neu Amplification Detected by Fluorescence in situ Hybridization in Cytological Samples from Breast Cancer

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4 HER-2/neu Amplification Detected by Fluorescence in situ Hybridization in Cytological Samples from Breast Cancer Cecilia Bozzetti

Introduction The proto-oncogene HER-2/neu (also called c-erbB-2) is located on chromosome 17q and encodes a 185-kDa transmembrane glycoprotein (p185) with tyrosine kinase activity that has partial homology with members of the epidermal growth factor (EGF) receptor family (Coussens et al., 1985). Amplification of the HER2/neu gene and/or p185 overexpression has been reported in 20–30% of primary breast cancer. Both alterations have been correlated with a more aggressive phenotype and worse disease-free and overall survival (Allred et al., 1992; Gullick, 1990). In light of this evidence, an innovative therapeutic approach targeting the receptor by monoclonal antibodies (MAb) to the HER-2/neu protein product has been promoted. Trastuzumab (Herceptin) is a highaffinity anti-HER-2/neu MAb developed by Genentech (San Francisco, CA) for the treatment of advanced breast disease. The results of the initial trastuzumab trials have shown clearly that this agent represents a Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 1: Molecular Genetics; Lung and Breast Carcinomas

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new specific therapy active in patients with advanced HER-2-positive breast cancer either alone or in combination with anthracycline and taxane-based chemotherapy (Vogel et al., 2002). Despite these results, the method of choice to determine whether a tumor is HER-2/neu positive is still under debate. Laboratory methods for assessing HER2/neu amplification or HER-2/neu protein overexpression include Southern, Northern, and Western Blot techniques, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). Amplification of the HER-2/neu gene is usually analyzed by FISH and HER-2/neu protein overexpression by IHC on tissue sections from the primary tumor. Assessment of HER2/neu by IHC is relatively simple and inexpensive, but the use of different antibodies as well as interpretative difficulties do not make this technique highly reproducible. Gene-based techniques, such as FISH, have less variability, but require special equipment and are more expensive. Nevertheless, FISH seems to be a more powerful technique. In some clinical situations, Copyright © 2004 by Elsevier (USA) All rights reserved.

268 such as preoperative chemotherapy in primary disease or unavailability of archival samples in metastatic patients, the determination of HER-2/neu status by FISH on cytological material is helpful. This approach allows the selection of the best treatment. The biology of HER-2/neu and its use as a target for antibodybased therapeutics, as well as current methodologies for HER-2/neu testing and its evolving role in the management of breast cancer has been reviewed by Ravdin (2000). Fine-needle aspiration, a well-established method in the diagnosis and biological characterization of breast cancer, has been successfully combined with molecular and cytogenetic analysis. Only a few studies concerning the feasibility of dual-color FISH on cytological material from primary breast cancer fine-needle aspirates (FNAs) has been published (e.g., Bozzetti et al., 2002). Though metastases are the target for trastuzumabbased therapy, HER-2/neu status is usually evaluated on the primary tumor, since metastatic lesions are rarely removed or biopsied to obtain a biological characterization. It has been postulated that breast cancer is a heterogeneous disease where, among different clones, an early stem line clone might evolve independently in the primary tumor and its metastasis, suggesting that clonal diversification and heterogeneity may account for cancer progression and metastatic dissemination (Kuukasjarvi et al., 1997). Tumor heterogeneity may explain why biomarkers of prognosis or of therapy responsiveness, evaluated exclusively from primary tumors, may not completely reflect all the biological properties of metastatic breast cancer. Amplification or overexpression of HER-2/neu may confer biological advantage to tumor cells, and genetic instability induced by amplification might generate a more aggressive cancer phenotype. Theoretically, this could explain the high proportion of patients that ultimately do not respond to trastuzumab-based therapies. A number of studies have shown a high level of concordance, although not complete, between HER-2/neu status evaluated on primary tumor and lymph node metastases by means of both IHC (Masood and Bui, 2000) and FISH (Xu et al., 2002). However, few studies have compared HER-2/neu status between primary tumors and paired distant metastases, showing an heterogeneous pattern of results (Gancberg et al., 2002). To our knowledge, none of the studies that evaluated HER-2/neu status on metastatic sites from breast cancer patients have been carried out on cytological material. In our experience, fine-needle aspiration biopsy (FNAB), possibly coupled with ultrasound for the sampling of deep lesions, is a relatively safe and less-invasive alternative to surgical biopsy to obtain cellular material for an updated definition of

IV Breast Carcinoma HER-2/neu status by FISH. This protocol circumvents many of the drawbacks related to immunocytochemistry of cytological samples (Nizzoli et al., 2003). In a previous publication (Bozzetti et al., 2002) we reported that HER-2/neu gene amplification can be reliably estimated by FISH on FNAs from primary breast cancer, and a good correlation was found between FISH cytology and FISH and/or IHC results from the corresponding histological sections. More recently, we demonstrated the feasibility of HER-2/neu evaluation by FISH on cytological samples obtained from distant metastatic lesions of breast cancer (Bozzetti et al., 2003). Our study emphasizes the feasibility and advantages of the two rapid and very informative techniques (FNAB and FISH). These techniques were performed to ascertain the malignant nature of a suspicious lesion and obtain predictive markers for response. Since the advent of trastuzumab, the characterization of the molecular profile in metastatic breast disease is becoming increasingly important for targeted therapies selection.

