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Luminiţa Leluţiu1, Rareş Buiga1, Liliana Resiga1, Alexandru Eniu1, Nicolae Ghilezan2, Corneliu D. Olinici2

1 Ion Chiricuţă Cancer Institute, Cluj-Napoca, Romania 2Iuliu Hatieganu University of Medicine and Pharmacy Cluj-Napoca, Romania

Background: The sample was chosen to contain all situations possible as in the guidance for authors. You can copy this sample and modify inside by Copy from your original document and Edit/Paste special/Unformatted text. Only docu-ments provided in Word 2003 will be accepted. Tables were chosen on one column or on two column. Patients and methods: The authors compared the specificity, simplicity of a procedure and method standardization, the simplicity of evaluation for the results for each method (immunohistochemistry and chromogenic in situ hybridization). 1255 cases of invasive breast carcinoma from surgically excised tumors and core needle biopsies were included in the study. The first step was the determination the HER2 status by immunohistochemistry. The cases with moderate (2+) and strong (3+) overexpression of HER2 protein were chosen for determining HER2 amplification by CISH. Results: False-posi-tive results are a significant problem when IHC is exclusively used to test for HER2 overexpression. The false-positives results are confined to the group 2+ positive and do not respond to targeted therapy. Concordance between IHC and CISH is high when immunostaining is interpreted as either negative or strongly positive. Many studies have suggested that CISH may better predict response to anti-HER2 therapy rather than IHC. Although IHC is less technically demand-ing it is more expensive than CISH. Conclusions: As a first step, IHC analysis of HER2 status in breast cancer is a use-ful predictor of response to therapy with Herceptin when it is strongly positive. In some cases IHC positive overexpres-sors are false-positives in CISH tests. Thus, screening of breast cancer with IHC and then confirmation 2+ positives IHC results by CISH is an effective evolving strategy for testing HER2 as a predictor of response to targeted therapy.Key words: Laryngeal Cancer, Lymph Node Involvement, Postoperative Radiotherapy.

Introduction1 Gene amplification is frequently detected in

human tumor cells and is thought to make an important contribution to tumorigenesis. Detailed analysis of altered regions of DNA has revealed complex DNA rearrangements often involving multiple genes and spanning several megabases in solid tumors. Many tumors show such a high degree of general DNA and chromosome rearrangement that some researchers argue the critical event in tumorigenesis is genomic instability with gene amplification being a consequence.

The human epidermal growth factor receptor-2 gene ERBB2 (commonly referred to as HER2/neu) is located on the long arm of chromosome 17q and encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor (EGFR) or HER family.This family of receptors is involved in cell-to-cell and cell-to-stroma

communication primarly through a process known as signal transduction.

HER2/neu gene amplification and/or protein overexpression has been identified in 18-20% of invasive breast cancer (early studies suggested that as many as 30% of breast cancers have HER2 overexpression).The presence of HER2/neu gene amplification is prognostically and therapeutically significant for patients with breast cancer. Overexpression of HER2 protein is associated with rapid tumour growth, increased risk of recurrence after surgery, poor response to conventional chemotherapy and shortened survival.

Testing to determine HER2 status has come into focus since the approval of transtuzumab (Herceptin) for the treatment of HER2-positive breast cancer. Transtuzumab (Herceptin) is a monoclonal antibody directed specifically against HER2 protein and was approved in 1998 by the US Food and Drug Administration for the treatment of metastatic disease.

1Journal of Radiotherapy & Medical OncologyMarch 2009 Vol. XV No. 1:29-35Address for correspondence: Luminiţa Leluţiu,

Ion Chiricuţă Cancer Institute, 34-36 Republicii Street, 400015 Cluj-Napoca, Romaniaemail: luminita_lelutiu@yahoo.com

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The benefit has been evidenced when transtuzumab is used with chemotherapy as a first treatment for metastatic breast cancer or as a single agent for patients with metastatic disease that progresses after initial chemotherapy.

The most commonly used methods for HER2 testing are immunohistochemistry (IHC), which measures HER2 protein in the cell membrane, and fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH), which evaluates HER2/neu gene amplification. Both methods are able to evaluate HER2/neu in formalin–fixed paraffin-embedded archival tissues.

