Polysomic Cases Research Articles

Results on quality assurance of fluorescence in situ hybridization (FISH) technique by reference centers for HER2 status determination in metastatic breast cancer (MBC): FISH 2002 study

22028 Background: The FISH 2002 Study evaluated concordance between IHC and FISH in local and regional centers. Primary MBC tumor samples were tested by IHC for HER2 status according to in-house techniques at 18 regional (n=871) and 81 peripheral (n=670) centers in France between 2002 and 2006. All samples were tested by FISH at regional centers [results presented SABCS 2006]. The second part of the study comprised a quality assurance study of the FISH techniques. Methods: 464 cases were selected according to protocol (see table) and were either re-assessed on archived pictures or retested by 3 reference centers (Clermont-Ferrand, Strasbourg & Paris-Curie). 246 samples were re-assessed and 166 were retested out of 395 re-evaluated cases. Results: Global discordance combining re-assessment and retesting results were 10.7% in the first half of the study and 5.1% in the second one (p=0.041). Conclusions: Concordance was excellent for negative and 2+ cases (97.5% to 100%). Among low amplification cases, 70% were concordant: discordant cases were mainly interpretation pitfalls of polysomic cases, emphasizing the importance of double stained FISH analyses. The overall concordance and quality of HER2 status determined by FISH has improved significantly over time. This improvement is likely to be related to the introduction of rigorous quality control procedures, training, and education, which should be continued. Type of case (n = 412) Cases selection Analyzable cases (n) Discordant cases (n) False positive (%) False negative (%) Concordant negative IHC/FISH * 10% random 45 0 – 0 Concordant positive IHC/FISH * 20% random 13 2 15.4 – IHC 2+ * All 173 6 11.1 0.8 Discordant IHC/FISH * All 18 2 25.0 0 Low amplification (≥6 and <8 copies) ** All 30 9 30.0 0 Poor FISH technical quality ** All 8 0 0 0 * FISH re-assessed on picture ** FISH retested Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Hoffmann-La Roche Hoffmann-La Roche Hoffmann-La Roche

Epidermal growth factor receptor gene mutation, amplification and protein expression in malignant pleural mesothelioma

Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial malignancies. In lung cancer cases, EGFR gene mutation at the kinase domain and EGFR gene amplification are reported to be predictors of the response to EGFR tyrosine kinase inhibitors. In malignant pleural mesothelioma (MPM), the role of EGFR is less clear. We studied EGFR gene mutation, amplification and protein expression in 25 Japanese patients with MPM. None had previously reported EGFR mutations detected by the TaqMan PCR assay. Using immunohistochemistry, 8/25 (32%) cases were positive for the EGFR protein. The cases of sarcomatous type and desmoplastic type were all negative. Fluorescence in situ hybridization analysis revealed three low polysomy cases and one high polysomy case. The low polysomy cases included one biphasic type and two epithelial types, and the high polysomy case was epithelial type. These four cases expressed EGFR protein. In MPM, EGFR seems to play a role in a limited subset of patients. To identify possible candidates for EGFR tyrosine kinase in inhibitor therapy, the information on the EGFR gene status may be valuable.

Central HER2 IHC and FISH analysis in a trastuzumab (Herceptin) phase II monotherapy study: assessment of test sensitivity and impact of chromosome 17 polysomy

Aims:To investigate the correlation between centrally assessed human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) results and response to treatment of patients with...

EGFR and HER2 gene expression status and their correlation in non-small cell lung cancer

To explore epidermal growth factor receptor (EGFR) and HER2 gene status, to assess the correlation between EGFR and HER2 gene status, and to investigate the role of copy number increase and amplification of EGFR gene and HER2 gene in the tumorigenesis and disease progression of non-small-cell lung cancer. Using Path Vysion kit and LSI EGFR SpectrumOrange/CEP7 Spectrum Green probes, EGFR gene and HER2 gene status were evaluated by fluorescence insitu hybridization (FISH) using formalin-fixed, paraffin-embedded samples from 31 patients with non-small-cell lung cancer, including 20 adenocarcinomas, 2 squamous cell carcinomas, 2 large cell carcinoma, 4 bronchoalveolar carcinomas and 3 adenosquamous carcinomas. The correlation between EGFR and HER2 gene status was analyzed. Six of thirty-one carcinomas showed EGFR gene amplification. Of 25 cases without EGFR gene non-amplification, four tetrasomy and 5 polysomy were detected. Overall, 15 out of 31 carcinomas demonstrated either EGFR gene copy number increase or gene amplification (15/31). HER2 gene amplification was seen in 2 of the 31 cases. Four trisomy, one tetrasomy and nine polysomy cases were found in 29 tumors that had no HER2 gene amplification. Overall, 16 of 31 cases showed either HER2 gene copy number increase and/or amplification (16/31). Synchronous EGFR and HER2 gene numerical changes, i.e. gene copy number increase and gene amplification, were found in 12 of 31 cases (12/31), and almost all such patients had either clinical stage III or IV tumor. EGFR gene numerical changes significantly correlated with HER2 gene abnormality (chi(2)(Adj) = 7.3045, P = 0.0069). EGFR or HER2 copy number increase is much more frequent than gene amplification in no-small-cell lung cancer. Our data based on gene alterations indicate, for the first time, that there is a significant correlation between EGFR alterations and HER2 abnormalities. Both genes are involved in the tumorigenesis and development of lung cancer. EGFR/HER2 dimer is one of the predominant heterodimerization types in lung cancer. The interactions between EGFR and HER2 may play a rule in the progression of non-small-cell lung cancer.

