Anthony Rhodes

American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer

PURPOSE To develop a guideline to improve the accuracy of human epidermal growth factor receptor 2 (HER2) testing in invasive breast cancer and its utility as a predictive marker. METHODS The American Society of Clinical Oncology and the College of American Pathologists convened an expert panel, which conducted a systematic review of the literature and developed recommendations for optimal HER2 testing performance. The guideline was reviewed by selected experts and approved by the board of directors for both organizations. RESULTS Approximately 20% of current HER2 testing may be inaccurate. When carefully validated testing is performed, available data do not clearly demonstrate the superiority of either immunohistochemistry (IHC) or in situ hybridization (ISH) as a predictor of benefit from anti-HER2 therapy. RECOMMENDATIONS The panel recommends that HER2 status should be determined for all invasive breast cancer. A testing algorithm that relies on accurate, reproducible assay performance, including newly available types of brightfield ISH, is proposed. Elements to reliably reduce assay variation (for example, specimen handling, assay exclusion, and reporting criteria) are specified. An algorithm defining positive, equivocal, and negative values for both HER2 protein expression and gene amplification is recommended: a positive HER2 result is IHC staining of 3+ (uniform, intense membrane staining of > 30% of invasive tumor cells), a fluorescent in situ hybridization (FISH) result of more than six HER2 gene copies per nucleus or a FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2; a negative result is an IHC staining of 0 or 1+, a FISH result of less than 4.0 HER2 gene copies per nucleus, or FISH ratio of less than 1.8. Equivocal results require additional action for final determination. It is recommended that to perform HER2 testing, laboratories show 95% concordance with another validated test for positive and negative assay values. The panel strongly recommends validation of laboratory assay or modifications, use of standardized operating procedures, and compliance with new testing criteria to be monitored with the use of stringent laboratory accreditation standards, proficiency testing, and competency assessment. The panel recommends that HER2 testing be done in a CAP-accredited laboratory or in a laboratory that meets the accreditation and proficiency testing requirements set out by this document.

American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer

PURPOSE To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. METHODS The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance. RESULTS Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in preanalytic variables, thresholds for positivity, and interpretation criteria. RECOMMENDATIONS The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials.

American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer

PURPOSE To develop a guideline to improve the accuracy of human epidermal growth factor receptor 2(HER2) testing in invasive breast cancer and its utility as a predictive marker. METHODS The American Society of Clinical Oncology and the College of American Pathologists convened an expert panel, which conducted a systematic review of the literature and developed recommendations for optimal HER2 testing performance. The guideline was reviewed by selected experts and approved by the board of directors for both organizations. RESULTS Approximately 20% of current HER2 testing may be inaccurate. When carefully validated testing is performed, available data do not clearly demonstrate the superiority of either immunohistochemistry(IHC) or in situ hybridization (ISH) as a predictor of benefit from anti-HER2 therapy. RECOMMENDATIONS The panel recommends that HER2 status should be determined for all invasive breast cancer. A testing algorithm that relies on accurate, reproducible assay performance, including newly available types of brightfield ISH, is proposed. Elements to reliably reduce assay variation (for example, specimen handling, assay exclusion, and reporting criteria) are specified. An algorithm defining positive, equivocal, and negative values for both HER2 protein expression and gene amplification is recommended: a positive HER2 result is IHC staining of 3 + (uniform, intense membrane staining of 30% of invasive tumor cells), a fluorescent in situ hybridization (FISH) result of more than six HER2 gene copies per nucleus or a FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2; a negative result is an IHC staining of 0 or 1 +, a FISH result of less than 4.0 HER2 gene copies per nucleus, or FISH ratio of less than 1.8. Equivocal results require additional action for final determination. It is recommended that to perform HER2 testing, laboratories show 95% concordance with another validated test for positive and negative assay values. The panel strongly recommends validation of laboratory assay or modifications, use of standardized operating procedures, and compliance with new testing criteria to be monitored with the use of stringent laboratory accreditation standards, proficiency testing, and competency assessment. The panel recommends that HER2 testing be done in a CAP-accredited laboratory or in a laboratory that meets the accreditation and proficiency testing requirements set out by this document.

American Society of Clinical oncology/college of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer

PURPOSE To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. METHODS The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance. RESULTS Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in preanalytic variables, thresholds for positivity, and interpretation criteria. RECOMMENDATIONS The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials.