MATERIALS Breast Cancer Specimens A first series of samples included FNAs from 66 primary breast cancer patients, which were stained with May-Grunwald Giemsa (MGG) stain for routine diagnostic cytology as well as HER-2/neu evaluation by FISH. After surgery, the corresponding sections of archival formalin-fixed, paraffin-embedded tissue were evaluated for HER-2/neu by FISH and IHC. A second series of samples included cytological samples from metastatic lesions obtained from 22 patients that presented, at different times after primary treatment, metastatic relapses in liver (12 patients), in pleura (three patients), in peritoneum (three patients), and in skin (four patients). Cytologic smears from metastases were submitted for routine diagnostic cytology as well as for HER-2/neu evaluation by FISH. Moreover, their corresponding primary breast tumors were evaluated by FISH either on paraffin histological sections, when available, or on destained archival cytological smears.

Laboratory Reagents 1. PathVysion HER-2 DNA Probe Kit. Components: Locus Specific Identifier (LSI) HER-2/neu SpectrumOrange/CEP 17 SpectrumGreen DNA Probe. 20X saline-sodium citrate (SSC) salts. NP-40.

4 HER-2/neu Amplification Detected by FISH in Cytological Samples from Breast Cancer DAPI counterstain (4,6-diamidino-2phenylindole) and 1,4-phenylenediamine in phosphate-buffered saline and glycerol. 2. 20X SSC (3 M sodium chloride, 0.3 M sodium citrate, pH 5.3): 66 g 20X SSC, 200 ml distilled water; adjust pH to 5.3 with concentrated HCl. Bring volume to 250 ml with distilled water. Filter through a 0.45-μm pore filtration unit. Can be stored at room temperature for up to 6 months. 3. Wash buffer (2X SSC/0.3% NP-40, pH 7.0–7.5): 100 ml 20X SSC, pH 5.3, 847 ml distilled water, 3 ml NP-40; adjust pH to 7.0–7.5 with 1 N NaOH. Bring volume to 1 L with distilled water. Filter through a 0.45-μm pore filtration unit. Discard used solution at the end of each day. Unused solution can be stored at room temperature for up to 6 months. 4. 70%, 85%, and 100% ethanol. 5. Proteinase K solution: 10 mg/ml proteinase K in PBS. Store 150-μl aliquots at −10°C to − 20°C for up to 6 months. 6. Xylene. 7. Carnoy’s solution: 60 ml ethanol 100%, 30 ml chloroform, 10 ml glacial acetic acid. 8. Rubber cement. 9. Immersion oil for microscopy.

Laboratory Equipment 1. HYBrite Denaturation/Hybridization System for FISH (Vysis). 2. Water bath (37°C ± 1°C, 72°C ± 1°C). 3. Vortex mixer. 4. Microcentrifuge. 5. pH meter. 6. Calibrated thermometer. 7. Coplin jars: vertical staining jars. 8. 22 mm × 22 mm glass coverslip. 9. 0.45-μm pore filtration unit.

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DNA is denaturated by heating to form single-stranded DNA. Hybridization is allowed to occur by overnight incubation at 37°C. Thereafter, the unbound labeled probe is removed by wash buffer and cells are counterstained with DAPI. The hybridized probe is detected by fluorescence microscopy. Our procedure is based on the application of a dualcolor technique, using a probe for HER-2/neu gene together with a probe that hybridizes to a pericentromeric region of chromosome 17. The LSI HER-2/neu DNA probe is directly labeled with Spectrum-Orange fluorophore and targets the DNA sequences ∼ 190 Kb in size spanning the HER-2/neu gene within band 17q11.2-q12 on long arm of chromosome 17. The Chromosome Enumerator Probe (CEP) 17 (alpha satellite) DNA probe is a 5.4-Kb probe targeting to the centromeric region of chromosome 17 (17p11.1-q11.1) and is directly labeled with SpectrumGreen fluorophore. Path-Vysion HER-2 DNA Probe Kit provides LSI HER-2/neu SpectrumOrange and CEP 17 SpectrumGreen DNA probes premixed and predenaturated in hybridization buffer. The probe mixture allows for the simultaneous determination of the HER-2/neu gene locus copy number and chromosome 17 copy number in interphase cells. Determination of the ratio of HER2/neu to chromosome 17 copy number is useful in the discrimination of aneusomy of chromosome 17 from true gene amplification of the HER-2/neu. This allows for proper assessment of amplification levels, especially low levels of amplification. In our procedure, both denaturation and hybridization take place in a HYBrite Denaturation/ Hybridization System for FISH (Vysis). This system is optimized for performing FISH assay by a reproducible denaturation/hybridization temperature control and an accurate timing of events. Slides are placed on a heating surface where denaturation and hybridization successively occur, simplifying standard FISH steps in particular by eliminating formamide-denaturation baths.