Evaluation of HER2 gene expression status in breast cancer by fluorescence in-situ hybridization

To deduce the protocol, scoring criteria and interpretive guidelines for assessment of HER2 gene expression status by fluorescence in-situ hybridization (FISH) and to compare the results with those obtained by immunohistochemistry. The HercepTest kit from Dako Cytomation was employed for immunohistochemistry. FISH for HER2 gene expression status was performed using PathVysion DNA probe kit on the archival paraffin-embedded sections of breast cancer tissues from 28 Chinese female patients with immunohistochemical staining scores of (3 +), (2 +), (1 +) and 0. Ten of the 12 patients with score (3 +) by immunohistochemistry were positive for HER2 by FISH, with 2 cases being polysomy. Two other cases with FISH-negative were also shown to be polysomy. Seven of the 10 patients with score (2 +) by immunohistochemistry showed HER2 gene amplification, with 1 case being polysomy. Two of the remaining 3 cases, which were FISH-negative, were shown to be polysomy. All the patients with scores (1 +, number = 3 ) or 0 ( number = 3) by immunohistochemistry failed to show amplification. One case of polysomy was noted in either group. Immunohistochemistry is useful as an initial screening tool for HER2 expression status. Because of the obvious discrepancies between protein expression and gene amplification, patients with score (2 +) by immunohistochemistry should undergo FISH testing as well. FISH is also required in selected examples with score (3 +) immunohistochemical results, especially in those with false-positive immunohistochemistry due to chromosome 17 aneuploidy.

Polysomy 17 in HER-2/neu Status Elaboration in Breast Cancer: Effect on Daily Practice

To assess the effect of chromosome 17 copy number on HER-2/neu status determination in breast cancers. HER-2/neu gene copy and chromosome 17 centromere numbers were evaluated on 893 breast carcinomas using double color fluorescence in situ hybridization (FISH). The net and chromosome 17 corrected (ratio) HER-2/neu copy numbers were compared and related to immunohistochemistry done according to the Food and Drug Administration (FDA)-approved scoring system (0, 1+, 2+, and 3+) as a first screening step in 584 cases. When a ratio > or = 2 was considered as criterion for FISH positivity, 49.3% (440 of 893) of cases showed amplification versus 56.2% (502 of 893) by using a net HER-2/neu gene copy number >4 as a alternative criterion; 14.8% (67 of 453) of cases having a ratio <2 had a net HER-2/neu gene copy number >4 and 1.1% (5 of 440) with a ratio > or = 2 had a net HER-2/neu gene copy number <4. Among discordant cases, 88.8% (64 of 72) were polysomic (>2.25 chromosomes 17/cell) and among polysomic cases, 12.8% (40 of 312) of the low polysomic (2.26-3.75 chromosomes 17/cell) and 36.9% (24 of 65) of the highly polysomic (>3.75 chromosomes 17/cell) cases showed discordance. In cases with a ratio <2, polysomy 17 incidences were 85.7% (6 of 7) in IHC 3+, 42.4% (79 of 186) in IHC 2+, 33.3% (15 of 45) in IHC 1+, and 29.1% (16 of 55) in IHC 0. A net increase in HER-2/neu gene copy number consecutive to polysomy 17 in the absence of specific gene amplification might lead to a strong protein overexpression in a small subset of breast carcinomas. HER-2/neu status determination by FISH is dependent on the criterion considered for positivity in clinical practice.