The Use of Cell Line Standards to Reduce HER-2/neu Assay Variation in Multiple European Cancer Centers and the Potential of Automated Image Analysis to Provide for More Accurate Cut Points for Predicting Clinical Response to Trastuzumab

Immunohistochemical analysis, the most efficient way of assaying for HER-2/neu overexpression in patients with invasive breast cancer, is subject to variation in sensitivity and evaluation when used in multiple laboratories. Cell lines with differing but constant levels of HER-2/neu expression have been advocated as standard material against which assay sensitivity can be gauged. Automated image analysis could provide more precise linear measurements of HER-2/neu expression than the subjective and categorical scoring system originally designed for the HER-2/neu clinical trials. Multiple European laboratories (range, 92-126) stained 7 cell line standards on 6 successive occasions using a variety of immunohistochemical assays for HER-2/neu. During the 2-year study period, a trend toward a standard sensitivity level was observed, with significant improvement in numbers of laboratories achieving appropriate results. Image analysis gave reproducible and significantly different linear values for a total of 621 HER-2/neu results on cell lines previously categorized manually as 3+, 2+, 1+, or 0. The use of cell line standards and image analysis have the potential to assist in standardizing immunohistochemical results for predictive markers and provide more accurate and quantifiable cut points for predicting clinical response to therapy, respectively.

Triple-negative breast cancer and PTEN (phosphatase and tensin homologue)loss are predictors of BRCA1 germline mutations in women with early-onset and familial breast cancer, but not in women with isolated late-onset breast cancer

IntroductionGiven that breast cancers in germline BRCA1 carriers are predominantly estrogen-negative and triple-negative, it has been suggested that women diagnosed with triple-negative breast cancer (TNBC) younger than 50 years should be offered BRCA1 testing, regardless of family cancer characteristics. However, the predictive value of triple-negative breast cancer, when taken in the context of personal and family cancer characteristics, is unknown. The aim of this study was to determine whether TNBC is a predictor of germline BRCA1 mutations, in the context of multiple predictive factors.MethodsGermline mutations in BRCA1 and BRCA2 were analyzed by Sanger sequencing and multiple ligation-dependent probe amplification (MLPA) analysis in 431 women from the Malaysian Breast Cancer Genetic Study, including 110 women with TNBC. Logistic regression was used to identify and to estimate the predictive strength of major determinants. Estrogen receptor (ER) and phosphatase and tensin homologue (PTEN) status were assessed and included in a modified Manchester scoring method.ResultsOur study in an Asian series of TNBC patients demonstrated that 27 (24.5%) of 110 patients have germline mutations in BRCA1 (23 of 110) and BRCA2 (four of 110). We found that among women diagnosed with breast cancer aged 36 to 50 years but with no family history of breast or ovarian cancer, the prevalence of BRCA1 and BRCA2 mutations was similar in TNBC (8.5%) and non-TNBC patients (6.7%). By contrast, in women diagnosed with breast cancer, younger than 35 years, with no family history of these cancers, and in women with a family history of breast cancer, the prevalence of mutations was higher in TNBC compared with non-TNBC (28.0% and 9.9%; P = 0.045; and 42.1% and 14.2%; P < 0.0001, respectively]. Finally, we found that incorporation of estrogen-receptor and TNBC status improves the sensitivity of the Manchester Scoring method (42.9% to 64.3%), and furthermore, incorporation of PTEN status further improves sensitivity (42.9% to 85.7%).ConclusionsWe found that TNBC is an important criterion for highlighting women who may benefit from genetic testing, but that this may be most useful for women with early-onset breast cancer (35 years or younger) or with a family history of cancers. Furthermore, addition of TNBC and PTEN status improves the sensitivity of the Manchester scoring method and may be particularly important in the Asian context, where risk-assessment models underestimate the number of mutation carriers.

The oestrogen receptor-negative/progesterone receptor-positive breast tumour: a biological entity or a technical artefact?

