This installment of Second Opinion examines the case of a patient with a new diagnosis of breast cancer presenting to our multidisciplinary breast cancer second opinion clinic.
A 52-year-old woman presented to our multidisciplinary clinic after a new diagnosis of invasive ductal carcinoma of the left breast. Suspicion of breast cancer was initially raised by abnormal screening mammography of the left breast 2 months prior to presentation. The woman was asymptomatic at that time, and mammography performed 15 months prior to the current presentation had been unremarkable. She was otherwise healthy.
The patient underwent lumpectomy and sentinel lymph node biopsy. Examination of the lumpectomy specimen revealed a combined histologic grade 2 invasive ductal carcinoma with a greatest tumor dimension of 7 mm and a closest resection margin of 2 mm. There was no lymphovascular invasion. The tumor was estrogen receptor (ER)-positive (60%) with low-level progesterone receptor (PR) expression (4%). HER2 immunohistochemistry (IHC) was performed and scored as 2+. Subsequent fluorescence in situ hybridization (FISH) showed a ratio of HER2 gene copy number to chromosome 17 centromere of 2.8. Three sentinel lymph nodes were negative for tumor involvement. The remainder of the disease staging work-up was negative, resulting in an overall stage of T1b, N0, M0 (stage I).
The woman presented to our clinic with this information seeking a recommendation regarding further treatment. Her physical examination and family history were unremarkable. On review of systems she noted continued menstrual bleeding, but her periods had become less frequent and more irregular over the preceding year.
Dr. Gabriel Brooks: This case of a perimenopausal woman with a small, node-negative, ER-positive, HER2-positive tumor presents considerable challenges with respect to risk assessment and selection of appropriate therapy. Our patient has multiple favorable prognostic factors, including a tumor diameter of less than 1 cm, positive ER expression (Figure 1), and negative sentinel lymph node biopsy. Her only adverse prognostic factor is HER2 amplification. We will begin with a discussion of the pathologic characteristics of this tumor.
Dr. Brooks: How do we interpret the results of HER2 testing?
Dr. Meenakshi Singh (pathology): Because HER2 status is used as a basis for making decisions regarding candidacy for both conventional chemotherapy and trastuzumab (Herceptin) treatment, it is essential to have a well-validated system for clinical determination of HER2 status. On the basis of joint guidelines from the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP), HER2-positive status is determined by either 3+ overexpression of the HER2 protein on IHC staining or a FISH ratio of greater than 2.2 for HER2 gene amplification (Figure 2). A HER2 IHC of 2+ or a HER2 FISH ratio between 1.8 and 2.2 is considered an equivocal result, and requires further analysis. The FISH ratio compares the number of HER2 gene copies to the number of chromosome 17 copies, as measured by the chromosome enumeration probe 17 (CEP17).
In this case, the sequence of an initial equivocal IHC result followed by FISH analysis was appropriate. This patient should be considered clinically positive for HER2 gene amplification, albeit at a low level.
Dr. Brooks: Are there any other factors that can interfere with the HER2 assay?
Dr. Singh: Appropriate tissue handling prior to testing is crucial to ensure reproducible results. The ASCO/CAP guidelines specify that time from tumor excision to fixation should be as short as possible and that tissue formalin fixation time should be at least 6 hours for excision specimens, at least 1 hour for needle-biopsy specimens, and not greater than 48 hours for either. At this institution, we ask our surgeons to record the exact times of specimen excision and placement into formalin. In the laboratory, we record the total time of specimen fixation so that we can monitor our compliance with the guidelines.
Additional factors that can confound HER2 results are polysomy of chromosome 17 and heterogeneity of HER2 amplification within a tumor. When three or more copies of chromosome 17 are present in a cell, the ratio of HER2 to CEP17 can be less than 1.8 despite a numeric increase in the number of HER2 copies. While elevated HER2 expression by IHC may be seen in this scenario, the majority of cases of polysomy 17 do not overexpress HER2. In cases of heterogeneity, tumors may have some areas of significant overexpression or amplification of HER2 and other areas of normal expression. Our patient did not have polysomy 17 or heterogeneity of HER2 expression.
Dr. Brooks: Our patient also has low-level expression of the progesterone receptor measured at 4%, with clearly positive estrogen receptor expression at 60%. What is the significance of low-level progesterone receptor expression?
Dr. Singh: In our laboratory, we do not report ER and PR status as negative or positive; rather, we report the percentage of cells that exhibited receptor staining by IHC, as was done in this case. The initial studies that validated PR expression as adding utility to ER expression in prediction of response to tamoxifen used quantitative radioligand binding assays to determine expression.[4,5] IHC is now the standard of care in assessing PR and ER expression, and it has been shown that response to tamoxifen improves continuously with increasing PR expression, so that low-level PR expression as observed in our patient can at least be seen to predict a better response to endocrine therapy than no expression at all. As with HER2 assessment, all ER and PR testing should be carried out in an accredited lab with positive and negative controls. The clone of HER2 antibody used and the method of analysis should be clearly documented in the pathology report.
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