METHOD Fluorescence in situ Hybridization—Principles of the Method The FISH technique is based on the ability of singlestranded DNA to anneal to complementary DNA. Detailed procedures for FISH have been reviewed (Le Beau, 1993). The target DNA is the nuclear DNA of interphase cells smeared on a glass microscope slide. The test probe is directly labeled with a fluorochrome. To allow hybridization of complementary sequences to occur, a formamide solution containing the predenaturated labeled DNA probe is applied to the microscope slide. The slide is coverslipped, sealed, and the cellular

HER-2/neu FISH on Cytologic Samples Collection of Samples

Samples from primary breast cancer and superficial metastases are obtained by multidirectional FNAB using a 22-gauge needle and 20-ml syringe. Samples from liver and other deep metastases are obtained by ultrasound-guided FNAB. Cell preparations from metastatic pleural and ascitic fluid are cytospins. Proceed as follows: 1a. Samples obtained by FNAB from superficial or deep lesion: smear the aspirated material on at least two glass slides, and air-dry.

270 1b. Samples obtained from pleural and ascitic fluid: cytocentrifuge the cellular suspension for 5 min at 600 rpm (45 g) onto glass slides, and air-dry. 2. Stain one or more slide with MGG stain for routine cytology, and keep the remaining slides unstained. 3. After cytologic diagnosis of malignancy, choose one unstained slide, representative of the lesion, for FISH assessment. 4. Mark the area to be hybridized with a diamondtipped scribe. The area should cover approximately a 22 mm × 22 mm portion of the slide. Store the slides in a container at room temperature until assay. Pretreatment of Cytologic Slides

Pretreatment is a necessary procedure for cytologic samples in the prehybridization steps. It helps increase the accessibility of the target DNA of the smeared cells to the DNA probe. Unstained slides are usually pretreated with wash buffer only, except for smears showing considerable thickness and overlapping cells, for which an alternative pretreatment with proteinase K is recommended. The latter treatment should be applied in case of a lack of hybridization, after that FISH procedure has been carried out on slides pretreated with wash buffer only. In this case, if more than one unstained cytologic slide is available for FISH procedure, choose a new slide to submit to proteinase K pretreatment. The steps for the pretreatment of MGG stained slides are described next. Pretreatment of no more than six slides at one time per Coplin jar is recommended to avoid decrease of baths temperature. Pretreatment with Wash Buffer

1. Fix the unstained smears twice in absolute methanol at room temperature for 5 min each, and air-dry. 2. Verify that the pH of the wash buffer is 7.0–7.5 at room temperature before use. 3. Add wash buffer (2X SSC/0.3% NP-40, pH 7.0–7.5) to a Coplin jar. 4. Prewarm the wash buffer by placing the Coplin jar in the water bath at 37°C until solution temperature has reached 37°C. 5. Incubate the samples in wash buffer for 30 min, and air-dry. 6. Dehydrate the samples in 70%, 85%, and 100% ethanol at room temperature for 2 min each, and air-dry. Pretreatment with Proteinase K

1. Dehydrate the samples in 70%, 85%, and 100% ethanol at room temperature for 2 min each, and air-dry.

IV Breast Carcinoma 2. Add 70 ml PBS to a Coplin jar. 3. Prewarm PBS by placing the Coplin jar in the water bath at 37°C. 4. When PBS temperature has reached 37°C, thaw an aliquot of the 10 mg/ml proteinase K solution and add 140 μl to 70 ml prewarmed PBS (20 μg/ml proteinase K). 5. Incubate the samples in 20 μg/ml proteinase K at 37°C for 5 min. 6. Rinse the samples in distilled water, and air-dry. 7. Dehydrate the samples in 70%, 85%, and 100% ethanol at room temperature for 2 min each, and air-dry. Pretreatment of Slides Stained with MGG Stain

1. Place the prestained slides in a Coplin jar with xylene until the coverslip could be removed easily. 2. After removing the coverslip, immerse the slides twice in clean xylene for 10 min each to remove the mounting medium. 3. Fix in Carnoy’s solution for 10 min at room temperature. Repeat twice using clean Carnoy’s solution. 4. Dehydrate the samples in 70%, 85%, and 100% ethanol at room temperature for 2 min each, and air-dry. 5. Pretreat slides with wash buffer or alternatively with proteinase K as previously described.

