Older age should not be a barrier to testing for somatic variants in homologous recombination DNA repair-related genes in patients with high-grade serous ovarian carcinoma.

BACKGROUND: Somatic pathogenic variants (PVs) in homologous recombination DNA repair (HR)-related genes found in high-grade serous ovarian carcinomas (HGSC) are not well-characterised in older patients (≥70 years). This may reflect low testing rates in older patients. METHODS: Data from 1210 HGSC patients in AACR Project GENIE and 324 patients in an independent dataset INOVATe were analysed. Cases where somatic variants could be distinguished from germline variants were included, and analysis was restricted to those with a somatic TP53 variant, to ensure cases were HGSC. RESULTS: Of 1210 patients in GENIE, 27% (n = 325) were aged ≥70 years at testing. Patients with somatic-only PVs in BRCA2 were older compared with BRCA1 (median 71 vs 60 years, p = 0.002). Median age for 21 patients with somatic-only PVs in 11 other HR-related genes ranged from 40 to 67 years. In older patients, 7% (n = 22) had somatic BRCA1/2 PVs, and 1% (n = 2) had PVs other HR-related genes; this rate was not significantly different to younger patients (<70 years), 7% (n = 62) BRCA1/2 and 2% (n = 19) other HR-related genes (p = 0.36). The overall frequency of somatic BRCA1/2 PVs was similar in INOVATe (n = 25; 7.7%) and somatic-only BRCA2 PVs were again found in older patients compared with BRCA1 (median age: at testing, 70 vs 63 years; at diagnosis, 68 vs 60 years). CONCLUSIONS: The overall frequency of somatic-only PVs in HR-related genes was similar in older and younger patients with HGSC, highlighting the importance of somatic testing irrespective of age. Limiting somatic testing by age may exclude patients who could benefit from maintenance poly(ADP-ribose) polymerase (PARP) inhibitors.

New therapeutic opportunities for women with low-grade serous ovarian cancer.

Low-grade serous ovarian cancer (LGSC) is a morphologically and molecularly distinct subtype of ovarian cancer, accounting for ~10% of serous carcinomas. Women typically present at a younger age and have a protracted clinical course compared with the more common, high-grade serous ovarian cancer. Currently, the primary treatment of LGSC is the same as other epithelial ovarian cancer subtypes, with treatment for most patients comprised of debulking surgery and platinum/taxane chemotherapy. Primary surgical cytoreduction to no visible residual disease remains a key prognostic factor; however, the use of platinum-based chemotherapy in both upfront and relapsed setting is being questioned due to low response rates in LGSC. Most LGSC expresses steroid hormone receptors, and selected patients may benefit from endocrine maintenance therapy following chemotherapy, in particular, those with evidence of residual disease at completion of surgery. In the recurrent setting, while hormonal therapies may offer disease stabilisation with relatively low toxicity, objective response rates remain low. Strategies to increase response rates, including combining with CDK4/6 inhibitors, are being investigated. LGSC has a high prevalence of activating somatic mutations in mitogen-activated protein kinase pathway genes, most commonly in KRAS, BRAF and NRAS. Trametinib, a MEK inhibitor, has shown efficacy over chemotherapy and endocrine therapy. The use of combination targeted therapies, immunotherapy and anti-angiogenic agents, remain active areas of investigation for the treatment of LGSC.

Strategies to enable large-scale proteomics for reproducible research.

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics.

Addressing the Challenges of High-Throughput Cancer Tissue Proteomics for Clinical Application: ProCan.

The cancer tissue proteome has enormous potential as a source of novel predictive biomarkers in oncology. Progress in the development of mass spectrometry (MS)-based tissue proteomics now presents an opportunity to exploit this by applying the strategies of comprehensive molecular profiling and big-data analytics that are refined in other fields of 'omics research. ProCan (ProCan is a registered trademark) is a program aiming to generate high-quality tissue proteomic data across a broad spectrum of cancer types. It is based on data-independent acquisition-MS proteomic analysis of annotated tissue samples sourced through collaboration with expert clinical and cancer research groups. The practical requirements of a high-throughput translational research program have shaped the approach that ProCan is taking to address challenges in study design, sample preparation, raw data acquisition, and data analysis. The ultimate goal is to establish a large proteomics knowledge-base that, in combination with other cancer 'omics data, will accelerate cancer research.

Tamoxifen-induced severe hot flashes and endoxifen levels: is dose reduction a safe and effective strategy?

