Michael Bilous

Current perspectives on HER2 testing: a review of national testing guidelines

Knowledge of HER2 status is a prerequisite when considering a patients eligibility for Herceptin (trastuzumab) therapy. Accurate assessment of HER2 status is essential to ensure that all patients who may benefit from Herceptin are correctly identified. There are several assays available to determine HER2 status: the most common in routine clinical practice are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Various factors can affect the results achieved with these assays, including the assay antibody/probe, the methodology and the experience of personnel. Many countries have implemented national testing guidelines in an attempt to standardize testing procedures and make results more accurate. These guidelines vary in the level of detail and the number of recommendations. This review looks at areas of consensus between the different national testing guidelines and highlights where errors may arise during the testing procedure. The key point underlined by this review is that whatever method is used to test for HER2 status, the technology must be validated first, and there must be regular internal and external quality control and quality assurance procedures.

Effect of margins on ipsilateral breast tumor recurrence after breast conservation therapy for lymph node-negative breast carcinoma.

Breast conservative surgery (CS) with radiotherapy (RT) is the most commonly used treatment for early‐stage breast carcinoma. However, there is controversy regarding the importance of the pathologic margin status on the risk of ipsilateral breast tumor recurrence (IBTR). The current study evaluated the effect of the pathologic margin status on IBTR rates in a cohort of women with lymph node‐negative breast carcinoma treated with CS and RT.

Emerging Technologies for Assessing HER2 Amplification

Patients with human epidermal growth factor receptor-2 (HER2)+ breast cancer are eligible for trastuzumab treatment; therefore, accurate assessment of HER2 status is essential. Until recently, only 2 methods were validated for determining the HER2 status of breast tumors in the routine diagnostic setting: immunohistochemical analysis and fluorescence in situ hybridization (FISH). Recently, bright-field in situ hybridization techniques such as chromogenic in situ hybridization (CISH) and silver-enhanced in situ hybridization (SISH), which combine features of immunohistochemical analysis and FISH, have been introduced for the determination of HER2 status. These new techniques use a peroxidase enzyme-labeled probe with chromogenic detection, instead of a fluorescent-labeled probe, allowing results to be visualized by standard bright-field microscopy. Thus, the histologic features and HER2 status of a specimen can be evaluated in parallel. Moreover, signals do not decay over time. This review discusses recent publications regarding CISH and SISH testing, including results scoring and concordance between FISH and immunohistochemical analysis.

Silver In Situ Hybridization (SISH) For Determination of HER2 Gene Status in Breast Carcinoma Comparison With FISH and Assessment of Interobserver Reproducibility

The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.

Sarcomas followig radiation therapy for breast cancer: A report of three cases and a review of the literature

Purpose: First to describe clinical and pathologic features of sarcomas arising after radiation therapy for breast cancer and to report three cases of sarcoma arising 7, 15, and 20 years following radiation therapy for breast cancer. Second, to review the literature on this treatment complication. Methods and Materials: Medline literature search. Results: The most frequent histology is osteosarcoma and bone is affected more commonly than soft tissue at a median latency of 11 years. The scapula is the most frequently affected bone. The most frequently affected soft tissue site is now the conserved breast with a median latency of 5.5 years. The aetiologic factors relating to these sarcomas are not fully defined with factors of beam energy, radiation dose, chemotherapy and regional edema being inconsistently reported. Conclusion: The frequency of radiation-induced sarcoma at 10 years of follow-up is approximately 0.2%. This is an overestimate by an unknown factor because of the description of sarcomas arising metachromously in breast cancer patients, in nonirradiated areas

Effect of an omental wrap on the healing and vascularity of compromised intestinal anastomoses

Adult Wistar rats were used to investigate the ability of an omental wrap to limit leakage from compromised intestinal anastomoses. Under ketamine anesthesia, a section of small bowel was divided and then reanastomosed using a “control” anastomosis, a “deficient” anastomosis, or an “ischemic” anastomosis, plus or minus the addition of a wrap of omentum. Initially 10 rats were randomly assigned to each group. Nineteen of the 20 rats with unwrapped compromised anastomoses died within six weeks, compared with five deaths in the rats protected by an omental wrap (Fishers exact test;P< 0.01). The experiment was then repeated with a sample of rats from each anastomotic group being sacrificed for histologic examination on days 2 to 7, 10, 14, and 42. At the time of sacrifice a dye was injected into the omental vasculature to determine its contribution to the healing anastomosis. An anastomosis could be demonstrated between omental and bowel wall vessels by the third postoperative day. At one week the infarcted bowel edges were being resorbed and the omentum formed a fibrotic cylinder aligning the separated ends of bowel wall. At six weeks the scar became more contracted and the bowel mucosa had started to grow onto its luminal surface. It is concluded from this study that the omental wrap is protective to a compromised anastomosis by providing a biologically viable plug to prevent early leakage and a source of granulation tissue and neovasculature for later wound repair.

