A comparative study of HER2/neu amplification and overexpression using fluorescencein situ hybridization (FISH) and immunohistochemistry (IHC) in 101 breast cancer patients

The HER2/neu protooncogene is expressed in the breast, ovarian, gastric and prostatic tumors. Studies done in a number of laboratories have demostrated that 25%–30% of breast cancer contain overexpression of HER2/neu gene. A comparative analysis of the amplification and overexpression of HER2/neu using fluorescencein situ hybridization (FISH) and immunohistochemistry (IHC) was performed to determine the correlation between both techniques. In this study, FISH with HER2/neu probe (Path Vysion) is compared to immunohistochemistry (rabbit anti-human c-erbB-2-DAKO) in a series of 101 prospective human breast cancer specimens. Among 25 patients with score of IHC 3+, 23 (92%) were detected amplified by FISH and in two cases we found overexpression (3+) but without gene amplification. Out of 46 cases with 2+ by IHC, we found 43 not amplified, two moderately amplified (<10 copies) and one highly amplified (>10 copies) (6.5%). No patient with IHC O or 1+, presented amplification of HER2/neu. A good correlation between both techniques was found. FISH technique should have clinical utillity overoat in cases with 2+.     

Trisomy 6 in basal cell carcinomas correlates with metastatic potential: a dual color fluorescence in situ hybridization study on paraffin sections

BACKGROUND: Most basal cell carcinomas (BCCs) are indolent lesions; a few become locally aggressive or even metastatic. Little is known about the molecular and genetic alterations in this malignant transformation. Conventional karyotyping in BCC has revealed a high frequency of nonclonal, structural rearrangements, with few cases that show multiple, unrelated, small clones suggestive of a multicellular origin. Trisomy 6 was described recently in a few BCCs, but the biologic significance of the appearance of trisomy 6 in BBCs was not clear. METHODS: Thirty cases including 4 metastatic, 4 locally aggressive, and 22 conventional nonaggressive BCCs were studied. Fluorescence in situ hybridization (FISH) was performed on 4 microm tissue sections, using alpha-centromeric enumeration probes for chromosome 6 (SpectrumGreen, Vysis Inc., Downers Grove, IL) and chromosome 4 (SpectrumOrange, Vysis Inc., Downers Grove, IL, used as disomic cell control). Trisomy 6 was semiquantitated within tumor cells and nonneoplastic cells in each case. RESULTS: Trisomy 6 was identified in all 4 metastatic BCCs within tumor cells and in corresponding BCCs at the primary cutaneous site in 2 of these 4 cases. Two locally aggressive BCCs, 1 of which had preceding radiation exposure, also showed trisomy 6. All nonaggressive BCCs and nonneoplastic cells were disomic for chromosome 6. CONCLUSIONS: Trisomy 6 has been identified as a cytogenetic aberration representative of tumor cells in aggressive and metastatic BCC. None of the nonaggressive BCCs in this study demonstrated trisomy 6. Acquisition of trisomy 6 by tumor cells in BCC may lead to the emergence of metastatic potential. Additional studies to define the underlying mechanisms may be valuable in preventing aggressive behavior in BCC.     

Response to dual HER2 blockade in a patient with HER3-mutant metastatic breast cancer

BackgroundHER3 activating mutations have been shown in preclinical models to be oncogenic and ligand-independent, but to depend on kinase-active HER2.Patients and methodsWhole-exome sequencing of the primary HER2-negative breast cancer and its HER2-negative synchronous liver metastasis from a 46-year-old female revealed the presence of an activating and clonal HER3 G284R mutation.ResultsHER2 dual blockade with trastuzumab and lapatinib as third-line therapy led to complete metabolic response in 2 weeks and confirmed radiological partial response after 8 weeks. Following the resection of the liver metastasis, the patient remains disease-free 40 weeks after initiation of the HER2 dual blockade therapy. Immunohistochemical analysis demonstrated a substantial reduction of phospho-rpS6 and phospho-AKT in the post-therapy biopsy of the liver metastasis.DiscussionThis is the first-in-man evidence that anti-HER2 therapies are likely effective in breast cancers harboring HER3 activating mutations.     