OBJECTIVES: Severe hot flash (HF) toxicity due to tamoxifen can compromise compliance. We previously found that HFs did not correlate with endoxifen level or CYP2D6 genotype. In this study, we reduced tamoxifen dose in patients with severe HFs to determine whether HFs were ameliorated whilst maintaining a purported therapeutic endoxifen level of >15 nM. MATERIALS AND METHODS: Twenty patients with severe HFs on 20 mg TAM had CYP2D6genotype, trough level tamoxifen and metabolites measured with Loprinzi HF scores (HFS) derived before and after DR of tamoxifen to 10 mg. Other data collected included demographics, smoking, alcohol, menstrual and breast cancer history, previous chemotherapies, concurrent medications, BMI and other tamoxifen toxicities. RESULTS: At the 20 mg tamoxifen dose, endoxifen levels were 25.6, 0-91.9 nM (median, range) with HFS 131, 22-1482 (median, range). Upon DR to 10 mg, median endoxifen level fell to 14.1, 0.6-71.9 nM (difference in means p = 0.11, two-tailed T test) with HFS 47, 5-864 (difference in means p = 0.24, two-tailed T test). Despite lacking statistical significance, 85% of patients reported subjective improvement of HFs with DR. After DR, the proportion of patients with endoxifen level <15 nM increased from 20% to 50%. HFS did not correlate with any other parameter. CONCLUSION: DR of tamoxifen from 20 mg to 10 mg daily resulted in halving of endoxifen levels and subjective improvement of HF. While half the dose-reduced patients were below a potential therapeutic level of endoxifen, other recent studies suggest that low endoxifen levels may not indicate reduced effectiveness of tamoxifen.

Accelerated Barocycler Lysis and Extraction Sample Preparation for Clinical Proteomics by Mass Spectrometry.

We have developed a streamlined proteomic sample preparation protocol termed Accelerated Barocycler Lysis and Extraction (ABLE) that substantially reduces the time and cost of tissue sample processing. ABLE is based on pressure cycling technology (PCT) for rapid tissue solubilization and reliable, controlled proteolytic digestion. Here, a previously reported PCT based protocol was optimized using 1-4 mg biopsy punches from rat kidney. The tissue denaturant urea was substituted with a combination of sodium deoxycholate (SDC) and N-propanol. ABLE produced comparable numbers of protein identifications in half the sample preparation time, being ready for MS injection in 3 h compared with 6 h for the conventional urea based method. To validate ABLE, it was applied to a diverse range of rat tissues (kidney, lung, muscle, brain, testis), human HEK 293 cell lines, and human ovarian cancer samples, followed by SWATH-mass spectrometry (SWATH-MS). There were similar numbers of quantified proteins between ABLE-SWATH and the conventional method, with greater than 70% overlap for all sample types, except muscle (58%). The ABLE protocol offers a standardized, high-throughput, efficient, and reproducible proteomic preparation method that when coupled with SWATH-MS has the potential to accelerate proteomics analysis to achieve a clinically relevant turn-around time.

Genome-wide association study of paclitaxel and carboplatin disposition in women with epithelial ovarian cancer.

Identifying single nucleotide polymorphisms (SNPs) that influence chemotherapy disposition may help to personalize cancer treatment and limit toxicity. Genome-wide approaches are unbiased, compared with candidate gene studies, but usually require large cohorts. As most chemotherapy is given cyclically multiple blood sampling is required to adequately define drug disposition, limiting patient recruitment. We found that carboplatin and paclitaxel disposition are stable phenotypes in ovarian cancer patients and tested a genome-wide association study (GWAS) design to identify SNPs associated with chemotherapy disposition. We found highly significant SNPs in ABCC2, a known carboplatin transporter, associated with carboplatin clearance (asymptotic P = 5.2 × 106, empirical P = 1.4 × 10-5), indicating biological plausibility. We also identified novel SNPs associated with paclitaxel disposition, including rs17130142 with genome-wide significance (asymptotic P = 2.0 × 10-9, empirical P = 1.3 × 10-7). Although requiring further validation, our work demonstrated that GWAS of chemotherapeutic drug disposition can be effective, even in relatively small cohorts, and can be adopted in drug development and treatment programs.

Profiling differential microRNA expression between in situ, infiltrative and lympho-vascular space invasive breast cancer: a pilot study.