FISH Procedure Denaturation and Hybridization

1. Allow the hybridization solution (LSI HER-2/neu SpectrumOrange/CEP 17 SpectrumGreen DNA Probe) to warm at room temperature for ∼10 min so that the viscosity decreases sufficiently to allow accurate pipetting. Vortex to mix. Centrifuge the tube for 2–3 sec in a bench-top microcentrifuge to bring the content to the bottom of the tube. 2. Apply 10 μl of the hybridization solution to the target area of the slide. 3. Immediately place a 22 mm × 22 mm glass coverslip over the probe mix and allow it to spread evenly under the coverslip. Gently tap the coverslip with forceps to ensure its adhesion to the slide and to avoid air bubbles. The remaining probe solution should be refrozen immediately after use. 4. To prevent drying of the specimen during hybridization, seal coverslip by ejecting a small amount of rubber cement around the perimeter of the coverslip, creating an air-tight seal. Keep slides in the dark long enough to allow the rubber cement to dry. 5. Denaturate samples at 70°C for 5 min and hybridize at 37°C overnight in a HYBrite Denaturation/ Hybridization System for FISH (Vysis).

4 HER-2/neu Amplification Detected by FISH in Cytological Samples from Breast Cancer Posthybridization Washes

1. Verify that the pH of the wash buffer is 7.0–7.5 at room temperature before use. 2. Add 70 ml of wash buffer to a Coplin jar. 3. Prewarm the wash buffer by placing the jar in the water bath until solution temperature has reached 73°C. 4. Note: Wash no more than six slides at once. For subsequent washes, ensure that the temperature of the wash solution is 73°C before adding slides. 5. Add 70 ml of wash buffer to a second Coplin jar and use at room temperature. 6. Remove the rubber cement seal from the slides by gently scraping with forceps. 7. Immerse slides in wash buffer at room temperature for 1–2 min until coverslip floats off and, if necessary, carefully use forceps to remove it. Keep slides in wash buffer until all coverslips have been removed. 8. Remove slides from wash buffer and drain excess liquid by touching the bottom edge of the slides to a blotter. 9. Verify with a calibrated thermometer that the temperature of the prewarmed wash buffer is 73°C. 10. Incubate slides in wash buffer at 73°C for 2 min. 11. Remove each slide from the wash bath and drain excess liquid by touching the bottom edge of the slides to a blotter and wiping the underside of the slide with a paper towel. 12. Air-dry the slides in the dark on a slide rack in an oblique position. 13. Apply 10 μl of DAPI counterstain to the target area of the slide and apply a 22 mm × 22 mm coverslip. Gently press the slide between two sheets of blotting paper, allowing DAPI to spread evenly under the coverslip. 14. Seal coverslip with nail enamel around the perimeter of the coverslip, creating an air-tight seal. Air-dry in the dark in a horizontal position. 15. Store the slides in the dark before signal enumeration. If slides are not viewed at once, store them at −20°C in the dark. After removing from −20°C storage, allow slides to reach room temperature prior to viewing, using fluorescence microscopy.

The same slide could show areas where hybridization successfully occurs, areas lacking signals, or where only red or green signals are present; this could be ascribed in case of fresh smears to particularly thick areas or in case of destained smears to areas where destaining unsuccessfully occurs. The background should appear dark or black and relatively free of fluorescent particles or debris. Avoid fields with clusters of cells exhibiting overlapping of nuclei, and avoid areas of necrosis and where the nuclear border are ambiguous. Counterstain with DAPI should allow for distinguishing between tumor cells and lymphocytes or normal epithelial cells that might be present in the smears, on the base of shape and dimension of cells. If any doubts arise about the nature of cells, verify it viewing the corresponding MGG stained slide. 3. Isolated cells or monolayered clusters are best for evaluation. Focus up and down to find all the signals present in the nucleus. Count split centromere signals as one. Exclude from the evaluation nuclei lacking hybridization signals. Score only nuclei with both green and red signals. As the level of gene amplification is heterogeneous among the nuclei of the same specimen, determine an average HER-2/neu gene copy number and an average centromere 17 copy number for each preparation, on the basis of the distribution of signals over the entire slide, or scoring at least 50 evaluable nuclei for each case. 4. Express results as a ratio of the number of copies of the HER-2/neu gene to the number of chromosome 17 centromeric markers. Classify the samples as: ▼

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Slide Evaluation

1. View samples using a 100X oil immersion fluorescence objective on a fluorescence microscope equipped with a 100W mercury lamp and a triple excitation/emission filter for simultaneous detection of SpectrumOrange, SpectrumGreen, and DAPI. 2. Visually scan the entire slide to find out areas where the signals are bright, distinct, and easily evaluable. Skip signals with weak intensity. There should be a complete, distinct separation between signals in order for them to be considered more than one signal.

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Unamplified: When two copies of HER-2/neu and two copies of chromosome 17 are found in the majority of cells and when the HER-2/neu to chromosome 17 ratio is lower than 2.0. Amplified: When HER-2/neu copy number per centromere 17 is greater than 2. On the basis of the average of HER-2/neu gene copy number, different levels of amplification can be defined as follows: Low level of amplification: When the number of HER-2/neu signals range from 5 to 10. Medium level of amplification: When the number of signals range from 11 to 20. High level of amplification: When there is a consistent presence of signal clusters or > 20 signals/cell. Polysomic: When an equal number of HER-2/neu and centromere 17 signals but greater than two in > 10% of cells is present. Deleted: When HER-2/neu copies are fewer than centromere 17 copies.