Prognostic utility of fluorescence in situ hybridization for determining HER2 gene amplification in breast cancer

An accurate investigation of the HER2 proto-oncogene is extremely important for the therapy and prognostication of breast cancer. Currently, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are standard methods for this purpose. The aim of this study was to detect the expression and amplification of HER2 in paraffin-embedded samples of breast cancer tissue and to investigate the relationship between HER2 amplification and various clinicopathological parameters in advanced breast cancers. We used FISH to examine the HER2 gene amplification and IHC to examine the expression of HER2 protein, estrogen receptor (ER) and progesterone receptor (PR) in 62 advanced breast cancers. HER2 gene amplification was detected by FISH in 12 breast cancers (19%) and HER2 protein expression with a score of 3+ was detected by IHC in 11 (17%). There was a significant correlation between the HER2 gene amplification and HER2 protein overexpression in breast cancers (P<0.0001). However, some mismatching was evident: 3 cases, negative for the HER2 gene, showed a HER2 protein expression score of 3+ and 2 cases, positive for HER2 gene amplification, had HER2 protein expression scores of 0 and 1+ (negative), respectively. ER and PR were expressed in 41 (66%) and 46 (74%) cancers, respectively. No correlation was observed between the HER2 gene amplification and any of the clinicopathological parameters examined, including age, histopathological type, TNM stage, tumor size, lymph node status, relapse and expression of PR. We observed three patterns among the 6 deceased cases: i) triple negativity for HER2, ER and PR, ii) positivity for HER2 gene amplification with a mismatching HER2 protein expression, and iii) positivity for the HER2 gene amplification with a matching HER2 protein expression score of 2+ or 3+. The triple negative cases and HER2 gene amplification positive cases with a mismatching HER2 protein expression had a poor outcome. These results suggest that in breast cancer, the detection of HER2 gene amplification by FISH is desirable compared with the HER2 protein expression determined by IHC. Moreover, triple negativity for HER2, ER and PR is a potentially very important prognostic marker.     

HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues

To analyze HER2 amplification in a large cohort of diagnostic breast cancer specimens, fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed on the same specimens with use of Food and Drug Administration&approved products. Procedures were standardized following the manufacturers' recommendations. Of 116,736 IHC specimens, 20% were positive for HER2. In 16,092 FISH specimens, 22.7% showed HER2 amplification. In the subset of 6556 tissues analyzed with IHC and FISH, however, 59% were positive on IHC and 23.6% were amplified on FISH. The increased frequency of positive test results is skewed by more frequent reflex FISH testing. In general, expression and amplification trended together, with the least amplification (4.1%) seen in IHC-negative cases, 7.4% amplification seen in IHC 1+ cases, 23.3% amplification seen in IHC 2+ cases, and 91.7% amplification seen in IHC 3+ cases. When FISH amplification ratios were stratified, the low FISH ratios (2.0-2.2) were most frequently seen in specimens with negative IHC results, high ratios (>5.0) were seen in IHC 3+ specimens, and intermediate levels of amplification were similar for all levels of IHC. The effect of changing the cutoff point was analyzed: removing cases with a ratio of exactly 2.0 decreased the FISH positivity rate to 22.2% in the combined IHC and FISH cohort. Sequentially moving the cutoff point to 2.2 and 2.5 affected cases at all IHC expression levels. Each change removed approximately 2% from the apparent positivity. This large database provides the distribution frequency of HER2 protein expression and gene amplification in invasive ductal and lobular breast cancer. The relationship between level of HER2 amplification and clinical outcome will require reanalysis of pivotal trial data.     