Ductal carcinoma in situ (DCIS), invasive breast cancer (IBC) and lympho-vascular invasion (LVI) represent distinct stages in breast cancer progression with different clinical implications. Altered microRNA (miRNA) expression may play a role in mediating the progression of DCIS to IBC and LVI. The aim of this pilot study was to investigate whether differential miRNA expression could play a role in breast cancer progression. Cancer cells from DCIS, IBC and LVI were microdissected from formalin fixed paraffin embedded (FFPE) tissue of five breast cancer samples. MiRNA profiling of extracted RNA was performed using the TaqMan® Array Human MicroRNA Cards A and B v3.0. Candidate miRNAs and gene targets were validated by qPCR. 3D culture of MCF10A, MCF10DCIS.com and T47D cells were used as models for normal, DCIS and IBC. Immunohistochemistry of candidate genes was performed on FFPE 3D cell cultures as well as on tissue microarray which included cores of DCIS and IBC samples. MiR-150, miR-126 and miR-155 were found to be more highly expressed in IBC and LVI compared to DCIS. Gene targets of these miRNAs, RhoA, PEG10 and MYB, were found to be more highly expressed in DCIS compared to IBC by qPCR and in MCF10A and MCF10DCIS.com cells compared to T47D cells by immunohistochemistry. There was no difference in intensity of staining of RhoA by immunohistochemistry in DCIS versus IBC samples on tissue microarray. In this pilot study, we found evidence to support a potential role for variation in miRNA levels in the transition from DCIS to IBC.

BRAF Mutations in Low-Grade Serous Ovarian Cancer and Response to BRAF Inhibition.

PURPOSE: Low-grade serous ovarian carcinoma (LGSC) responds poorly to chemotherapy and is characterized by activating mutations in the Ras sarcoma-mitogen-activated protein kinase (RAS-MAPK) pathway, including oncogenic BRAF. However, response to BRAF inhibitors is tumor-type specific. Significant improvement in survival is seen in patients with BRAF-mutant melanoma, but other cancer types, such as colorectal cancers, are generally less sensitive. We examined the frequency and characteristics of BRAF-mutated LGSC and described the response to treatment with BRAF inhibitors. PATIENTS AND METHODS: Mutations were assessed in LGSC (N = 65) by using targeted, exome, and whole-genome sequencing. Patient characteristics, treatment, and clinical outcome were assessed, and the median follow-up time was more than 5 years. BRAF inhibitors were trialed in two patients with a somatic BRAF V600E mutation: one patient received dabrafenib monotherapy and was monitored clinically, biochemically (cancer antigen [CA]-125 levels), and with positron emission tomography (PET) imaging. Expression of the BRAF V600E protein in this patient was assessed by immunohistochemistry. RESULTS: Among patients with LGSC, nine (13.8%) of 65 had a somatic BRAF mutation. Of the nine patients with BRAF mutation-positive LGSC, four experienced progressive disease that did not respond to conventional chemotherapy. Two of the patients experienced progression quickly and died as a result of disease progression, and two received targeted treatment. Two patients with BRAF V600E mutation received BRAF inhibitors at relapse and both achieved durable responses. CONCLUSION: BRAF mutations are not uncommon in patients with LGSC and should be routinely tested, because BRAF inhibitors can be an effective treatment for these patients. The results highlight the need for targeted treatment in this rare tumor type, and a prospective study is needed to formally assess the response rate and clinical benefit.

Serous ovarian and primary peritoneal cancers: A comparative analysis of clinico-pathological features, molecular subtypes and treatment outcome.

OBJECTIVE: Primary peritoneal cancer is rare and considered equivalent to stage III/IV ovarian cancer, but questions remain concerning its underlying biology, prognosis and optimal management. METHODS: Clinico-pathological and treatment details of primary peritoneal (n=120) and ovarian cancer (n=635) were obtained on women recruited to the Australian Ovarian Cancer Study. Log-rank test was used to compare survival and cox proportional hazards models were fitted to obtain hazard ratios and 95% confidence intervals, both unadjusted and adjusted for age, grade, FIGO stage, residual disease and treatment with neoadjuvant chemotherapy. Molecular subtype was determined by gene expression profiling using published data. RESULTS: Compared with advanced serous ovarian cancer, primary peritoneal cancer patients were older (mean age 65.5 vs. 60.2years, p<0.001), more often treated with neoadjuvant chemotherapy (38.4% vs. 11.4%, p<0.001). Gene expression profiling classified a substantially higher proportion of primary peritoneal carcinomas as C1 (mesenchymal, reactive stromal infiltration) subtype (70.6% vs. 32.1%, p=0.029), which was associated with lower complete surgical resection rate. Women with primary peritoneal cancer had significantly shorter progression-free (11.6 vs. 13.6months, p=0.007) and overall survival (31.7 vs. 39.8months, p=0.012). In multivariate analysis, residual disease and neoadjuvant chemotherapy were both independently associated with increased risk of progression and death. CONCLUSIONS: Primary peritoneal cancer patients were more frequently treated with neoadjuvant chemotherapy and had inferior survival. Different tumor biology characterized by activated stromal fibrosis in primary peritoneal cancer may underlie the differences in treatment and clinical outcome.