Chromosome 17 polysomy and Her-2/neu status in metastatic breast cancer patients indicated to trastuzumab therapy

9584 Background: Numeric and structural aberrations of chromosome 17 (C17) are common findings in breast cancer patients. Aneusomy of C17 is often correlated with bad prognosis. Polysomy of C17 may explain the immunohistochemical (IHC) positivity in fluorescent in situ hybridization (FISH) negative cases. Although polysomic cases represent only 10–20% of breast cancer, at least 5–7% of them are not indicated to trastuzumab treatment because they do not fill criteria of Her-2/neu: C17 ratio >2,0 and efficacy of trastuzumab in polysomic patients has not been confirmed yet. In Czech Republic, such patients are indicated for trastuzumab treatment only when they are IHC 3+ positive (high positivity). Methods: In the Czech National Centre for Her-2/neu evaluation at Palacky University, 541 samples of breast cancer tumours were tested by FISH assay (PathVision) with locus specified probes for detection of Her-2/neu gene (LSI Her-2/neu; 17q11.2-q12) and centromeric region of C17 (CEP 17; 17p11.1-q11.1). In tumors with polysomy of C17 (>2.5 centromeric signals/nucleus), numeric aberrations of C17 were further confirmed using FISH probes covering p-arm of C17: 17p11.2, 17p12 and 17p13. Results: Her-2/neu gene (H) amplification was found in 44% cases (H/C17>2.0) and 6.3% patients showed borderline amplification (H/C17=1.5–2.0). Polysomy of C17 (C17/nucleus>2,5) was found in 15.3% of cases. In these polysomic cases, 70.2% had amplification (H/C17>2.0), 11.7% borderline amplification (H/C17=1.5–2.0) and 15.6% showed normal status (H/C17 <1,50) of Her-2/neu. However, only 58% of polysomic tumors were true polysomic when examined by probes covering p-arm of C17. Further investigations showed that false polysomic tumors were partially due to cross-hybridization of C17 centromeric probes to centromeres of other chromosomes, but also due to amplification and/or translocation of C17 centromeric signals. Conclusions: Re-examination of polysomic tumors with FISH probes covering p-arm of C17 better reflects status of C17 versus Her-2/neu in breast cancers and increases percentage of patients who may profit from trastuzumab based therapies. This work was supported by grants IGA MZCR 7495- 3 and 7506–3. No significant financial relationships to disclose.

Cytogenetic analyses of hepatocellular carcinoma by in situ hybridization with a chromosome-specific DNA probe.

Numerical chromosome analysis has been established in solid tumors by using in situ hybridization (ISH) with a chromosome-specific probe. We analyzed human hepatocellular carcinoma (HCC) by ISH for chromosome 17 and investigated the correlation of its copy number with histologic malignancy, proliferative activity, p53 mutation, and DNA ploidy. Chromosome 17 was hybridized with a pericentromere-specific DNA probe directly on the tumor cells isolated from paraffin blocks of 25 surgically resected HCCs. Proliferative activity was measured by Ki-67 immunohistochemistry, p53 mutation was analyzed by p53 immunohistochemistry, and DNA ploidy was estimated by cytofluorometry. Forty-four percent of the 25 HCCs showed numerical abnormality of chromosome 17. Many disomic cases had a less malignant histology, whereas many polysomic cases had a more malignant histology. The Ki-67 positive index of polysomic cases was higher than that of disomic cases. In 22 cases (88.0%), the copy number of chromosome 17 was well matched with DNA ploidy. However, the numerical abnormality of chromosome 17 did not show a significant correlation with p53 mutation. Two of four HCCs that showed histologic heterogeneity were also heterogenous on ploidy pattern and the copy number of chromosome 17. Conversely, there was one case in which only ISH could demonstrate heterogeneity, although the other features exhibited homogeneity. Numerical chromosome abnormalities correlated with the increase of histologic malignancy proliferative activity, and DNA ploidy. Moreover, ISH analysis was useful in assessing the intratumoral heterogeneity in HCC, especially when current methods failed to detect it. Thus, ISH provides information on important biologic features, such as malignant potential and intratumoral heterogeneity, in HCC.

Parental origin of the extra chromosomes in polysomy X

Five polymorphic index markers were analyzed by polymerase chain reaction (PCR) to ascertain the parental origin of the extra X chromosomes in seven polysomic cases (one 49,XXXXX, three 49,XXXXY, two 48,XXXY, and one 48,XXYY). All four X chromosomes in 49, X polysomies were maternal in origin and the extra X chromosomes in 48 X polysomies were paternal. In each case the multiple X chromosomes were contributed by a single parent. Taken together with previously reported cases, these data support a single mechanism of sequential nondisjunction during either maternal or paternal gametogenesis as the cause of higher order sex chromosome polysomy.