Cardiac toxicity in breast cancer patients treated with dual HER2 blockade

Although dual HER2 blockade shows promising results in patients with HER2‐positive breast cancer it is unclear whether this treatment strategy increases the risk for cardiac adverse events. We conducted a meta‐analysis of randomized trials to investigate the risk of cardiac adverse events when a combination of anti‐HER2 therapies compared to anti‐HER2 monotherapy. We searched Medline, the Cochrane library, as well as the electronic abstract databases of the major international congresses' proceedings to identify randomized trials that evaluated the administration of anti‐HER2 monotherapy (lapatinib or trastuzumab or pertuzumab) versus anti‐HER2 combination (pertuzumab plus trastuzumab or trastuzumab plus lapatinib) therapy in breast cancer. The trials were considered eligible if the only systematic difference between the study arms was the type of anti‐HER2 therapy used. Study outcomes were the congestive heart failure (CHF) grade ≥3 and left ventricular ejection fraction (LVEF) decline <50% or more than 10% from baseline. Six trials were considered eligible. Overall incidence results for CHF in the combined anti‐HER2 therapy and the anti‐HER2 monotherapy were 0.88% (95% CI: 0.47–1.64%) and 1.49% (95% CI: 0.98–2.23%). The incidence of LVEF decline was 3.1% (95% CI: 2.2–4.4%) and 2.9% (95% CI: 2.1–4.1%), respectively. The OR of CHF between anti‐HER2 combination and monotherapy was 0.58 (95% CI: 0.26–1.27, p‐value= 0.17) while the OR of LVEF decline was 0.88 (95% CI: 0.53–1.48, p‐value= 0.64). This meta‐analysis provides evidence supporting comparable cardiac toxicity between anti‐HER2 combination therapy and anti‐HER2 monotherapy.     

HER-2 gene amplification by fluorescence in situ hybridization (FISH) compared with immunohistochemistry (IHC) in breast cancer: a study of 528 equivocal cases

The concordance rate between fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) on evaluating HER-2/neu status is still controversial, especially for the IHC 2+/equivocal cases. In this study, we investigated the amplification of HER-2/neu gene by FISH in IHC (2+)/equivocal specimens to clarify the correlation between HER-2/neu and clinicopathologic features of breast cancers. HER-2/neu amplification was determined by FISH on 528 specimens of breast carcinomas with equivocal expression of HER-2/neu protein by IHC detection. 65.5 % of IHC 2+ patients were negative for HER-2/neu amplification, 29.0 % were positive and the remaining was equivocal. A statistically significant inverse association was found between hormone receptor expression and HER-2/neu amplification (P < 0.05). Furthermore, polysomy of CEP17 was detected in 60 % of breast carcinomas. The results highlight the necessity of FISH test for further categorization when breast carcinoma cases are scored 2+ by IHC.     

Among triple-negative breast cancers, HER2(0) breast cancer shows a strong tendency to be basal-like compared with HER2(1+) breast cancer: preliminary results

Background

Diagnosis of triple-negative breast cancer (ER-negative, PgR-negative, HER2-negative; TNBC) is performed by means of immunohistological staining. HER2-negative includes HER2(0) and HER2(1+), based on differences in the staining intensity, but there have been no reports on comparison of these two types in TNBC. Accordingly, this study was designed to investigate the possible differences in the biological characteristics of HER2(0) breast cancer and HER2(1+) breast cancer in TNBC.

Methods

Tissue specimens from 89 TNBC patients were immunohistochemically stained for CK5/6, EGFR, p53, Ki67, E-cadherin, TOP2A and Bcl-2. The expressions of these markers and the clinicopathological findings were compared between the HER2(0) patient group and the HER2(1+) patient group. When either CK5/6 or EGFR was positive, the specimen was judged to be the basal-like phenotype of breast cancer.

Results

The percentages of CK5/6- and/or EGFR-positive specimens in the HER2(0) and HER2(1+) groups were 44.9 and 16.8%, respectively, showing that there was a significantly greater number of basal-like phenotype patients in the HER2(0) group (p?<?0.01). The percentage of E-cadherin-positive specimens in the HER2(0) group was 66.6%, which was significantly greater than the 40.0% recorded in the HER2(1+) group (p?<?0.05). The respective percentages of TOP2A-positive specimens in the HER2(0) and HER2(1+) groups were 55.0 and 30.0%, and the difference was statistically significant (p?<?0.05).

Conclusion

In TNBC, HER2(0) breast cancer showed a strong tendency to include more of the basal-like phenotype compared with HER2(1+) breast cancer. The staining results indicated the possibility that HER2(0) breast cancer and HER2(1+) breast cancer have different characteristics.     

Correlation of <Emphasis Type="Italic">HER2</Emphasis> gene status assessment by fluorescence in situ hybridization between histological sections and cytological specimens of breast cancer

Background

While HER2 gene detection in cytological specimens using fluorescence in situ hybridization (FISH) has been reported, the appropriate criteria for such specimens remain controversial.