Dose Escalation of Tamoxifen in Patients with Low Endoxifen Level: Evidence for Therapeutic Drug Monitoring-The TADE Study.

PURPOSE: Endoxifen is the major mediator of tamoxifen effect and endoxifen levels <15 nmol/L may be associated with increased risk of breast cancer recurrence. We increased tamoxifen dose in breast cancer patients with low endoxifen levels and assessed the influence of various parameters on reaching 15 nmol/L and 30 nmol/L endoxifen levels. EXPERIMENTAL DESIGN: Tamoxifen dose was increased in those with endoxifen levels below 30 nmol/L. Toxicity, including hot flash score, was measured. CYP2D6 metabolizer status was classified as ultra-rapid (UM), extensive (EM), intermediate (IM), or poor (PM) based genotype of somatic DNA. RESULTS: Dosage was escalated in 68 of 122 participants. On 20 mg tamoxifen, 24% had endoxifen levels below 15 nmol/L and this reduced to 6% following dose escalation. In over 50% of cases, there was no identified cause for low endoxifen. Low baseline endoxifen level, and not CYP2D6 metabolizer status, independently predicted reaching threshold targets for both the 15 nmol/L and 30 nmol/L targets (P = 0.04 and 0.003 respectively). The 15 nmol/L target was reached in all UM/EM and IM patients, 63% of PM patients, and 58% of those with baseline endoxifen of <10 nmol/L. There was no correlation between hot flash score and genotype or any tamoxifen metabolite level including endoxifen (R = 0.07). CONCLUSIONS: Low endoxifen on standard dose tamoxifen was the only independent predictor of failure to achieve potentially therapeutic levels. Trials examining tamoxifen dose escalation and breast cancer outcome should be guided by endoxifen levels alone, without reference to CYP2D6 genotype or presence of hot flashes. Clin Cancer Res; 22(13); 3164-71. ©2016 AACRSee related commentary by Hertz and Rae, p. 3121.

Paclitaxel sensitivity in relation to ABCB1 expression, efflux and single nucleotide polymorphisms in ovarian cancer.

ABCB1 (adenosine triphosphate-binding cassette transporter B1) mediates cellular elimination of many chemotherapeutic agents including paclitaxel, which is commonly used to treat ovarian cancer. A significant association between common single nucleotide polymorphisms (SNPs) in ABCB1 and progression-free survival has been reported in patients with ovarian cancer. Variable paclitaxel clearance due to genotype specific differences in ABCB1 activity in cancer cells and/or normal tissues may underlie the association. Using cell-based models, we evaluated the correlations between ABCB1 expression, polymorphisms, transporter activity and paclitaxel sensitivity in ovarian cancer (n = 10) and lymphoblastoid (n = 19) cell lines. Close associations between ABCB1 expression, transporter function and paclitaxel sensitivity were found in lymphoblastoid cell lines, although we could not demonstrate an association with common SNPs. In ovarian cancer cell lines, ABCB1 expression was low and the association between expression and function was lost. These results suggest that ABCB1 related survival difference in ovarian cancer patients is more likely to be due to differential whole body paclitaxel clearance mediated by normal cells rather than a direct effect on cancer cells.

The prognostic value of Ki67 in systemically untreated patients with node-negative breast cancer.