Most HER2/neu studies have been performed by IHC, which detect overexpression of the HER2 protein product on the cell membrane of tumor cells. The technique is widely accessible and easy to perform at a reasonable cost. This semi-quantitative method is beset by technical artifacts, sensitivity differences between different antibodies, and subjective interpretation, resulting in interobserver variability between pathologists. For IHC stain intensity 2+ additional testing, with FISH or CISH is recommended.

FISH is a quantitative procedure for detecting HER2/neu gene amplification on the nuclei of tumor cells. The FISH method is a more complex and more expensive technique; a disadvantage of FISH is the requirement of expensive equipment for the analysis(a fluorescent microscope with suitable filters, a sensitive CCD camera).Moreover, fluorescent signals tend to fade in a few weeks or months and it does not allow for a simultaneous histological examination of tissue structure beyond basic tissue identification.

CISH is a new technique used in HER2/neu gene detection. The concordance between CISH and FISH can range from 85% to as high as 100%. The advantages of CISH are: it requires an ordinary microscope; the method is more economical; the signal intensity is permanent.

Patients and methodsOur study was focused on 1255 breast

carcinoma tissue samples with final histopathologic

diagnosis of invasive ductal, lobular carcinoma and other special types. From a total number of 1255 cases, 960 cases were diagnosed and treated between January 2006 and September 2008 at the Ion Chiricuţă Cancer Institute Cluj-Napoca and 295 samples were received from other hospitals only for HER2 testing by IHC or CISH.

All 960 tumors were removed by mastectomy or core needle biopsies (breast-conserving therapy), and axillary dissection was performed.

The patients ages were grouped as either premenopausal (less than 50 years) or postmenopausal (50 years and more). The age of the patients with breast cancer ranged from 26 to 81 years with a median age of 52 years.

Histologic typing was performed according to the World Health Organization classification and grading according to the Elston and Ellis modified criteria of Bloom and Richardson (Table I).

The pathologists reviewed the HER2 results of all IHC cases in the period covered, reassessing them in accordance with the US Food and Drug Administration-approved HER2 Herceptin scoring guidelines.

Immunohistochemical AnalysisFor each case, all H&E-stained slides were

reviewed and 1 or 2 representative tumor tissue blocks were selected for study. Immunohistochemical analysis were performed on 4 μm sections according to standard procedures. We cut 4 μm sections from archival tissue that had been fixed in buffered formalin and embedded in paraffin, and mounted on silanized slides (SuperFrost Plus).

The IHC-stained slides were interpreted and scored on the scale of 0 to 3+ according to the FDA-approved guidelines for HercepTest (Table II). Areas with intraductal carcinoma were excluded from the evaluation.

Some cases with 2+ and 3+ IHC results were further evaluated by CISH, for HER2/neu gene amplification.

Our laboratory participates regularly in an international UK-based quality assurance system (NEQUAS) for immunohistochemistry.

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Luminiţa Leluţiu et al: HER2 Testing in Breast Cancer Using Immunohistochemical Analysis…

Table I: Tumor and histological characteristics

Tumor his-tological

grade

Age G1 G2 G3 Grand totalNo % No % No % No %

Ductal < 50 53 5.52 144 15.00 131 13.65

328 34.17

≥ 50 73 7.60 247 25.73 212 22.08

532 55.42

Total ductal 126 13.13 391 40.73 343 35.73

860 89.58

Lobular < 50 5 0.52 6 0.63 3 0.31 14 1.46≥ 50 14 1.46 11 1.15 1 0.10 26 2.71

Total lobular 19 1.98 17 1.77 4 0.42 40 4.17Ductal+Lo

bular< 50 2 0.21 9 0.94 4 0.42 15 1.56≥ 50 3 0.31 14 1.46 18 1.88 35 3.65

Total D+L 5 0.52 23 2.40 22 2.29 50 5.21Mucinous < 50 1 0.10 0 0 0 0 1 0.10

≥ 50 4 0.42 0 0 1 0.10 5 0.52Total mucinous 5 0.52 0 0 1 0.10 6 0.63

Apocrine < 50 0 0 0 0 2 0.21 2 0.21≥ 50 0 0 0 0 1 0.10 1 0.10

Total apocrine 0 0 0 0 3 0.31 3 0.31Micro

papillary< 50 0 0 1 0.10 0 0 1 0.10≥ 50 0 0 0 0 0 0 0 0

Total micro 0 0 1 0.10 0 0 1 0.10Grand total 155 16.15 432 45.00 373 38.8

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Table II: Recommended immunohistochemical scoring method