Methods

Fine needle aspiration (FNA) samples collected from surgically resected breast cancer specimens were rinsed in a cytopreservative solution containing fixative. Then, slides of the FNA samples were prepared by liquid-based cytology (LBC) (ThinPrep system, Hologic) according to the manufacturer’s instructions, and a PathVision HER2 DNA probe kit (Abbott) was used for FISH staining. The results were evaluated using an automated MetaCyte imaging system (MetaSystems, Altlussheim, Germany). HER2 gene amplification was scored using the HER2/chromosome enumeration probe 17 (CEP17) signal count ratio as follows: amplified, >2.2; equivocal, 1.8–2.2; and unamplified, <1.8. The cytology results were compared with the histology results from concordant cases.

Results

Successful results were obtained in 98 of 100 cases, and results from the FNA specimens were in agreement with those from the histological sections in 97 of these 98 cases (accuracy rate, 99 %; kappa, 0.962).

Conclusions

FISH-based assessment of the HER2 gene status is consistent between histological sections and cytological specimens of breast cancer.

     

A systematic review of dual targeting in HER2-positive breast cancer

Background

Human epidermal growth factor receptor 2 (HER2) is overexpresed in 15–20% of all breast cancers. Treatment with trastuzumab has led to an improved outcome and prolonged survival of HER2-positive breast cancer patients and today the drug is established as standard of care in both the adjuvant and metastatic settings. However, trastuzumab resistance is common and a major focus in the treatment of HER2-positive breast cancer has been developing therapeutic agents to either potentiate the effect of trastuzumab or to target cells which have become resistant to trastuzumab. The present review addresses efficacy and toxicity of dual targeting in HER2-positive breast cancer.

Materials and methods

A computer-based literature search was carried out using PubMed; data reported at international meetings and clinicaltrials.gov was included.

Results

This paper describes efficacy and safety of lapatinib, pertuzumab or trastuzumab-DM1 in combination with trastuzumab in the (neo)adjuvant and metastatic settings. Furthermore, combinations of trastuzumab and drugs targeting the downstream pathway are described.

Conclusion

Dual blockade is likely to represent a substantial advance for patients with HER2-positive breast cancer. However, the relevant subpopulation remains to be defined and side effects including cardiotoxicity might be a limiting factor to the use. There is an urgent need for prospective biomarker-driven trials to identify patients for whom dual targeting is cost-effective.     

Characterization of HER1 (c-erbB1) status in locally advanced breast cancer using fluorescence in situ hybridization and immunohistochemistry.

Epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane glycoprotein encoded by the HER1 protooncogene, located at 7p12. This receptor is related to the pathogenesis of breast cancer. The aim of this study was to analyze the status of HER1 using fluorescence in situ hybridization (FISH) and immunohistochemistry in a series of 48 patients with locally advanced breast cancer (LABC). Before neoadjuvant chemotherapy, core biopsies were taken from patients with LABC and were processed into paraffin blocks. Biopsies were then studied using FISH with a HER1 probe (Vysis, Downers Grove, Ill., USA). They were also analyzed immunohistochemically using two different EGFR antibodies from DakoCytomation (Denmark, A/S) and from Zymed (San Francisco, Calif., USA). HER1 amplifications were not found, although 31% of the cases presented aneusomy of chromosome 7. Only 2 cases presented EGFR expression. LABC presented a low level of EGFR expression. HER1 amplification was not present in LABC, although the polysomy of chromosome 7 was a common finding.     

Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked immuno-sorbent assay

c-erb-2 amplification and overexpression are currently attracting a great deal of attention because a new adjuvant therapy using an antibody against the c-erbB-2 gene product, trastuzumab (Herceptin; Genentech, Inc., South San Francisco, CA), has proved effective in treating breast cancer with amplification and/or overexpression of c-erbB-2. Aberrations of c-erbB-2 have also been detected in ovarian, endometrial and gastric carcinomas at varied frequencies. Amplification of the c-erbB-2 locus (17q12-q21.32), overexpression of c-erbB-2 protein (p185) and serum levels of soluble c-erbB-2 protein fragments (p105) were examined in gastric cancer patients using fluorescence in situ hybridization (FISH), immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. Overexpression of c-erbB-2 protein was found in 29 (8.2%) of the 352 gastric carcinomas analyzed. In FISH analysis, all tumors with 3+ immunostaining and 1 of 5 tumors with 2+ staining showed high-level amplification of c-erbB-2. Pre-operative serum p105 was quantified in serum specimens from 129 patients with gastric cancer and 28 patients with benign diseases. There were no significant differences in the serum p105 levels among 11 patients with c-erbB-2-overexpressing carcinomas, 118 patients with c-erbB-2 non-overexpressing carcinomas and 28 controls, although a single case of gastric carcinoma overexpressing c-erbB-2 with extensive liver metastasis had a higher level than the cut-off value. The mechanisms of overexpression of p185 and high-level amplification of c-erbB-2 in gastric adenocarcinomas seem similar to those well-established in breast cancers. Patients having gastric adenocarcinoma with c-erbB-2 amplification are potential candidates for a new adjuvant therapy using humanized monoclonal antibody.     

