Prognostic profiling
Conventional histopathological evaluation, including size, grade, stage, and mitotic frequency, is supplied by Medical Solutions through a specialist team of pathologists led by Professor Ian Ellis, and provides a basic assessment of a breast cancer patient’s tumour characteristics.
Oestrogen Receptor (ER) and Progesterone Receptor (PR)
The value of morphological assessment can be enhanced by the use of a range of tissue based assays performed in our Reference Laboratory. These provide further information to aid in assessing prognosis, and perhaps more importantly in assessing adjuvant treatment suitability. Two such tests, recommended by NICE guidance, which we are providing to the NHS and private healthcare sectors, are ER/PR and HER2 tests.
Oestrogen and progesterone receptor status is determined immunohistochemically. While providing some prognostic information, these paired tests are also used routinely in a treatment decision role, to determine suitability for hormone therapies such as Tamoxifen or aromatase inhibitors. Medical Solutions utilise our own in-house standardised and validated tests, as well as commercially available assays for both Oestrogen and Progesterone receptors, thus providing a comprehensive and analytically robust service to the clinician.
HER2 protein and gene analysis
Over–expression of the membrane associated receptor protein HER2 (erbB2) as a consequence of gene amplification occurs in 10-20% of breast cancers, and importantly is associated with a poorer prognosis. Immunohistochemical demonstration of HER2 protein over-expression in a breast tumour (or confirmation of gene amplification by in situ hybridisation in borderline cases) is a prerequisite for use of HER2-targeted therapies such as trastuzumab (Herceptin™). Medical Solutions offers the FDA-approved, standardised and validated DAKO HercepTest™ assay for assessment of HER2 protein expression, and the Abbott/Vysis PathVysion™ assay for assessment of HER2 gene amplification status. Both methods of evaluation follow UK guidelines.
In addition to our Essential Portfolio, a Comprehensive Portfolio of tests offering the most up-to-date classification, prognostic and treatment-option information is available to the clinician.
Topoisomerase IIa (TOP2A)
Topoisomerase IIa is a key enzyme in DNA replication and also a target for various chemotherapeutic agents, in particular the anthracycline drugs. The TOP2A gene is located in close proximity to the HER2 gene on chromosome 17q21, and a significant percentage of patients with HER2 amplification demonstrate abnormalities in the TOP2A gene. Anthracyclines, one of the most widely used class of cytotoxic agents for treatment of breast cancer, inhibit TOP2A by trapping the DNA strand break intermediates, leading to persistent DNA cleavage. TOP2A gene amplification and subsequent protein over-expression is associated with improved response anthracycline-based chemotherapy.
BRCA1 mutation carrier status prediction
The International Breast Cancer Linkage Consortium Group have demonstrated recently that protein expression phenotype of breast cancer can be used to predict the chance of BRCA1 mutation carrier status in patients with breast cancer. On the basis of ER/PR, HER2 and cytokeratin phenotyping by IHC, a BRCA1 mutation carrier status risk assessment can be provided for the patient. Identification of high risk is an indication for formal BRCA 1 mutation testing.
Protein expression class typing
Recent molecular studies of breast cancer have demonstrated distinct classes of breast cancer, based on gene expression profiles. These findings have been extrapolated to protein-based techniques, identifying various classes of breast cancer, including luminal type-A and -B, HER2 and basal groups, based upon immunohistochemical evaluation. These results provide the clinician and histopathologist with the information for determining the molecular class and characteristics of the patient’s tumour.
Lymphovascular invasion identification
Lymphovascular invasion is a negative prognostic feature, associated with early recurrence, reduced disease-free survival and a poor outcome in breast cancer. Identification of tumour cells within lymphatic channels is often difficult as part of routine histopathological evaluation. However, the recent development of immunohistochemical tests for revealing the lymphatic channels of tumours has significantly enhanced the ability to identify this phenomenon, and added to the predictive power of this prognostic indicator.
Women who carry certain mutations in the BRCA1 gene are three times more likely to develop breast cancer than the general population. Furthermore this risk is heritable and may be passed to the carrier’s children. Current testing involves an expensive and complex test which looks for mutations in the BRCA1 gene. However, the International Breast Cancer Linkage Consortium Group have recently demonstrated that protein expression phenotype (ie the pattern of expression of a particular set of proteins) in the tumour can be used to predict the chance of a BRCA1 mutation in patients with breast cancer.