IV Breast Carcinoma

272 Aneuploidy of chromosome 17 was excluded as a source of increased HER-2/neu copy number. A positive control slide is included in each run and consists of a cytologic slide known to be amplified for the HER-2/neu gene because it was assayed by FISH in a previous run.

HER-2/neu FISH on Paraffin Sections Formalin-fixed, paraffin-embedded tissue was cut into 4-μm-thick sections that were heated overnight at 56°C. Deparaffinization, pretreatment, enzyme digestion, and fixation of slides were performed using the Vysis Paraffin Pretreatment Kit (Vysis) according to the manufacturer’s recommended protocol. Denaturation and hybridization were carried out in a HYBrite Denaturation/Hybridization System for FISH (Vysis). Ten microliters of HER-2/neu probe mix were applied to the tissue sections that were denaturated at 72°C for 2 min and hybridized overnight at 37°C. The slides were then washed in posthybridization wash buffer at 72°C for 2 min and counterstained with DAPI. For each specimen, at least 100 cells were scored for both HER-2/neu and chromosome 17 signals by image analysis. Images were processed at 1250X magnification using an Olympus MX60 fluorescence microscope with a 100W mercury lamp. Separate narrow band pass filters were used to detect SpectrumOrange, SpectrumGreen, and DAPI. Images were processed with Software Quips (Applied Imaging, Newcastle, UK; Olympus Distributor). Amplification of the HER-2/neu gene was indicated by a ratio of HER-2/neu to chromosome 17 copy number greater than two. For polysomic and deleted cases the same criteria of cytologic samples were applied.

HER-2/neu Immunohistochemistry Sections of archival formalin-fixed, paraffinembedded tissue (5 μm) were placed on slides coated with poly-L-lysine. After deparaffinization and blocking of endogenous peroxidase, HER-2/neu immunostaining was performed using rabbit anti-human c-erbB-2 oncoprotein as primary antibody (Dako, Copenhagen, Denmark) at 1/100 dilution. Binding of the primary antibody was revealed by means of the Dako QuickStaining, Labeled Streptavidin-Biotin System (Dako LSAB), followed by the addition of diaminobenzidine (DAB) as a chromogen. The immunohistochemical expression of HER-2/neu was evaluated in a semiquantitative way. The tumor samples were scored as 3+ when > 10% of the cells showed a specific dark-brown border associated with the cell membrane. Scores of 0, 1+, and 2+ were assigned to negative, weak, or moderate membrane staining, respectively.

RESULTS HER-2/neu on Primary Breast Cancer Amplification of the HER-2/neu gene was evaluated by FISH on 66 primary breast cancer FNAs. Twentythree paired paraffin sections were tested by FISH and 36 by IHC. Figure 38 shows an example of a highly amplified primary breast cancer FNA. HER-2/neu FISH on Cytologic Smears

Forty-eight (73%) of the 66 primary breast carcinomas evaluated for HER-2/neu on FNAs were unamplified and 18 cases (27%) were amplified. In unamplified

Figure 38 Photomicrograph of fine-needle aspirate from primary breast cancer hybridized with a HER-2/neu oncogene probe; multiple red signals in a cluster pattern indicate HER2/neu amplification. Original magnification 1250X.

4 HER-2/neu Amplification Detected by FISH in Cytological Samples from Breast Cancer cases, the most frequent pattern consisted of two red and two green signals; one case (1.5%) was polysomic and seven cases (11%) showed a HER-2/neu deletion. Of the 18 amplified tumors, one, six, and 11 cases showed low, medium, and high levels of amplification, respectively. Highly amplified tumors showed HER2/neu spot numbers ranging from 20 to 100, and often distributed in clusters. Samples with gene amplification showed centromere 17 disomy in nine cases and chromosome 17 polysomy in nine cases, with a number of centromere 17 signals up to 10. With regard to the feasibility of FISH on cytology, hybridization was successful in 80% of the samples pretreated with wash buffer only. Twenty percent of samples were submitted to a further digestion with proteinase K. HER-2/neu FISH on Paraffin Sections

Among 66 breast cancer FNAs, 23 also had HER2/neu FISH evaluated on paired paraffin-embedded sections out of the 66 previously mentioned primary breast carcinomas. One case was not evaluable because of the lack of hybridization. Five of 22 evaluable cases (23%) were amplified and 17 (77%) unamplified. Among the unamplified cases, four were classified as polysomic. Amplified tumors showed a percentage of amplified cells ranging between 50–100%, and often distributed in clusters.