AIM: To evaluate the utility of Ki67 as a prognostic marker in a series of patients with node-negative breast cancer untreated with adjuvant systemic therapy. METHODS: The cohort consisted of 203 cases treated with breast conserving surgery and radiation only; median follow-up was 183 months (range 156-277 months). An immunohistochemical panel of oestrogen receptor (ER), progesterone receptor (PR), cytokeratin (CK)5/6 and Ki67 and human epidermal growth factor 2 in situ hybridization (HER2-ISH) was performed on the tumour samples. Ki67 scores were evaluable in 193/203 patients (95.1%). The primary outcome was breast cancer specific survival (BCSS). RESULTS: Of the cohort, 29 (14.2%) died of breast cancer. A cut off of 10% separated tumours into a 'Ki67-low' (n=70) or 'Ki67-high' group (n=123). The breast cancer specific survival was 97.1% and 77.6% for Ki67-low and Ki67-high groups, respectively. Univariate analysis showed that in this lymph node-negative cohort, the predictors for BCSS were tumour size, Ki67, LVI, age and histological grade 3. Multivariable analysis showed that Ki67 index and lymphovascular space invasion were independent predictors of breast cancer death. To examine the utility of Ki67 in assignment of immunohistochemically molecular subtypes, cases were assigned into Luminal A (ER-positive, HER2-negative, Ki67 ≤14%), Luminal B (ER-positive, HER2-negative, Ki67 >14%) and triple negative (ER/PR-negative and HER2-negative, any Ki67). The 15-year breast cancer specific survival was 91.7%, 79.4% and 75.8%, respectively. CONCLUSIONS: A statistically significant difference in breast cancer specific survival is seen in groups defined using Ki67 and receptor status, whereas histological grading was not a significant predictor of survival. Ki67 immunostaining provides prognostic information beyond traditionally assessed clinicopathological variables.

Prohibitin expression is associated with high grade breast cancer but is not a driver of amplification at 17q21.33.

AIMS: In a study of ductal carcinoma in situ of the breast, we identified five genes at chromosome 17q21.33 that were over-expressed in high grade cases, and showed a correlation between expression and gene copy number. The aim of this study was to investigate potential drivers of genomic amplification at 17q21.33. METHODS: Analysis of high resolution comparative genomic hybridisation and published data specified a minimum region of amplification at 17q21.33. Prohibitin (PHB) expression was examined by immunohistochemistry in 285 invasive breast cancers. Gene copy number was examined by fluorescence in situ hybridisation. RESULTS: The minimum region of amplification at 17q21.33 included ten genes with PHB selected as a candidate driver. Increased PHB expression was associated with higher grade breast cancer and poorer survival. Amplification of PHB was detected in 13 of 235 cases (5.5%) but was not associated with PHB expression. PHB amplification was most common in the ERBB2+ breast cancer subtype, although high expression was most prevalent in basal-like and luminal B cancers. CONCLUSIONS: Amplification at 17q21.33 is a recurrent feature of breast cancer that forms part of a 'firestorm' pattern of genomic aberration. PHB is not a driver of amplification, however PHB may contribute to high grade breast cancer.

Ki67 and proliferation in breast cancer.

New approaches to the prognostic assessment of breast cancer have come from molecular profiling studies. A major feature of this work has been to emphasise the importance of cancer cell proliferation as a key discriminative indicator of recurrence risk for oestrogen receptor positive breast cancer in particular. Mitotic count scoring, as a component of histopathological grade, has long formed part of a routine evaluation of breast cancer biology. However, there is an increasingly compelling case to include a specific proliferation score in breast cancer pathology reports based on expression of the cell cycle regulated protein Ki67. Immunohistochemical staining for Ki67 is a widely available and economical test with good tolerance of pre-analytical variations and staining conditions. However, there is currently no evidence based protocol established to derive a reliable and informative Ki67 score for routine clinical use. In this circumstance, pathologists must establish a standardised framework for scoring Ki67 and communicating results to a multidisciplinary team.

Characteristics of HER2-positive breast cancer diagnosed following the introduction of universal HER2 testing.

The aim of this study was to determine the impact of universal HER2 testing on the clinico-pathologic profile of HER2+ breast cancer. Data were extracted from breast cancer pathology reports spanning two periods: before (2003/4, n = 379), and after (2008/9, n = 560) the introduction of universal testing. In 2003/4, 43.3% of breast cancers were tested for HER2 with 16% of tested cases HER2+. In 2008/9, 98.4% of cases were tested with 14.7% HER2+. In 2008/9, HER2+ status was associated with younger age, higher grade, increased tumour size, lymph node involvement, negative oestrogen and/or progesterone receptor status. HER2+ cases diagnosed in 2003/4 were not significantly different in respect of these features. The rate of HER2+ breast cancer amongst screen detected cases in 2008/9 was 8.3%. The phenotype of HER2+ breast cancer was stable following the introduction of universal testing. The overall rate of HER2+ breast cancer was influenced by screen detection.

High-risk estrogen-receptor-positive breast cancer: identification and implications for therapy.