Tumours associated with BRCA1 mutations frequently demonstrate a basal cell morphology and express markers associated with that cell type, namely cytokeratins 5/6 and 14. They also frequently demonstrate the so-called “triple negative” phenotype, usually being oestrogen and progesterone receptor, and HER2 negative. Therefore, on the basis of ER/PR, HER2 and cytokeratin CK5/6 and -14 phenotyping by IHC, a BRCA1 mutation carrier status risk assessment can be provided for the patient. Identification of high risk is an indication for formal BRCA 1 mutation testing. A typical high risk profile would look like this:
- ER/PR - negative
- HER2 – negative
- Cytokeratin 5/6 – positive
- Cytokeratin 14 – positive
Immunostaining for BRCA1 mutation carrier risk status has been shown to be more sensitive and have a higher positive predictive value than family history.
Evaluation
A risk assessment is provided by a registered pathologist based upon by-eye evaluation of staining intensities and distributions for the five antibodies listed above.
The presence of circulating tumour cells (CTC) in the peripheral blood is associated with poor prognosis in patients with metastatic breast cancer. The FDA has approved the use of the CellSearch™ Circulating Tumour Cell System for in vitro diagnostic purposes for this specific indication. In this assay, a CTC count of 5 or more tumour cells per 7.5 mL of blood is predictive of shorter progression-free and overall survival.
The CellSearch™ System is the first platform to be designed to automate and standardize the capture, enrichment, identification and enumeration of circulating tumour cells of epithelial origin (based upon a CD45-, EpCAM+, and cytokeratin 8+, 18+, and/or 19+ phenotype) in peripheral blood.
Specialist staining can also be carried out on the isolated cells, allowing the enumeration of cells bearing markers such as HER2, EGFR and other cluster differentiation (CD) antigens. Indeed any FITC labelled commercially available antibody can be validated and utilised in parallel with the CellSearch™ System.
Medical Solutions is the only UK commercial reference laboratory providing CTC analysis.
Evaluation
Fresh blood samples (7.5ml) are taken and run on the CellSearch platform at Medical Solutions’ laboratories. The presence of more than 5 circulating tumour cells per 7.5ml sample is regarded as a negative prognostic sign.
EGFR is a validated anticancer target against which there are in excess of 70 new agents in various stages of development. In different cancer types, certain subsets of patients have been shown to benefit from these therapies. While the picture remains unclear in breast cancer, some relationships have been established. EGFR (erbB1/HER1) signalling is believed to be associated with the development of hormone resistance in some breast cancer cases. Over-expression of EGFR protein is also seen in a proportion of primary breast carcinoma cases. While its utility in breast cancer remains unproven, immunohistochemical detection of the protein target is still recognised as a prerequisite for use of the new EGFR-targeted therapies, which includes small-molecule inhibitors such as gefitinib (Iressa™) and erlotinib (Tarceva™).
It is also recognised that some patients may respond to newer agents targeted specifically at EGFR, while not over-expressing EGFR protein, and there remain questions about which test is appropriate for demonstrating utility of these agents. A considerable body of evidence indicates that amplification or increased copy number of the EGFR gene, somatic mutations, and a limited number of polymorphisms, may impact on the response of these tumours to EGFR-targeted therapies, although they have little prognostic value per se.
Medical Solutions offers a validated standardised EGFR FISH test, which can be used to demonstrate EGFR gene amplification or increased copy number due to polysomy. Amplification is frequently, but not always, associated with increased protein expression.
We also offer a multiplexed test for the 29 most common somatic mutations found in the EGFR gene, several of which are known to affect sensitivity to small molecule inhibitors of this receptor.
There is a growing body of evidence that certain inherited polymorphisms in the EGFR gene may be linked to EGFR amplification in breast tumours, and subsequent protein over-expression. Testing for this polymorphism is also available through Medical Solutions.
Evaluation
Breast tumour samples immunostained for EGFR are assessed visually by a registered pathologist and assigned a semi-quantitative score based upon criteria of staining intensity and distribution, using an FDA-approved algorithm.
FISH evaluation to identify EGFR gene amplification is assessed visually over a number of representative areas within the sample to account for heterogeneity. The sample is assigned a score in the form of a ratio, relating the number of copies of the EGFR gene to the number of copies of chromosome 7. A score of >2 indicates gene amplification.
The results of mutation tests are reported as positive or negative for the presence of a particular mutation and the relative frequency of that mutation in the tumour sample.
The proliferative activity of neoplastic cells influences the clinical course of certain types of human tumour. In breast cancer a high proliferative index is generally associated with a poorer clinical outcome.
