2+ disease eligible for adjuvant Trastuzumab over 2005–2008 were reviewed. Disease-free survival (DFS) and overall survival (OS) were calculated with the Kaplan–Meier method using SPSS (version 16.0.01 for Windows, SPSS, Chicago, IL, USA).

Results:

A total of 239 of 338 (70.7%) eligible HER2+ patients received treatment. At 3 years, the DFS of the treated group was 90.3% vs 73.3% and the OS was 98.5% vs 87.6%. In all, 47 of 92 stage I patients received Trastuzumab. Despite a trend towards worse prognostic factors in the treated group the DFS was 100% vs 84.1% and the OS was 100% vs 93.3%.

Conclusion:

Our results are comparable to those from landmark Trastuzumab trials. As evidence continues to emerge that smaller HER2+ cancers may behave aggressively our analysis of stage I tumours adds further support to the use of Trastuzumab in these patients.     

Loss of PTEN is associated with elevated EGFR and HER2 expression and worse prognosis in salivary gland cancer

Background:

Activity of the tumour-suppressor gene PTEN is reduced in different types of cancer and implicates non-responsiveness to targeted therapy. This study evaluates the gene and protein status of PTEN in salivary gland carcinomas.

Methods:

A total of 287 carcinomas of the major and minor salivary glands were investigated for phosphatase and tensin homologue located on chromosome 10 (PTEN) deletion and loss of PTEN expression using fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Results were correlated to clinicopathological parameters, long-term survival, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (IHC and FISH) status of the tumours.

Results:

Hemizygous deletions of PTEN were found in 35 out of 232 (15.1%) carcinomas, while homozygous deletions were observed in 17 out of 232 (7.3%) tumours. Phosphatase and tensin homologue located on chromosome 10 deletion was common in certain histological subtypes and especially homozygous deletion was associated with high-grade malignancy, lymph node metastases and unfavourable long-term prognosis (P<0.001). Loss of PTEN expression was present in 59 out of 273 (21.6%) carcinomas and was significantly correlated to genomic PTEN deletion, high-grade malignancy (P<0.001), increased tumour size (P=0.036), lymph node metastases (P=0.007) and worse disease-specific survival (P=0.002). Genomic PTEN deletion, in particular homogenous deletion (P<0.001) predominantly occurred in tumours with increased gene copy number of EGFR (60.0%) and/or amplification of HER2 (63.6%). Loss of PTEN expression was frequently found in tumours overexpressing EGFR (28.6%) and/or HER2 (52.6%).

Conclusion:

PTEN function is reduced in different types of salivary gland cancer indicating unfavourable prognosis. Its association with EGFR and HER2 signalling might affect targeted therapy.     

Detection of HER2 amplification in circulating free DNA in patients with breast cancer

Background:

Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20–25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer.

Methods:

Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control.

Results:

We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry.

Conclusions:

The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer.     

Addition of GM-CSF to trastuzumab stabilises disease in trastuzumab-resistant HER2+ metastatic breast cancer patients

Background:

One of the proposed mechanisms of trastuzumab-induced regression of human epidermal growth factor receptor 2-positive (HER2+) tumours includes facilitation of antibody-dependent cell-mediated cytotoxicity (ADCC). Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates ADCC. We presented our pilot study of adding GM-CSF to trastuzumab in patients with trastuzumab-resistant HER2+ metastatic breast cancer.

Methods:

Patients with HER2+ metastatic breast cancer that progressed after trastuzumab +/− chemotherapy were continued on trastuzumab 2 mg kg^–1 intravenous weekly and GM-CSF 250 μg m^–2 subcutaneous daily. Patients were assessed for response every 8 weeks. Treatment was continued until disease progression or intolerable toxicity.

Results:

Seventeen patients were evaluable (median age 48 years, range 27–75 years). The median number of metastatic sites was 2 (range 1–3); the most common site was the liver (n=10). The median number of prior regimens for metastatic disease was 2 (range 1–5). No objective disease response was observed, but five patients (29%) had stable disease for a median duration of 15.8 (range 10–53.9) weeks. The most common adverse event was rash at the injection site. No grade 4 or irreversible adverse event was seen.

Conclusion:

The addition of GM-CSF to trastuzumab alone had a modest clinical benefit and acceptable safety profile in heavily pretreated patients with trastuzumab-resistant HER2+ metastatic breast cancer.     

Combination of osteopontin and activated leukocyte cell adhesion molecule as potent prognostic discriminators in HER2- and ER-negative breast cancer

Background:

To analyse the discriminative impact of osteopontin (OPN) and activated leukocyte cell adhesion molecule (ALCAM), combined with human epidermal growth factor 2 (HER2) and oestrogen receptor (ER) in breast cancer.

Methods:

Osteopontin, ALCAM, HER2 and ER mRNA expression in breast cancer tissues of 481 patients were analysed (mRNA microarray analysis, kinetic RT–PCR). Hierarchical clustering was performed in training cohort A (N=100, adjuvant treatment) and validation cohorts B (N=200, no adjuvant treatment, low-risk) and C (N=181, adjuvant treatment, high-risk).