Score HER2 protein assesst-ment

Staining pattern

0 Negative No staining is seen or membrane staining is less that 10% invasive tumour cells

1+ Negative Faint/barely perceptive membrane staining detected in more than 10% invasive tumour cells

2+ Equivocal Weak to moderate complete membrane staining in more than 10% invasive tumour cells or < 30% with strong complete mem-brane staining

3+ Positive Strong, uniform complete membrane stain-ing in more than 30% invasive tumour cells

Chromogenic In Situ Hybridization (CISH) For chromogenic in situ hybridization standardization (CISH) analysis, we selected 56 cases

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Prognostic Value of Lymph Node Involvement in Laryngeal Carcinoma

with moderate (2+) and strong (3+) overexpression of HER2 protein detected by IHC in our laboratory and in another hospital center, which were clinically eligible for treatment with transtuzumab.

We cut 4 μm sections from the archival tissue that had been fixed in buffered formalin and embedded in paraffin. Then, the sections were deparaffinized and heated in CISH pretreatment buffer (SPOT-Light tissue pretreatment kit, Zymed Laboratories). Immunodetection was performed by using a commercial SPOT-Light HER2 CISH Detection Kit (Zymed) according to the manufacturer’s instructions.

HER2 gene signals were scored in histologic sections in at least 200 neoplastic cells with a microscope Nikon E-1000 using a 40x objective.

The CISH assays were run by one technologist following the protocol of Zymed. CISH results were read by the same pathologist who was blinded to the previous IHC results.

CISH was assessed on invasive ductal carcinoma areas only; foci of intraductal carcinoma were excluded from the analysis.

Tumors were classified depending on the number of gene copies in the nuclei as follows Table III (the recommended CISH scoring by Zymed):

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E.Ciuleanu et a

Tabel III: Evaluation of HER2 gene status using Chronogenic in Situ Hybridization (CISH)

Amplification HER2/neu gene statusHigh-level amplification > 10 dots, or large cluster, or mixture of

multiple dots and large clusters, of HER2 gene per nucleus in > 50% tumor cells

Low-level amplification 6-10 dots, or small clusters, or mixture of multiple dots and small clusters of HER2 gene per nucleus in > 50% tumor cells

No amplification Polysomy: 3-5 dots of HER2 per nucleus of 50% tumor cellsDiploid: 1-2 dots of HER2 per nucleus of 50% tumor cells

Imunohistochemical (IHC) ResultsA total of 1255 breast cancer specimens were

tested for HER2 status: 960 (76.49%) from the Cancer Institute and 295 (23.51%) from other hospitals. From these 960 cases, 860 (89.58%) were invasive ductal carcinoma (126 cases with SBR grade 1, 391 cases with SBR grade 2, and 343 cases with SBR grade 3), 40 (4.17%) were invasive lobular carcinoma, 50 (5.21%) were invasive ductal + lobular carcinoma and 10 cases (1.04%) other special types (mucinous carcinoma, invasive micropapillary carcinoma, apocrine carcinoma).

The 591 (61.56 %) of 960 specimens evaluated with the HercepTest were scored 0,154 (16.04%) were scored 1+, 75 (7.81%) were scored 2+ and 140 (14.58%) were scored 3+ (Table IV).

Table IV: Imunohistochemcal Analysis of Breast Tumor Specimens for HER2 Testing

IHC score No. (%) (N=960)01+2+3+

591(61.56)154 (16.04)75 (7.81)140 (14.58)

All 40 cases with final histopathological diagnosis of invasive lobular carcinoma were scored negative (0,1+) by IHC.