Ki-67 is a nuclear protein that appears in all active phases of the cell cycle (G1, S, G2, and M), and has been shown to be a valuable indicator of proliferation and prognosis in breast cancer. Ki-67 in the nuclei of proliferating cells in paraffin-embedded tumour samples can be readily detected by immunohistochemistry using the MIB-1 antibody.
Evaluation
MIB-1-positive cells are scored over a number of fields and the result expressed as a percentage of total tumour cells, i.e. a proliferative fraction. Numerous median cut-off values are to be found in the literature. Medical Solutions will use an appropriate cut-off at the medican value, and report the tumour as having a high (>median value) or low (<median value) growth fraction.
Over–expression of the membrane-associated receptor protein HER2 (erbB2), as a consequence of gene amplification, occurs in 10-20% of breast cancers, and importantly is associated with a poorer prognosis.
Immunohistochemical demonstration of HER2 protein over-expression in a breast tumour is a prerequisite for use of HER2-targeted therapies such as trastuzumab (Herceptin™). Medical Solutions offers the FDA-approved, standardised and validated DAKO HercepTest™ assay for assessment of HER2 protein expression, and the Abbott/Vysis PathVysion™ assay for assessment of HER2 gene amplification status.
Evaluation
Specimens immunostained for HER2 protein are assessed visually by a registered pathologist, who will assign a score based upon an FDA-approved algorithm, of 0 to 3+. A score of 3+ is regarded as positive for HER 2 protein overexpression. Scores of 0 and 1+ are considered negative. Specimens achieving a score of 2+ are considered borderline, and are referred for FISH HER 2 gene analysis. FISH scores are expressed as ratios of HER2 gene copy number to copy number of chromosome 17. A score greater than 2 is considered evidence of HER2 amplification.
Patients achieving a score of 3+ by immunohistochemistry, or who are shown to be amplified by FISH, are considered eligible for Herceptin™ treatment.
Breast cancers frequently spread to distant organs via lymphatic vessels, therefore lymphatic invasion is considered a negative prognostic feature, and is associated with early recurrence, reduced disease-free survival and a poor outcome. Pathologists generally examine cancers for evidence of tumour cells within lymphatic vessels, however identification of these vessels is often difficult as part of routine histopathological evaluation.
In addition to identification of LVI based upon morphological criteria alone, Medical Solutions offers an objective immunohistochemical test based upon the novel antibody D2-40, which reveals lymphatic channels in tumours. Recent studies have shown that the use of D2-40, which specifically identifies lymphatic endothelium, provides a robust means of identifying lymphatic invasion, and furthermore is an accurate predictor of outcome for patients with invasive ductal carcinomas of the breast.
Evaluation
Immunostaining with the D2-40 antibody is evaluated visually by a registered pathologist, who will scan representative areas of the specimen for evidence of tumour cells residing within vessels accurately identified by this test.
The value of morphological assessment can be enhanced by the use of a range of tissue based assays performed in our Reference Laboratory. These provide further information to aid in assessing prognosis, and perhaps more importantly in assessing adjuvant treatment suitability. Two such tests, recommended by NICE guidance, which we are providing to the NHS and private healthcare sectors, are ER/PR and HER2 tests.
Oestrogen and progesterone receptor status is determined immunohistochemically. While providing some prognostic information, these paired tests are also used routinely in a treatment decision role, to determine suitability for hormone therapies such as Tamoxifen or aromatase inhibitors. Medical Solutions utilises our own in-house, standardised and validated tests, as well as commercially available assays for both Oestrogen and Progesterone receptor, providing a comprehensive and analytically robust service to the clinician.
Evaluation
Immunostaining for ER and PR is evaluated visually by pathologists and laboratory scientists, and the result presented in the form requested by the client, as percentage positive cells, H-score or Allred/Quick Score.
The H-score is a semi-quantitative system for assessing the intensity and distribution of positive staining with a range between 0 and 300. A score of 300 is achieved when 100% of the tumour cells show the highest level of intensity (scored as 0-3). A score above 10 is considered positive. A negative result for ER/PR is considered a poor prognostic sign, while a positive is an indicator for tamoxifen or other hormonal therapies.
The Quick score is a similar system with a range between 0 and 8. Scores of over 2 are considered positive.
The Oncotype DX™ test is the first of a new generation of multivariate index tests using gene expression technology which examines 16 genes associated with proliferation, ER status, invasion, HER2 and other features of a breast cancer. The value of this test lays in its claimed predictive power which quantifies the likelihood of breast cancer recurrence in women with newly diagnosed, early stage breast cancer. In addition to predicting distant disease recurrence, Oncotype DX™ also assesses the benefit from certain types of chemotherapy. This test has been validated for use in breast cancer patients whose disease is newly diagnosed, stage I or II, lymph node-negative, ER-positive, and who are eligible for hormone-based therapy.