Chromogenic in situ hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridisation and immunohistochemistry.

Aims: The objective of this study was to evaluate the accuracy, ease of use and reproducibility of chromogenic in situ hybridisation (CISH) for HER2 testing by studying its inter‐laboratory concordance in five Australian pathology laboratories. Methods: The HER2 status of 49 breast cancers was determined by CISH twice in two different laboratories. Each sample had previously been tested by immunohistochemistry (IHC; 2+ and 3+ cases selected) and fluorescence in situ hybridisation (FISH). Participating laboratories were blinded to these test results. Oestrogen receptor (ER) status was also evaluated for each cancer. Results: High correlation was observed between FISH and CISH results. No cases showing high gene amplification by FISH were scored as non‐amplified by CISH (kappa coefficient = 1). High correlation was observed between IHC and CISH, all IHC 3+ samples showing amplification by CISH. Inter‐laboratory CISH concordance was also good (kappa coefficient = 0.67). Fifty‐six per cent of HER2‐amplified samples tested ER positive, while 42% of ER‐positive cases showed HER2 gene amplification, confirming that HER2 testing should not be confined to ER‐negative breast cancers. Conclusions: These findings demonstrate that CISH is a robust test to assess HER2 status in breast cancer and therefore is an important addition to the HER2 testing algorithm.

Carcinomas of the renal pelvis associated with smoking and phenacetin abuse: p53 mutations and polymorphism of carcinogen-metabolising enzymes

Phenacetin abuse and smoking are established risk factors for transitional cell carcinomas of the urinary tract. In the present study, we analysed exposure and the clinical course of patients who underwent nephrectomy for transitional cell carcinoma of the renal pelvis. PCR‐SSCP of archival, paraffin‐embedded histological sections followed by direct DNA sequencing revealed that 29 of 89 (33%) renal pelvic carcinomas contained a p53 mutation. Double mutations were found in 4 tumours and triple mutations in 1 tumour. The incidence of p53 mutations was significantly higher in tumours with grades 3 and 4 than in those with grades 1 and 2 and higher in invasive than in non‐invasive tumours. Furthermore, patients with carcinomas carrying a p53 mutation showed poorer survival than those without mutation. The type of p53 mutation in renal pelvic carcinomas was similar to that reported for bladder cancer, G:C→A:T transition mutations being most frequent (45%, 33% of these at CpG sites), followed by G:C→T:A and G:C→C:G transversions. The incidence and type of p53 mutation did not differ significantly in patients with a history of phenacetin abuse, smoking or neither of these habits. This was also true for G:C→T:A transversions (17.5% of mutations), which are considered typical of smoking‐induced carcinomas at other sites, e.g., lung, oral cavity and oesophagus. Our results indicate that the frequency and pattern of p53 mutations are similar in transitional cell carcinomas of the bladder and the renal pelvis and do not reflect exposure to phenacetin and/or smoking. The frequency of genetic polymorphism in genes coding for carcinogen‐metabolising enzymes (CYP1A1, NAT1, GSTT1 and GSTM1) was also independent of exposure. Although the sample size of our study does not allow definite conclusions, these data are compatible with chronic tissue damage as a causative factor in the evolution of urothelial carcinomas rather than pointing to a direct mutagenic effect of phenacetin and tobacco‐specific carcinogens. Int. J. Cancer (Pred. Oncol.) 79:531–536, 1998.© 1998 Wiley‐Liss, Inc.

Her2 Testing recommendations in Australia

Summary HER2 is the target of a new treatment for metastatic breast cancer using the humanised monoclonal antibody trastuzumab (Herceptin). Since only around 20% of breast cancers carry the overexpressed HER2 receptor protein to which this treatment is directed, patient selection is very important in determining eligibility for the drug. Currently, immunohistochemistry and fluorescence in situ hybridisation are the main tests used for HER2 detection, and these testing recommendations have been developed based on national and international data.

Immunocytochemistry and in situ hybridisation of epidermal growth factor receptor and relation to prognostic factors in breast cancer.

The breast tumour distribution of epidermal growth factor receptor (EGFR) was studied in 193 patients with primary breast cancer by immunocytochemistry on frozen sections. EGFR was correlated (P = 0.0009) with growth fraction assessed by Ki-67, and negatively correlated with oestrogen receptor (ER, P = 0.0001) and progesterone receptor (PR, P = 0.0001) status. In 47 patients, in-situ hybridisation for EGFR mRNA showed good agreement with the immunocytochemically assessed EGFR protein. There were, however, several tumours in which EGFR mRNA could be detected in the absence of EGFR protein and there were differences between the ER and PR status of those tumours in which translation of EGFR mRNA was not seen. The cause of these differences is unclear, but these findings may represent a clue as to the differential control of breast cancer cell receptors.