The estrogen receptor (ER) has long been recognized as a key discriminative feature of breast cancer, which carries profound implications for management. However, recent advances in the understanding of breast cancer heterogeneity have demonstrated the importance of biologic context to the interpretation of ER as a prognostic and predictive factor. The use of tumor subtyping methods and prognostic indicators based on molecular profiling of tumor tissue is now helping to delineate high-risk ER-positive cancer types that have distinct risk and treatment response profiles. These new approaches to breast cancer classification will have a major impact on the conduct of clinical trials and individual patient assessment in the future.

Development of a data entry auditing protocol and quality assurance for a tissue bank database.

Human transcription error is an acknowledged risk when extracting information from paper records for entry into a database. For a tissue bank, it is critical that accurate data are provided to researchers with approved access to tissue bank material. The challenges of tissue bank data collection include manual extraction of data from complex medical reports that are accessed from a number of sources and that differ in style and layout. As a quality assurance measure, the Breast Cancer Tissue Bank (http:\\www.abctb.org.au) has implemented an auditing protocol and in order to efficiently execute the process, has developed an open source database plug-in tool (eAuditor) to assist in auditing of data held in our tissue bank database. Using eAuditor, we have identified that human entry errors range from 0.01% when entering donor's clinical follow-up details, to 0.53% when entering pathological details, highlighting the importance of an audit protocol tool such as eAuditor in a tissue bank database. eAuditor was developed and tested on the Caisis open source clinical-research database; however, it can be integrated in other databases where similar functionality is required.

Electronic biorepository application system: web-based software to manage receipt, peer review, and approval of researcher applications to a biobank.

The importance of suitably characterized and preserved biospecimens for research is acknowledged, yet providing information about the availability of biospecimens and associated data, responding to enquiries from researchers, processing applications for material, and seeking independent scientific review of proposed projects are complex and time-consuming processes. Most biorepositories operate as not-for-profit entities; therefore, cost containment is a major consideration. We identified that online systematizing and automation of all of the tasks associated with application management could reduce the administrative workload and therefore reduce costs and improve the efficiency of a biobank. Accordingly, we have developed a Web-based electronic Biorepository Application System (eBAS) that allows researchers to search for suitable material from the biobank database, submit an online expression of interest, and complete all the information required for a full application. Peer review is also managed through eBAS. Implementation of eBAS has streamlined application management and external peer review of researcher applications, and has facilitated automated record storage and management. This approach has potential to reduce the costs and complexities of administering researcher applications. We have also linked eBAS to an open-source clinical research and specimen management database, Caisis.

HER2 discordance between primary breast cancer and its paired metastasis: tumor biology or test artefact? Insights through meta-analysis.

The proto-oncogene, HER2, has prognostic and predictive relevance in invasive breast cancer (IBC). HER2 testing of primary IBC guides treatment selection and is assumed to reflect HER2 status of associated metastases, although HER2 discordance between IBC and metastasis has been reported. Systematic review and meta-analysis of HER2 status in IBC and its paired loco-regional or distant metastasis were done. Quality appraisal considered whether (within-subject) testing conditions were maintained for paired primary and metastasis. Random effects logistic regression models were used to estimate pooled within-subject HER2 discordant proportions and to examine study-level covariates, including tumor-related and testing-related variables, potentially associated with HER2 discordance differences across (between) studies. Modelled paired HER2 data for primary and metastatic cancer (2520 subjects, 26 studies) showed a pooled HER2 discordance of 5.5% (3.6-8.5%). Sensitivity analysis, excluding the only study not maintaining same conditions for paired testing, gave a pooled estimate of 5.2% (3.5-7.8%). Pooled discordant proportion was not associated with differences between studies in test type, test scoring or interpretation criteria, subjects' median age, study time-frame, or HER2 positivity in primary cancer (all P > 0.05). However, type of metastasis was significantly associated with estimated HER2 discordance (P = 0.0017): studies of primary tumor paired with distant metastases had higher discordance [11.5% (6.9-18.6%)] than studies of primary paired with lymph node metastases only [4.1% (2.4-7.2%)], or those paired with nodal or various metastases [3.3% (2.0-5.6%)]; P < 0.01. HER2 discordant proportion was higher where paired metastases were metachronous relative to synchronous to primary IBC (P = 0.0024). Sensitivity analysis provided weak evidence (P = 0.074) that discordance in the direction of change from HER2-negative primary cancer to HER2-positive paired metastasis was more likely than the reverse. Study-level meta-analysis suggests factors associated with the type of metastasis as underlying mechanisms for observed HER2 discordance between primary IBC and paired metastasis. Test-related factors did not account for differences across studies in the HER2 discordant proportion.