Results:

Negative/low ER and HER2, high OPN and low ALCAM mRNA expression helped to identify patients at particularly high risk, showing shorter DFS, P<0.001, and OAS, P=0.001. Although both validation cohorts showed diverse risk and treatment profiles, this marker constellation was concordantly associated with shorter DFS and OAS (P<0.001 and P=0.075 for cohort B and P=0.043 and P<0.001 for cohort C, respectively). In multivariate analysis, this algorithm was the main independent prognostic factor. Cohort B: DFS, P=0.0065, OAS, not significant; cohort C: DFS, P=0.026, OAS, P<0.001.

Conclusion:

Activated leukocyte cell adhesion molecule and OPN mRNA expression has a strong discriminative impact on survival within cancer patients with low or negative expression of ER and HER2, so called ‘high-risk'' breast cancers, and might help in identifying patients who could benefit from new treatment approaches like targeted therapies in the adjuvant setting.     

Increased mortality in HER2 positive, oestrogen receptor positive invasive breast cancer: a population-based study

Background:

This study assessed the impact of human epidermal growth factor receptor 2 (HER2) status on the outcomes in an unselected population of breast cancer patients who did not receive HER2-targeted therapy.

Methods:

HER2 status by immunohistochemistry and fluorescence in situ hybridisation was compared with clinicopathological data, overall survival (OS) and disease-free survival (DFS) for all patients presenting with breast cancer over 3 years.

Results:

In 865 patients (median follow up 6.02 years), HER2 positivity was identified in 13.3% of all cancers and was associated with higher tumour grade (P<10⁻⁸), lymphovascular invasion (P<0.001) and axillary nodal metastasis (P=0.003). There was a negative association with oestrogen-receptor (ER) and progesterone-receptor expression (P<10⁻⁸), but the majority (57%) of HER2+tumours were ER+HER2 positivity was associated with poorer OS (P=0.0046) and DFS (P=0.0001) confined to the lymph node-positive (LN+) and ER+ subgroups.

Conclusion:

HER2-positive cancers were less common in this population-based cohort than most selected series. The association of HER2 positivity with poor prognosis was confined to the ER+ and LN+ subgroups. The survival deficit for the 7.5% of patients with ER+/HER2+ cancer compared with ER+/HER2– patients points to a significant subgroup of women who may not (currently) be considered for HER2-directed therapy.     

Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling

Background:

The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers.

Methods:

The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERα, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs.

Results:

The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r²=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab.

Interpretation:

Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.     

Prognostic and predictive values of EGFR overexpression and EGFR copy number alteration in HER2-positive breast cancer

Background:

Epidermal growth factor receptor (EGFR) is overexpressed in a subset of human epidermal growth factor receptor 2 (HER2)-positive breast cancers, and coexpression of HER2 and EGFR has been reported to be associated with poor clinical outcome. Moreover, interaction between HER2 and EGFR has been suggested to be a possible basis for trastuzumab resistance.

Methods:

We analysed the clinical significance of EGFR overexpression and EGFR gene copy number alterations in 242 HER2-positive primary breast cancers. In addition, we examined the correlations between EGFR overexpression, trastuzumab response and clinical outcome in 447 primary, and 112 metastatic HER2-positive breast cancer patients treated by trastuzumab.

Results:

Of the 242 primary cases, the level of EGFR overexpression was 2+ in 12.7% and 3+ in 11.8%. High EGFR gene copy number was detected in 10.3%. Epidermal growth factor receptor overexpression was associated with hormone receptor negativity and high Ki-67 proliferation index. In survival analyses, EGFR overexpression, but not high EGFR copy number, was associated with poor disease-free survival in all patients, and in the subgroup not receiving adjuvant trastuzumab. In 447 HER2-positive primary breast cancer patients treated with adjuvant trastuzumab, EGFR overexpression was also an independent poor prognostic factor. However, EGFR overexpression was not associated with trastuzumab response, progression-free survival or overall survival in the metastatic setting.

Conclusions:

Epidermal growth factor receptor overexpression, but not high EGFR copy number, is a poor prognostic factor in HER2-positive primary breast cancer. Epidermal growth factor receptor overexpression is a predictive factor for trastuzumab response in HER2-positive primary breast cancer, but not in metastatic breast cancer.     

Long-term survivor characteristics in HER2-positive metastatic breast cancer from registHER

Background:

Data characterising long-term survivors (LTS) with human epidermal growth factor receptor 2 (HER2)–positive metastatic breast cancer (MBC) are limited. This analysis describes LTS using registHER observational study data.

Methods:

A latent class modelling (LCM) approach was used to identify distinct homogenous patient groups (or classes) based on progression-free survival (PFS), overall survival, and complete response. Demographics, clinicopathologic factors, first-line treatment patterns, and clinical outcomes were described for each class. Class-associated factors were evaluated using logistic regression analysis.

Results:

LCM identified two survivor groups labelled as LTS (n=244) and short-term survivors (STS; n=757). Baseline characteristics were similar between groups, although LTS were more likely to be white (83.6% vs 77.8%) with oestrogen receptor–positive (ER+) or progesterone receptor–positive (PgR+) disease (59.4% vs 50.9%). Median PFS in LTS was 37.2 (95% confidence interval (CI): 32.9–40.5) vs 7.3 months (95% CI: 6.8–8.0) in STS. Factors associated with long-term survival included ER+ or PgR+ disease, metastasis to node/local sites, first-line trastuzumab use, and first-line taxane use.

Conclusions:

Prognostic variables identified by LCM define a HER2-positive MBC patient profile and therapies that may be associated with more favourable long-term outcomes, enabling treatment selection appropriate to the patient''s disease characteristics.