by both FISH and immunohistochemistry on corresponding histologic sections. Among 66 cases examined, matched results from FISH cytology and FISH histology were obtained in 22 cases. Five cases were amplified and 15 unamplified, according to FISH on both cytological and histological samples. Four of five amplified cytological specimens showed a high level and one a medium level of amplification. Two cases, moderately amplified on FISH cytology, were classified as polysomic on FISH histology. Concordance between FISH cytology and histology was 91%. FISH Cytology versus Immunohistochemistry

Paired results according to cytology by FISH and histology by immunohistochemistry were obtained in 36 cases (Table 19). Eight paired cases were both amplified and overexpressed. All were strongly immunostained; four were moderately and four highly amplified. Twenty-five cases were negative for both amplification and overexpression. Three of 36 cases were discordant. Two of them, moderately amplified on FISH cytology, were not overexpressed and were polysomic by FISH histology. The other discordant case, unamplified on FISH cytology, showed a medium level of overexpression. Concordance between FISH cytology and immunohistochemistry was 92%.

HER-2/neu Immunohistochemistry

Immunohistochemistry was performed on 36 paired paraffin-embedded sections out of the 66 primary breast carcinomas. Nine cases (25%) showed overexpression: eight cases had a percentage of intensively stained (3+) tumor cells ranging between 90–100% and one case showed moderate (2+) staining intensity. The remaining 27 cases (75%) showed normal HER-2 protein expression. FISH Cytology versus FISH Histology

Table 19 shows the relationship between HER-2/neu results evaluated by FISH on cytologic specimens and

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HER-2/neu on Metastatic Lesions Table 20 shows the HER-2/neu results obtained by FISH in primary breast tumor and in corresponding distant metastatic lesion. The time period between primary tumor diagnosis and cytologic diagnosis of metastatic lesion ranged from 1 month to 13 years. HER-2/neu was assessed on 22 cytologic samples from metastatic sites: 14 were unstained and eight were MGG destained smears. Seven out of 22 (32%) metastases were amplified. Amplification was observed in 4 of 12 liver metastases, in 1 of 3 ascitic fluids and in 2 of 4 skin metastases. Among amplified cases, one

Table 19 HER-2/neu Results Comparing Fluorescence in situ Hybridization on Cytological Samples With FISH and Immunohistochemistry on Corresponding Histological Sections Histology FISH FISH Cytology

Amplified

Unamplified

Amplified Unamplified Concordance

5 0 20 of 22 (91%)

2 15

IHC Overexpressed 8 1 33 of 36 (92%)

Normal 2 25

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Table 20 HER-2/neu Status Assessed by Fluorescence in situ Hybridization (FISH) on Primary Breast Cancer and on Cytological Samples from Metastatic Sites Case

Primary Tumor

Date Primary/metastasis

Site

HER-2/neu Metastatic Site

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Not evaluable Amplified Amplified Polysomic Amplified* Unamplified Unamplified Unamplified Unamplified* Unamplified Unavailable Unavailable Unavailable Unavailable Not evaluable* Amplified Not evaluable Unamplified Unamplified* Unamplified Unavailable Amplified (low level)

May 1986/June 1998 May 1996/Aug 1998 June 1997/March 2002 March 2000/Aug 2002 Nov 2001/Oct 2002 March 2000/Feb 2001 May 1995/June 2000 March 2000/May 2002 Sept 2002/Oct 2002 April 1989/June 2001 Feb 1994/June 2001 June 1988/Feb 2002 June 1997/April 2002 Nov 1995/July 2002 Feb 1998/Jan 2002 Jan 1993/Dec 2002 Dec 1997/Dec 2002 Sept 1999/Jan 2003 April 1995/May 2001 May 1997/July 2002 June 1998/March 2002 Feb 1993/Nov 2002

Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Pleura Pleura Pleura Peritoneum Peritoneum Peritoneum Skin Skin Skin Skin

Amplified* (low level) Amplified* Amplified* Polysomic Amplified Unamplified* Unamplified Unamplified Unamplified Unamplified Unamplified Unamplified Unamplified Unamplified Unamplified* Amplified Unamplified* Unamplified* Unamplified Polysomic Amplified Amplified (low level)

*FISH on destained cytological smears.

liver and one skin metastasis resulted poorly amplified. In all three pleural fluids, HER-2/neu was unamplified. One out of 12 liver and 1/4 of skin metastases were polysomic. Seventeen out of 22 metastatic cases also had HER2/neu assessed on the primary tumor. In 5 of 22 primary tumors, neither histologic nor cytologic specimens were retrieved because patients had been surgically treated elsewhere. Thirteen out of 17 cases were evaluated by FISH on archival histologic sections; 4 cases had HER-2/neu evaluation on MGG destained cytologic smears obtained at the time of diagnosis. In 3 of 17 (18%) cases, HER-2/neu was not evaluable: one histologic sample because of technical pitfalls and two samples, one cytologic and one histologic, because of lack of hybridization. Among the 14 evaluable primary tumors, five were amplified, eight were unamplified, and one polysomic. Among the five amplified cases, one resulted poorly amplified. Paired FISH results on primary and corresponding metastatic tumor were obtained in 14 cases. All five matched amplified metastatic lesions showed amplification also in their primary tumor. Of the two polysomic metastases, one had the primary tumor polysomic and one unamplified. In the remaining seven cases, no amplification was detected at metastatic site and in the corresponding

primary tumor. Overall, HER-2/neu evaluation on primary and metastatic lesions yielded concordant results in all cases submitted for comparison.