Conventional histopathological evaluation, including size, grade, stage, and mitotic frequency, is supplied by Medical Solutions through a specialist team of pathologists led by Professor Ian Ellis, and provides a basic assessment of a breast cancer patient’s tumour characteristics.
These methods are comprehensive and comply with WHO, EU, RCPath Minimum Dataset® and CAP guidance. Furthermore this form of evaluation underlies “prognostic profiling”, which predicts survival.
Specific algorithms are used to generate scores that are in turn related back to historic US and UK clinical datasets for breast cancer. At Medical Solutions we utilise the Nottingham Prognostic index (NPI), which is a well-established tool in the UK. NPI predicts survival based upon tumour grade, size and stage, however it is of lesser value in making treatment decisions.
A more recent development is Adjuvant Online, an online service which generates survival probability figures based upon similar input data to the NPI but also calculates the benefit to be expected from adjuvant treatment (hormonal or chemotherapy). This programme has recently been developed further to factor in genomic information, such as the recurrence score generated by the Oncotype DX™ assay, before arriving at a result.
These types of prognostic scores, based on clinicopathological features, will be the central component of your report, providing both physician and surgeon with a relatively accessible and comprehensible evaluation of disease outcome.
Recent studies of gene profiling in breast cancer using cDNA microarray technology have identified biologically distinct su-groups with clinical and prognostic relevance. These findings have now been extrapolated to protein-based techniques, using immunohistochemistry with well-characterised antibodies against a range of biomarkers related to epithelial cell lineage, differentiation, hormone and growth factor receptors and other gene products known to be altrered in breast cancer. Using modern computer methods, 5 distinct groups, with characteristic protein expression patterns, have been identified. These are referred to as luminal type-A and -B, two HER2-positive groups, and a basal phenotype group.
This type of analysis has demonstrated significant differences between the above sub-groups with respect to not only the established prognostic markers such as tumour grade, size and histology, but also in terms of patient outcome with overall and disease-free survival.
While Herceptin™ is recognised as an effective treatment for breast cancers with overexpression of the HER2 protein or amplification of the HER2 gene, only a minority of patients respond to this targeted agent. Therefore while HER2 overexpression is a prerequisite for Herceptin™ prescription it may not be sufficient alone to predict response in all cases.
Recent studies show that Herceptin™ activates a phosphatase enzyme called PTEN ("Phosphatase and Tensin homolog mutated in multiple advanced cancers 1"), which blocks tumour cell survival, thereby controlling unwanted cell growth. Cancer cells that have lost PTEN expression are therefore more resistant to Herceptin™. Furthermore, reduced PTEN expression has been shown to predict poor response to the hormonal therapy Tamoxifen. Immunohistochemical evaluation of levels of PTEN expression can be used to predict likely response to HER2 and other treatments in breast cancer.
This is a new test, and as such there is no agreed standardised method of evaluation. Our pathologist will provide a written evaluation of the staining and guidance on interpretation.
Topoisomerase IIa is a key enzyme in DNA replication and also a target for various chemotherapeutic agents, in particular the anthracycline drugs. The TOP2A gene is located in close proximity to the HER2 gene on chromosome 17q21, and a significant percentage of patients with HER2 amplification demonstrate abnormalities in the TOP2A gene.
Anthracyclines, one of the most widely used class of cytotoxic agents for treatment of breast cancer, inhibit the protein product of the TOP2A gene by trapping the DNA strand break intermediates, leading to persistent DNA cleavage. TOP2A gene amplification and subsequent protein over-expression is associated with improved response to anthracycline-based chemotherapy.
Evaluation
TOP2A gene amplification or deletion is assessed by FISH (Fluorescent In Situ Hybridisation). The result of this test is expressed as a ratio of TOP2A gene copies to copies of chromosome 17. A ratio of 0.8-1.9 is considered normal (non-amplified), a ratio of 2.0 or greater is considered amplified, and a ratio of 0.7 or less indicative of deletion of the TOP2A gene. Amplification of the TOP2A gene is significantly associated with a favourable response to anthracycline-based chemotherapy, whereas deletion of this gene may be associated with resistance to the same agents.
Note: The TOP2A test as performed by Medical Solutions incorporates the HER2 FISH test carried out simultaneously on the same tissue sample. The results of the HER2 assessment will be reported together with that for TOP2A.
For information on how to order any of the above tests or portfolios please contact
our Business Development Manager.