All cases (295) from other hospitals were invasive ductal carcinoma: 61 cases (20.67%) were IHC positive (2+, 3+) and 234 (79.32%) were IHC negative (0, 1+).

IHC positive specimens (invasive ductal carcinoma with 2+ and 3+ IHC results) were futher evaluated by CISH (56 cases in our laboratory).

Table V shows the IHC assay in relation to the tumor histologic grades.Of the 960 cases, 215 (22.39%) were IHC positive and 745 (77.60%) were IHC negative. From the Cancer Institute most of the tumors IHC positive tumors were invasive ductal carcinoma with histologic grade 3 (50.23%), followed by grade 2 (42.79%), then grade 1 (6.97%). Most of the patients with histologic grade 3 and 2 were in postmenopausal status (50 years or older). No significant association was noted between histologic grade and IHC scores (P = 0.928). Similarly, no association was observed between age and histologic grade (P=0.929).

Many patients more than 50 years old and older (postmenopausal status) seemed to be HER2 positive and amplified, whereas more patients less than 50 years old (premenopausal status) seemed to be HER2 negative and non-amplified (Table VI).However, these associations were not statistically significant (P=0.962)

Chronogenic in Situ Hybridization (CISH) results

All 56 tests were successfully performed with the Zymed protocol for CISH.At least two detectable amplicons per tumor nucleus were observed, and these served as the internal control.In addition, a highly amplified positive control was run simultaneously with each test.

CISH result were evaluated with the light microscope at low-power and high-power magnification. Amplified cases showed at least six brown signals per nucleus; distinct clustering of amplicons was also obtained. Those cases reported as having no amplification or low amplification were not verified further for chromosome 17 aneuploidy.

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Journal of Radiotherapy & Medical Oncology, March 2009 Vol. XIV No. 1:29-35

Table VII shows the CISH assay outcomes of the 56 cases (cases from our laboratory with 2+ and 3+ IHC results and cases that were tested by IHC in other center) in relation to the IHC category.Of the 56 cases, 36 were IHC 2+ positive and 20 were IHC 3+

positive.With the CISH assay, 23 (66.66%) of the 36 cases with IHC 2+ showed HER2/neu gene amplification, and 12 (33.33%) no amplification. All 20 cases with IHC 3+ showed high amplification with CISH assay.

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Table V: Breast tumor samples and their corresponding immunohistochemistry (IHC) and tumor histologic grade

Tabe l

VI: Comparison of age (menopausal status) and immunohistochemistry results

Parameter Age <50 Age ≥ 50 TotalImmunohistochem-istry

Negative (0, 1+) 275 (36.91%) 470 (63.08%) 745Positive (2+) 35 (46.66%) 40 (53.33%) 75Positive (3+) 51 (36.42%) 89 (63.57%) 140

Total 361 599 960

Tabel VII: Chromogenic in situ hybridization (CISH) results

IHC Score 2+ Score 3+Total cases 36 20No amplification 12 (33.33%) 0With amplification 24 (66.66%) 20 (100%)

• high-level 9 20• low-level 15 0

DiscussionIn the present study, 276 (21.99%) of 1255

cases were IHC positive and 979 (78%) were IHC negative.

From the Cancer Institute most of the IHC positive tumors were invasive ductal carcinoma with histologic grade 3 (50.23%), followed by grade 2 (42.79%), then grade 1 (6.97%). Most of the patients with histologic grade 3 and 2 were in postmeno-pausal status (50 years or older).

Histological grading by itself is not accepted as a prognosis factor. However, several studies have proved that 3+ histological graded is associated with IHC markers, related to poor prognosis.

All 40 cases with final histopathological diagnosis of invasive lobular carcinoma were scored negative (0,1+) by IHC, and these results are in concordance with the previous reports.