A recent study by Rakha et al 1 shows that breast tumours with single hormonal receptor positivity are biologically and clinically distinct groups and particularly that oestrogen receptor (ER)-negative/progesterone receptor (PR)-positive tumours exhibit more aggressive behavioural characteristics than double-receptor-positive tumours. However, there is also an increasingly prevalent opinion, that the ER−/PR+ phenotype does not exist and that the ER-negativity in these cases is due to inadequate tissue fixation or technical failure of the immunohistochemical assay.2–4 This is an important dichotomy to resolve because, if ER−/PR+ tumours simply represent an artefact of the method of assessment, then they are essentially positive for both the receptors, which may have implications for how these patients are managed. To investigate this, we reviewed data from …

Loss of PTEN expression is associated with IGFBP2 expression, younger age, and late stage in triple-negative breast cancer

OBJECTIVES To investigate the association between PTEN loss and IGFBP2 expression in a series of triple-negative breast cancers and to relate this expression to basal cytokeratin expression and clinicopathologic features. METHODS One hundred and one formalin-fixed and paraffin-processed triple-negative breast cancer cases from the University of Malaya Medical Centre were tested immunohistochemically for cytokeratins 5/6 and 14, PTEN, and IGFBP2. The resulting slides were scored for proportion and intensity of staining. RESULTS Loss of tumor nuclear and cytoplasmic staining for PTEN occurred in 48.3% of cases and was significantly associated with younger age at diagnosis (47 years compared with 57 years in those without PTEN loss; P = .005). Independent predictors of PTEN loss were late stage at presentation (P = .026), cytokeratin 5/6 positivity (P = .028), and IGFBP2 expression (P = .042). High levels of IGFBP2 expression were seen in 32% of cases; an independent predictor of high levels was cytokeratin 14 negativity (P = .005). PTEN loss and high levels of IGFBP2 expression were associated with poorer survival, but neither of these trends was significant. CONCLUSIONS PTEN loss is a frequent event in triple-negative breast cancers and is significantly associated with younger age at onset of breast cancer, late stage, and IGFBP2 expression.

Technical variations in prostatic immunohistochemistry: need for standardisation and stringent quality assurance in PSA and PSAP immunostaining

Aims: To assess variations in prostate specific antigen (PSA) and prostate specific acid phosphatase (PSAP) immunohistochemistry with particular reference to the antibody type (monoclonal or polyclonal) and the tissues used for optimising immunostaining conditions and as external positive controls. Methods: A questionnaire was sent to all laboratories registered with the UK National External Quality Assurance Scheme for immunohistochemistry enquiring about the immunohistochemical methods routinely used for the diagnosis of prostate cancer. Results: Responses were received from 220 (68%) laboratories. All UK respondents routinely performed PSA immunostaining but PSAP immunostaining was available in only 57% of these laboratories. Monoclonal anti-PSA, polyclonal anti-PSA, monoclonal anti-PSAP, and polyclonal anti-PSAP were used by 40%, 60%, 29%, and 27% of UK respondents, respectively. Benign prostate tissue was most commonly used to determine optimal antibody dilutions and as external quality control for PSA/PSAP, with only 6% and 3% of respondents, respectively, including high grade prostate cancer in the tissues used for these purposes. Conclusions: The wide variation in the methods used highlights the need for standardisation and more stringent quality assurance of the immunohistochemical staining techniques used for PSA and PSAP. The widespread use of benign prostate tissue to determine optimal antibody dilutions and as an external positive control for PSA and PSAP immunostaining is of particular concern because this approach may result in a method that is not sufficiently sensitive to detect the reduced PSA and PSAP expression associated with high grade prostate cancer.

The Reliability of Rabbit Monoclonal Antibodies in the Immunohistochemical Assessment of Estrogen Receptors, Progesterone Receptors, and HER2 in Human Breast Carcinomas

The reliability of the rabbit monoclonal antibodies SP1, SP2, SP3, and 4B5 was immunohistochemically assessed on a range of 96 invasive breast carcinomas and the results compared with those achieved with established antibody markers for estrogen receptors (6F11), progesterone receptors (PgR636), and HER2 (polyclonal A0485 and clone CB11), with HER2 status validated by fluorescence in situ hybridization (FISH) and silver in situ hybridization. Optimal results depended on the duration of microwave antigen-retrieval time and the use of a high pH buffer for rabbit and mouse estrogen receptor antibodies (SP1 and 6F11), although only on antigen-retrieval duration for the progesterone receptors SP2 and PgR636. The highest rate of concordance between HER2 overexpression and HER2 gene amplification was with the rabbit monoclonal antibodies (SP3 and 4B5) and FISH. Rabbit monoclonal antibodies are reliable alternatives to established antibody markers for the immunohistochemical testing of estrogen receptors, progesterone receptors, and HER2 in breast cancer.