DISCUSSION Overexpression of the HER-2/neu protooncogene is associated with breast cancer progression and poor patient prognosis (Press et al., 1997; Slamon et al., 1997). However, until now the prognostic role of this marker had limited value in clinical decisions, mainly because most studies did not find HER-2/neu to be a prognostic indicator in the case of lymph node-negative patients (Ottestad et al., 1993). On the other hand, HER-2/neu has an increasingly established role in the area of predictive molecular markers. Apparently, a uniform population of breast cancers can be divided, as far as their sensitivity to medical treatments is concerned, into subgroups based on their likely or unlikely response to given treatments. Patients that overexpress HER-2/neu were repeatedly reported to be less responsive to the cyclophosphamide, methotrexate, fluorouracil (CMF)-containing adjuvant chemotherapy regimens (Miles et al., 1999), although with a few contrasting results (Menard et al., 2001), but they might preferentially benefit from the anthracycline-based

4 HER-2/neu Amplification Detected by FISH in Cytological Samples from Breast Cancer adjuvant chemotherapy regimens (Thor et al., 1998). As far as endocrine therapies are concerned, only a limited proportion of HER-2/neu positive breast cancers are estrogen receptor positive. In these patients, the efficacy of adjuvant tamoxifen seems to be adversely influenced, according to some authors (Carlomagno et al., 1996), whereas response to oophorectomy plus tamoxifen adjuvant therapy in premenopausal women may be favored (Love et al., 2003). Considering the neoadjuvant endocrine therapy model, treatment with tamoxifen in a randomized clinical trial was confirmed as unfavorably influenced by HER-2/neu positivity, whereas treatment with the new aromatase inhibitor letrozole was reported as more likely associated with objective response (Ellis et al., 2001). The meaning of the predictive value of HER-2/neu molecular marker was greatly enhanced by the availability of trastuzumab, a high-affinity humanized antiHER-2/neu antibody. The specific efficacy of this agent was confirmed in a few pivotal trials in metastatic disease in HER-2/neu-overexpressing patients. Administered as single agent in patients pretreated with 1–2 lines of chemotherapy, a limited but definite proportion of them responded to treatment (Cobleigh et al., 1999). Administered as a single agent in nonpretreated patients, more than one-third showed an objective response (Vogel et al., 2002). Addition of this agent to chemotherapy increased response rates, time to disease-progression, and survival duration (Slamon et al., 2001). Difficulties in interpreting and comparing data from different series aimed at assessing the prognostic and predictive value of HER-2/neu amplification/overexpression in clinical specimens might be due to the variety of reagents and techniques that had been employed. The availability of a specific treatment such as trastuzumab increases the need to select patients that will get the maximum benefit from this treatment and avoid any undue toxicity either in metastatic disease and, possibly in the future, in the adjuvant setting. In fact, although generally well-tolerated, trastuzumab-based therapy is correlated with a significant risk of cardiotoxicity. In any case, in the next years there is likely to be an expanding role of HER2/neu assessment, either in the selection of type of chemotherapy (anthracycine or not), or in the selection of patients for trastuzumab treatment. The possibility of determining HER-2/neu status not only from histological samples but also on cytological specimens may help clinicians in treatment decisions. It has been reported that HER-2/neu positivity of primary tumor incompletely predicts the response to trastuzumab, possibly because of a change of HER2/neu status during metastatic progression. It has been

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previously demonstrated that recurrent breast cancer is actually an heterogeneous disease where a biologically dominant clone could eventually overcome the others and evolve independently in both primary cancer and metastases (Kuukasjarvi et al., 1997). In this light, it would be worth ascertaining the concordance of HER2/neu status between primary and metastatic lesion. Nevertheless, few reports concern HER-2/neu assessment on distant metastatic lesions, whereas the majority compares HER-2/neu status in primary tumor and in regional concurrent lymph node metastases. However, concurrent regional lymph node metastases should not be assumed to be equivalent to distant ones; there is actually a genetic heterogeneity that underlies the development of a distant lesion, years after the primary cancer. Moreover, cells that metastatize via lymphatics could display different biological properties from those traveling to distant sites, due to the invasion by blood vessels. Up to now, few studies have evaluated the feasibility of detecting HER-2/neu amplification by FISH in breast cancer FNAs (McManus et al., 1999). Results obtained by FISH on FNAs from surgical samples or on FNAs from breast cancer patients have been correlated with results obtained by immunohistochemistry on the corresponding frozen sections or cytological smears. To our knowledge, only one report has correlated the detection of HER-2/neu amplification by FISH both on breast cancer FNAs and on the corresponding frozen section (Klijanienko et al., 1999). In one study (Bozzetti et al., 2002), we evaluated HER-2/neu by FISH on breast cancer FNAs and by both FISH and IHC on the corresponding paraffin sections. A good concordance was obtained between FISH cytology and FISH histology (91%) as well as between FISH cytology and IHC (92%). In our opinion, the discrepancies between FISH cytology and FISH histology could mainly be ascribed to the fact that FISH spots on cytological preparation are more easily visualized than those on the corresponding tissue section, as previously reported by Klijanienko et al. (1999). The presence of monolayered and isolated cells allows for a more accurate signal enumeration on FNAs than on histological sections where fluorescence signal clusters are often prevalent. For this reason, although cytological samples are classified by visual evaluation only, the use of image analysis is recommended for histological samples. On the other hand, difficulties in detecting and counting the signals on cytological samples that showed considerable thickness and overlapping cells could be overcome by proteinase K pretreatment. The FISH method on FNAs allows for the visualization of HER-2/neu on a cell-bycell basis. Counterstaining with DAPI permits the