From total IHC positive results, 56 tests selected were successfully performed with the Zymed protocol for CISH.The concordance between 3+ IHC and CISH-amplified cases was 100% (20 out 20), denoting all gene amplified cases to be overexpressing the HER2 protein.In contrast, the agreement on 2+ IHC and CISH-amplified cases was only 66.66%, which is lower than previously reported in other study. The agreement on IHC positive and CISH-amplified cases was 78.57%. The

IHC result Age G1 G2 G3 Grand totalHER 0 < 50 47 (21.86%) 86 (40.00%) 82 (38.14%) 215

≥ 50 77 (20.48%) 172 (45.74%) 127 (33.78%) 376Total HER 0 124 (20.98%) 258 (43.65%) 209 (35.36%) 591

HER 1+ < 50 5 (8.33%) 35 (58.33%) 20 (33.33%) 60≥ 50 11 (11.70%) 47 (50.00%) 36 (38.30%) 94

Total HER 1+ 16 (10.39%) 82 (53.25%) 56 (36.36%) 154HER 2+ < 50 7 (20.00%) 12 (34.29%) 16 (45.71%) 35

≥ 50 2 (5.00%) 15 (37.50%) 23 (57.50%) 40Total HER 2+ 9 (12.00%) 27 (36.00%) 39 (52.00%) 75

HER 3+ < 50 2 (3.92%) 27 (52.94%) 22 (43.14%) 51≥ 50 4 (4.49%) 38 (42.70%) 47 (52.81%) 89

Total HER 3+ 6 (4.29%) 65 (46.43%) 69 (49.29%) 140Grand total 155 (16.15%) 432 (45.00%) 373 (38.85%) 960

33.33% (12 of 36) cases IHC-positive/CISH-non-amplified tumors in this study, all of which had 2+ IHC scores, is higher than the 17% false positive reported by the literature. In this case, false-positive results are a significant problem when IHC is exclusively used to test for HER2 overexpresion. A significant percentage (5-15%) of breast carcinomas

show moderate HER2 membrane staining without evidence of amplification, and they most represent false positive staining (highly sensitive nonspecific staining). It may be caused by alternative transcriptional or post-transcriptional mechanisms controlling HER2 expression.

Fig. 1: HER2 protein testing in breast cancer (IHC) : (a) No staining is seen (score=0); (b) Faint perceptive membrane staining detected in more than 10% invasive tumour cells (score=1+);

(c) Weak to moderate complete membrane staining in more than 10% invasive tumour cells (score=2+); (d) Strong, uniform complete membrane staining in more than 30% invasive tumour cells(score=3+)

Fig. 2: Chromogenic in situ hybridization (CISH) analysis shows: (a) One or two dots can be seen in each nucleus, indicating the absence of HER2/neu gene amplification.(b) Small clusters and multiple dots can be seen, indicating a low

level amplification of HER2/neu gene.(c) and (d).Large clusters of hybridised HER2 probe, indicating the presence of a high level of HER2/neu gene amplification.

Some cases, received from other hospitals that were scored with moderate overexpression (2+) had in fact the score 1+ (negative). This aspect can explain discrepancies between the IHC and CISH results in our study.

A test for chromosome 17 aneuploidy was not performed on non-amplified or low-amplified CISH assay, because recent studies have shown that it makes the analysis more costly and time consuming without adding relevant data.

ConclusionsIn this study HER2 protein by IHC and

HER2/neu gene amplifications by CISH was successfully evaluated as a molecular biology procedure that is easily integrated into routine testing in our laboratory. This method is a practical alternative to FISH that can be used in conjunction with IHC, which remains the first screening procedure of choice.

Immunohistochemistry is easy to perform, a relatively inexpensive method and able to detect the majority of breast cancer patients whose tumor has a IHC-negative score (0, 1+) or a 3+ IHC score, all of which have complete concordance with CISH.

Because discrepancies between IHC and CISH were observed in the 2+ IHC category, all cases with 2+ IHC results will be tested by CISH before transtuzumab therapy. This association IHC-CISH not only identifies the IHC false-positive subset that will not benefit from transtuzumab, but also keeps testing costs for HER2 status at a reasonable minimum.

It is very important that the HER 2 testing by IHC and CISH is performed in the same laboratory, to exclude the IHC false-positive/CISH negative results.

