Richard J. Cote

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.

Radical Cystectomy in the Treatment of Invasive Bladder Cancer: Long-Term Results in 1,054 Patients

PURPOSE To evaluate our long-term experience with patients treated uniformly with radical cystectomy and pelvic lymph node dissection for invasive bladder cancer and to describe the association of the primary bladder tumor stage and regional lymph node status with clinical outcomes. PATIENTS AND METHODS All patients undergoing radical cystectomy with bilateral pelvic iliac lymphadenectomy, with the intent to cure, for transitional-cell carcinoma of the bladder between July 1971 and December 1997, with or without adjuvant radiation or chemotherapy, were evaluated. The clinical course, pathologic characteristics, and long-term clinical outcomes were evaluated in this group of patients. RESULTS A total of 1,054 patients (843 men [80%] and 211 women) with a median age of 66 years (range, 22 to 93 years) were uniformly treated. Median follow-up was 10.2 years (range, 0 to 28 years). There were 27 (2.5%) perioperative deaths, with a total of 292 (28%) early complications. Overall recurrence-free survival at 5 and 10 years for the entire cohort was 68% and 66%, respectively. The 5- and 10-year recurrence-free survival for patients with organ-confined, lymph node-negative tumors was 92% and 86% for P0 disease, 91% and 89% for Pis, 79% and 74% for Pa, and 83% and 78% for P1 tumors, respectively. Patients with muscle invasive (P2 and P3a), lymph node-negative tumors had 89% and 87% and 78% and 76% 5- and 10-year recurrence-free survival, respectively. Patients with nonorgan-confined (P3b, P4), lymph node-negative tumors demonstrated a significantly higher probability of recurrence compared with those with organ-confined bladder cancers (P <.001). The 5- and 10-year recurrence-free survival for P3b tumors was 62% and 61%, and for P4 tumors was 50% and 45%, respectively. A total of 246 patients (24%) had lymph node tumor involvement. The 5- and 10-year recurrence-free survival for these patients was 35%, and 34%, respectively, which was significantly lower than for patients without lymph node involvement (P <.001). Patients could also be stratified by the number of lymph nodes involved and by the extent of the primary bladder tumor (p stage). Patients with fewer than five positive lymph nodes, and whose p stage was organ-confined had significantly improved survival rates. Bladder cancer recurred in 311 patients (30%). The median time to recurrence among those patients in whom the cancer recurred was 12 months (range, 0.04 to 11.1 years). In 234 patients (22%) there was a distant recurrence, and in 77 patients (7%) there was a local (pelvic) recurrence. CONCLUSION These data from a large group of patients support the aggressive surgical management of invasive bladder cancer. Excellent long-term survival can be achieved with a low incidence of pelvic recurrence.

Bioassay of prostate-specific antigen (PSA) using microcantilevers.

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 μg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein–protein binding, DNA hybridization, and DNA–protein interactions, as well as drug discovery.

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.

ACCUMULATION OF NUCLEAR P53 AND TUMOR PROGRESSION IN BLADDER CANCER

BACKGROUND We have previously demonstrated a strong association between nuclear accumulation of p53 protein, as determined by immunohistochemical analysis, and mutations in the p53 gene. The purpose of this study was to determine the relation between nuclear accumulation of p53 and tumor progression in transitional-cell carcinoma of the bladder. METHODS Histologic specimens of transitional-cell carcinoma of the bladder (stages Pa, noninvasive disease, to P4, disease with direct extension into adjacent organs or structures) from 243 patients who were treated by radical cystectomy were examined for the immunohistochemical detection of p53 protein. Nuclear p53 reactivity was then analyzed in relation to time to recurrence and overall survival. RESULTS The detection of nuclear p53 was significantly associated with an increased risk of recurrence of bladder cancer (P < 0.001) and with decreased overall survival (P < 0.001). In patients with cancer confined to the bladder, the rates of recurrence for stage P1, P2, and P3a tumors that had no detectable nuclear p53 reactivity at five years were 7, 12, and 11 percent, respectively, as compared with 62, 56, and 80 percent, respectively, for tumors that had p53 immunoreactivity. Similar results were obtained when the presence or absence of p53 in the nuclei of the tumor cells was studied in relation to overall survival. In a multivariable analysis stratified according to grade, pathological stage, and lymph-node status, nuclear p53 status was an independent predictor (and in cancer confined to the bladder, the only independent predictor) of recurrence and overall survival (P < 0.001). CONCLUSIONS In patients with transitional-cell carcinoma confined to the bladder, an accumulation of p53 in the tumor-cell nuclei detected by immunohistochemical methods predicts a significantly increased risk of recurrence and death, independently of tumor grade, stage, and lymph-node status. Patients with transitional-cell carcinoma confirmed to the bladder that demonstrates nuclear p53 reactivity should be considered for protocols of adjuvant treatment.

Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype.

Purpose: The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44+CD24−/low phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC. Experimental Design: We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK+ from early breast cancer patients. CK+ cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging. Results: We detected the putative stem cell–like phenotype in all CK+ specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in <10% of cells. Conclusions: This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

The antigen retrieval (AR) technique, which is predominantly based on high-temperature heating of tissues, is used as a non-enzymatic pretreatment for immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections. It has been widely applied in pathology and analytical morphology. The existence of a growing body of literature on the AR technique raises a number of interesting issues for the further development of AR. These issues include the use of a “test battery” and the concept of “maximal retrieval” applied to the selection of optimal test protocols for the standardization of AR. (J Histochem Cytochem 45:327–343, 1997)

Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases.

We used monoclonal antibodies to identify occult micrometastases in the bone marrow of 49 patients with operable (stage I and II) breast carcinoma. Follow-up (mean, 29 months; median, 30 months) revealed that 12 patients recurred. The presence of bone marrow micrometastases (BMM) was significantly associated with early recurrence (P less than .04). The estimated 2-year recurrence rate for patients with no BMM detected (BMM-) was 3%; in patients with BMM, the 2-year recurrence rate was 33%. When BMM and axillary lymph node (LN) status were combined, groups of patients at low risk (LN-, BMM-; 2-year recurrence rate, 0%) and high risk (LN+, BMM+; 2-year recurrence rate, 42%) for early recurrence were identified. Bone marrow tumor burden was related to early recurrence. Among patients with BMM, those who did not recur had on average fewer extrinsic cells in their marrow than those who recurred (15 v 43 cells, respectively). Multivariate analysis comparing BMM, LN+ versus LN-, and tumor size (less than or equal to 2 cm v greater than 2 cm) revealed no factor independently associated with early recurrence. Peripheral tumor burden of BMM (0 or less than 10 extrinsic cells v greater than or equal to 10 extrinsic cells) was the only independent predictor of early recurrence (P less than .003). In conjunction with conventional prognostic factors, particularly axillary LN status, evaluation for BMM might be used to stratify patients for adjuvant treatment programs. Because this pilot study involved few patients with short-term follow-up, the results should be interpreted with caution. The examination of bone marrow for micrometastases remains an experimental procedure; the clinical usefulness of the test will be established through larger studies with long-term follow-up.

Antigen retrieval techniques: current perspectives.

Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology. (J Histochem Cytochem 49:931–937, 2001)

Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients.

PURPOSE This study was performed to evaluate the potential of specific mRNA markers to detect micrometastases by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis of sentinel lymph nodes (SNs) and blood from patients with breast cancer. PATIENTS AND METHODS We assessed the specificity of carcinoembryonic antigen (CEA), cytokeratin-19 (CK-19), CK-20, gastrointestinal tumor-associated antigen-733.2 (GA733.2), and mucin-1 (MUC-1) in the blood of healthy donors (n = 13) and lymph nodes from patients without cancer (n = 3) by RT-PCR assay. The sensitivity of the RT-PCR assay for the target mRNA markers was assessed in breast cancer cell lines (n = 4), primary breast tumors (n = 8), and the frozen sections of SNs (n = 22) from 22 patients with American Joint Committee on Cancer (AJCC) stages I to IIIA breast cancer. RESULTS CK-20 was the only mRNA marker not detected in lymph nodes or blood from patients without cancer. Both the blood and lymph nodes from patients without cancer expressed CEA, CK-19, GA733.2, and MUC-1 mRNA. All four breast cancer cell lines and six of eight primary breast tumors expressed all five mRNA markers. Expression of mRNA by the RT-PCR assay in the frozen-section SNs (n = 12) without metastases by conventional histopathology ranged from 8% (CK-20) to 92% (GA733.2). Detection of RT-PCR cDNA products in frozen-section SNs was increased with Southern blot analysis compared with ethidium bromide gel electrophoresis (EtBr) for all mRNA markers except CK-19. CONCLUSION CEA, CK-19, GA733.2, and MUC-1 show no diagnostic value as mRNA markers for the detection of micrometastases by the RT-PCR assay because they are expressed in the blood and lymph nodes of patients without cancer. Further studies are needed to assess the sensitivity of CK-20 to detect micrometastases by the RT-PCR assay in the blood and frozen-section SNs of patients with breast cancer.