A predictive model of pathologic response based on tumor cellularity and tumor-infiltrating lymphocytes (CelTIL) in HER2-positive breast cancer treated with chemo-free dual HER2 blockade

BackgroundThe presence of stromal tumor-infiltrating lymphocytes (TILs) is associated with increased pathologic complete response (pCR) and improved outcomes in HER2-positive early-breast cancer (BC) treated with anti-HER2-based chemotherapy. In the absence of chemotherapy, the association of TILs with pCR following anti-HER2 therapy-only is largely unknown.Patients and methodsThe PAMELA neoadjuvant trial treated 151 women with HER2-positive BC with lapatinib and trastuzumab [and hormonal therapy if hormone receptor (HR)-positive] for 18 weeks. Percentage of TILs and tumor cellularity were determined at baseline (N = 148) and at day 15 (D15) of treatment (N = 134). Associations of TILs and tumor cellularity with pCR in the breast were evaluated. A combined score based on tumor cellularity and TILs (CelTIL) measured at D15 was derived in PAMELA, and validated in D15 samples from 65 patients with HER2-positive disease recruited in the LPT109096 neoadjuvant trial, where anti-HER2 therapy-only was administer for 2 weeks, then standard chemotherapy was added for 24 weeks.ResultsIn PAMELA, baseline and D15 TILs were significantly associated with pCR in univariate analysis. In multivariable analysis, D15 TILs, but not baseline TILs, were significantly associated with pCR. At D15, TILs and tumor cellularity were found independently associated with pCR. A combined score (CelTIL) taking into account both variables was derived. CelTIL at D15 as a continuous variable was significantly associated with pCR, and patients with CelTIL-low and CelTIL-high scores had a pCR rate of 0% and 33%, respectively. In LPT109096, CelTIL at D15 was found associated with pCR both as a continuous variable and as group categories using a pre-defined cut-off (75.0% versus 33.3%).ConclusionsOn-treatment TILs, but not baseline TILs, are independently associated with response following anti-HER2 therapy-only. A combined score of TILs and tumor cellularity measured at D15 provides independent predictive information upon completion of neoadjuvant anti-HER2-based therapy.Clinical trial numberNCT01973660.     

Serum HER2 extracellular domain in metastatic breast cancer patients treated with weekly trastuzumab and paclitaxel: association with HER2 status by immunohistochemistry and fluorescence in situ hybridization and with response rate.

PURPOSE: We explored the relationship between circulating HER2 extracellular domain (ECD) and tissue HER2 status as determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). We also examined its predictive value in a cohort of metastatic breast cancer patients treated with weekly trastuzumab and paclitaxel. METHODS: Eligible patients had pre- and post-treatment stored serum specimens and were treated on a previously reported phase II trial. Retrospective analysis evaluated: the association between pretreatment serum HER2 ECD and tissue HER2 status by IHC and FISH; and the association between change in serum HER2 ECD after 12 weeks of therapy and response proportion. RESULTS: Stored serum samples were available for 55/95 (58%) patients. Statistically significant associations were found between HER2 status as assessed by IHC and FISH, and baseline serum HER2 ECD level. Patients whose ECD normalized after 12 weeks of therapy had a higher response proportion compared with patients with persistently high ECD levels (68% versus 15%, P=0.005). A relative decline of over 55% from baseline HER2 ECD predicted response to therapy. CONCLUSION: A statistically significant association was observed between pretreatment serum HER2 ECD and tissue HER2 status as assessed by IHC and FISH. A decrease in serum HER2 ECD level was a significant predictor of response to trastuzumab-based therapy.