CISH is emerging as a practical, cost-effective, and valid alternative to FISH in testing for gene alteration, especially in centers primarily working with IHC.

References:1. Wolff A, Hammond E, Schwartz J, et al. American

Society of Clinical Oncology/College of American Pathologist guideline recommandations for human epidermal

growth factor receptor 2 testing in breast cancer. J Clin Oncol 2007; 25: 118-145.

2. Arnould L, Arveux P, Couturier J, et al. Pathologic complete response to transtuzumab- based neoadjuvant therapy is related to the level of HER-2 amplification. Clin Cancer Res 2007; 13: 6404-6409.

3. Di Palma S, Collins N, Faulkes C, et al. Chromogenic in situ hybridization(CISH) should be an accepted method in the routine diagnostic evaluation of HER-2 status in breast cancer.J Clin Pathol 2007; 60: 1067-1068.

4. Dendukuri N, Khetani K, McIsaac M, Brophy J. Testing for HER2-positive breast cancer:a systematic review and cost-effectiveness analysis.CMAJ 2007; 176: 1429-1434.

5. Ellis CM, Dyson M J, Stephenson TJ, Maltby EL. HER-2 amplification status in breast cancer: a comparison between immunohistochemical staining and fluorescence in situ hybridization using manual and automated quantitative image analysis scoring techniques.J Clin Pathol 2005; 58:710-714.

6. Hoff ER, Tubbs RR, Myles JL, Procop GW. HER2/neu amplification in breast cancer: stratification by tumor type and grade. Am J Clin Pathol 2002; 117: 916-921.

7. Ross JS, Fletcher JA, Bloom KI, et al. Targeted therapy in breast cancer: the Her2/neu gene and protein.Mol Cell Proteomics 2004; 3: 379-398.

8. Walker R, Bartlett J, Dowsett M, et al. HER2 testing in the UK : further update to recommandations.J Clin Pathol 2008; 61: 818- 824.

9. Tubbs RR, Pettay JD, Roche PC, Stoler MH, Jenkins RB, Grogan TM. Discrepancies in clinical laboratory testing of eligibility for transtuzumab therapy: apparent immunohistochemical false-positive do not get the message. J Clin Oncol 2001; 19: 2714-2721.

10. O’Malley FP, Thomson T, Julian J, et al. HER2 testing in a populatinon –based study of patients with metastatic breast cancer treated with transtuzumab. Arch Pathol Lab Med 2008; 132: 61-65.

11. Cayre A, Mishellany F, Lagarde N, Penault-Llorca F. Comparison of different commercial kits for HER2 testing in breast cancer: looking for the accurate cutoff for amplification. Breast Cancer Res 2007, 9:R64

12. Thomsom TA, Hayes MM, Spinelli JL, et al. HER2/neu in breast cancer: interobserver variability and performance of immunohistochemistry with 4 antibodies compared with fluorescent in situ hybridization.Mod Pathol 2001; 14: 1079-1086.

13. Ferrero-Pous M, Hacene K, Bouchet C, Le Doussal V, Tubiana-Hulin M, Spyratos F. Relationship between c-erb-B2 and other tumor characteristics in breast cancer prognosis.Clin Cancer Res 2000; 6: 4745-4754.

14. Sinzak-Kuta A, Tomaszewska R, Rudnicka-Sosin L, Okon K, Stachura J. Evaluation of HER2/neu gene amplification in patients with invasive breast carcinoma. Comparison of in situ hybridization methods. Pol J Pathol 2007; 58: 41-50.

15. Bilous M, Dowsett M, Hanna W, et al. Current perspectives on HER2 testing: a review of national testing guidelines. Mod Pathol 2003; 16: 173-182.

The authors ndicated no potential conflicts of interestAuthor Contributions:

Conception and design: Luminiţa Leluţiu, C.D. OliniciProvision of study material or patients: Luminiţa Leluţiu, Rareş Buiga, Alex. EniuCollection and assembly of data: Luminiţa LeluţiuData analysis and interpretation: Luminiţa Leluţiu, N. GhilezanManuscript writing: Luminiţa LeluţiuFinal approval of manuscript: C.D. Onilici