—  SHORT COURSE #59  —

In Situ Hybridization in Diagnostic Pathology

Case 6 - Ductal Breast Carcinoma with Lymph Node Metastases and HER-2/neu Amplification

Ricardo V. Lloyd and Arie Perry


Clinical History
This 52-year old woman presented with an enlarging breast mass. Needle biopsy revealed an invasive ductal carcinoma and Her2/neu immunostaining was interpreted as 2+ positive. The patient was treated with neoadjuvant chemotherapy followed by mastectomy and axillary lymph node dissection 3 months later.

Gross Description
The original diagnosis of invasive ductal carcinoma was established from an ultrasound guided needle biopsy of the left breast. A modified radical mastectomy performed after neoadjuvant chemotherapy (3 months later) revealed a prior biopsy site without grossly detectable tumor.


Case 6 - Figure 1

Case 6 - Figure 2

Case 6 - Figure 3
HER2/neu


Case 6 - Figure 4
CEP17 (green); HER2/neu (pink)
Case 6 - Figure 5
CEP17 (green); HER2/neu (pink)

Microscopic Description
Sections from the original biopsy demonstrated an invasive ductal carcinoma, grade II /III (Bloom-Richardson score 7), with minimal tubule formation, moderate nuclear atypia, and moderate mitotic activity. In the mastectomy specimen, only a microscopic focus of residual carcinoma was identified in the region of the prior biopsy tract. However, twelve of sixteen axillary lymph nodes were involved by metastatic carcinoma (12/16). There was 2+ immunoreactivity for Her-2/neu, utilizing the Dako HercepTest antibody in the biopsy material and gene amplification was confirmed by FISH analysis.

Discussion
Of all the applications of FISH analysis in anatomic pathology, the assessment of HER-2/neu status in breast cancer has attracted the greatest attention and application. This topic has been recently reviewed in great detail [1, 2, 3, 4, 5, 6] . Although there is now agreement that HER-2/neu assessment provides clinically useful information, the optimal diagnostic approach is still widely debated. The HER-2/neu gene ("HER-2" for human EGF receptor 2, "neu" for rat neuroblastoma / glioblastoma-associated oncogene, a.k.a. c-erb-B2) on chromosome 17q21 is a member of the EGFR family of tyrosine kinases and encodes a 185 kD transmembrane protein implicated in the regulation of cell growth. It has no known ligand and normally requires heterodimerization with other ErbB co-receptors, such as EGFR (ErbB1) or ErbB3 for activation.

HER-2/neu protein overexpression, primarily due to gene amplification is thought to be involved in the malignant progression of several tumor types and has been identified in 20-35% of breast carcinomas, primarily of high-grade ductal type [1, 2, 3, 4, 5, 6, 7, 8, 9] . The association is even greater with the comedocarcinoma form of DCIS, where it is encountered in roughly 90% of cases [10, 11] . HER-2/neu gene amplification and protein expression status is thought to provide both prognostic and therapeutic information and the current rationale for HER-2/neu testing is based on the following observations:
  1. Overexpression / amplification is an independent prognostic variable, associated with reduced patient survival, especially in lymph node positive, but also in node negative cases [7, 8, 12, 13] .

  2. Overexpression / amplification is associated with increased responsiveness to adriamycin-based therapeutic regimens [4].

  3. Overexpression / amplification is associated with increased responsiveness to Herceptin (trastuzumab), which specifically targets the overexpressed surface protein [4, 14, 15] .

  4. Overexpression / amplification is associated with decreased responsiveness to radiation therapy, cyclophosphamide, methotrexate, 5-FU, hormonal therapy, and taxol (unless administered with Herceptin) [4].

  5. There is significant risk of cardiotoxicity with adriamycin and Herceptin therapy and this combination should therefore, be avoided in patients with low probability of response [15].

Presently, the two primary modalities for assessing HER-2/neu status in routinely processed breast cancer biopsies are immunohistochemistry and FISH. Each technique has its advantages and disadvantages and both FDA-approved antibodies and DNA probes are now commercially available. As such, these assays are also generally reimbursable. Although immunohistochemistry is simpler, cheaper, and more widely available than FISH [16, 17, 18, 19] , concerns have been raised regarding the variable antibody specificities and sensitivities, as well as the subjectivity involved in semiquantitative interpretations, such as the 0-3+ scale recommended with the FDA-approved Dako HercepTest antibody. Due to the increased objectivity, reproducibility, accuracy, and associations with clinical behavior [8, 12, 19, 20, 21, 22, 23, 24, 25] , FISH is now playing an increasing role in these assessments. The most commonly advocated approaches are:
  1. Immunohistochemistry alone. Fewer pathologists are using this approach today, given the concerns with false positives and false negatives.

  2. Immunohistochemistry as an initial screen, with FISH confirmation of 2+ immunoreactive cases.

  3. Immunohistochemistry as an initial screen, with FISH confirmation of all immunopositive (2+ or 3+) cases.

  4. FISH on all cases.

Utilizing the Dako HercepTest antibody, the majority of our 2+ cases have subsequently been interpreted as negative for HER-2/neu amplification, and only 10-15% were positive by FISH as in the current case. This is similar to the experience of many others, though some have reported fewer false positives using other antibodies [24]. Although most have reported excellent FISH concordance for cases with 3+ reactivity, others have detected significant numbers of false positives even within this group, suggesting that all positive cases should be confirmed by FISH. Lastly, although there is only a small percentage of false negatives (0-1+) identified by FISH, the fact that these immunonegative cases represent the largest group (70-75%), even 1-2% discrepancies amount to a large number of patients, for which the clinical management might be radically altered based on HER-2/neu status. Therefore, some are now advocating FISH analysis on all cases and this is the approach most recently adopted by our Anatomic Pathology Division at Washington University.

Currently available commercial FISH probes/kits for analysis of HER-2/neu amplification include the PathVysion assay from Vysis (www.vysis.com), the INFORM assay from Ventana (www.oncor.com/ventanatoc.htm), and a CISH assay from Zymed (www.zymed.com) [26]. Tubbs et al. have similarly developed a reliable CISH test based on gold-facilitated autometallographic in situ hybridization (GOLDFISH) [27]. The PathVysion and INFORM assays are FDA approved and offer supervised training for the initial use and interpretation of their products. With the INFORM assay, a single HER-2/neu probe is applied and >4 signals per nucleus is considered a positive reading. Only the Vysis assay includes a second probe for CEP17, in order to correct for chromosome 17 polysomy (>4 copies of chromosome 17). They require a HER-2/neu to CEP17 signal ratio >2 for a determination of gene amplification. Although good concordance has been reported between the PathVysion and INFORM assays [19], Pauletti and colleagues [8] found Her-2/neu gains (>4 copies) in 46% of breast carcinomas, with only 21% classified as "amplified" after correcting for polysomy 17. Similarly, McCormick et al. found 17 polysomic carcinomas in their set of 215 cases that would have been incorrectly scored as amplified by absolute HER-2/neu copy numbers alone [28]. This suggests a significant risk of overinterpreting amplification with the systems using only a single probe for HER-2/neu.

References

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