276 recognition of some nuclear details for the differentiation between epithelial cells and host elements. The application of dual-color FISH technique, using a probe for the HER-2/neu gene together with a probe for a pericentromeric region of chromosome 17, has the advantage of providing an accurate evaluation of gene copy number alterations, allowing one to distinguish between tumors with normal HER-2/neu gene content, tumors with a gain of only a few extra copies of the HER-2/neu gene, tumors highly amplified, and tumors with a HER-2/neu deletion. At present, few studies have compared HER-2/neu status in primary breast cancer with paired distant metastases. In a preliminary report by Edgerton et al. (2000), overall 25% discordance was found in HER2/neu status between 193 primary tumors and 68 paired local recurrence, 32 lymph nodal, and 93 distant metastases. Nevertheless, a note of caution should be applied due to the lack of further details regarding this work. On the contrary, other studies based on FISH or IHC assessment, and carried out on small series of patients, have reported a stable, although not always complete, HER-2/neu status congruence between primary and distant metastases (Tanner et al., 2001). A wider series, recently published by Gancberg et al. (2002), found 94% and 93% of concordance between paired primary tumors and distant metastatic lesions when analyzed by IHC or FISH, respectively. Actually, our data suggest that HER-2/neu status is mostly stable in primary breast cancer and in the corresponding distant metastatic sites. In our analysis, one case was found polysomic on skin metastasis and unamplified on the paired primary histologic sample. As discussed by Wang et al. (2002), in primary breast cancer, aneusomy 17 is a common feature occurring in absence of HER-2/neu amplification; even high polysomy 17 is not sufficient to produce a significant increase in gene transcription, eventually leading to HER-2/neu protein overexpression. Interestingly, a low-level gain of only a few extra copies of HER-2/neu gene was found in one case, both in primary and in the metastatic lesion. This finding must be distinguished from extra gene copies due to the formation of sister chromatids in S- or G2-phase cells, mostly arranged in pairs. Criteria for a low-level gene copy number increase are the presence of extra signals in a major subpopulation and their random distribution in the nuclei. The biologic and clinical significance of a gain of a few gene copies is still unclear. In our opinion, testing HER-2/neu status exclusively on primary breast cancer specimen could be a safe policy for using trastuzumab in metastatic disease, especially when results are obtained by FISH methodology. Reasons other than a possible change in HER-2/neu

IV Breast Carcinoma status could be advocated to explain the lack of response to trastuzumab therapy. Many methodological limitations may interfere with HER-2/neu assessment either by IHC or FISH in primary breast cancer, particularly when dealing with archival samples collected years before. Previous studies have pointed out that, in terms of feasibility and accuracy, FISH provides a tempting alternative to HER-2/neu evaluation by IHC, whose specificity and sensitivity problems have been described (Press et al., 1994). On the other hand, hybridization may be eventually compromised by Bouin fixation in archival histological samples subsequently assessed by FISH; this may account for the fact that, in the study of Gancberg et al. (2002), a high rate of samples was not evaluable by FISH. Similarly, in our experience on HER-2/neu evaluation on distant metastases, among archival series, two of the 13 histological samples and one of the 12 cytological destained smears were not assessable by FISH. To our knowledge, none of the studies that evaluated HER-2/neu status on metastases from breast cancer patients have been carried out on cytological material. In our experience, FNAB, coupled with ultrasound methodology for hepatic lesions, is a relatively easy and safe method to obtain cellular material for FISH analysis, which has been proved to circumvent many of the shortcomings due to molecular and immunocyto/histochemical techniques both on frozen tissue sections and fresh aspirates (Nizzoli et al., 2003; Pauletti et al., 2000). The FISH technique on cytological specimens, including smears, FNAs, and imprint preparations, allows for a rapid and reproducible quantitative analysis of DNA alterations in single cells (Wolman, 1997), thus avoiding many of the drawbacks of tissue sections. We suggest that FNAB, performed on a suspicious breast cancer metastatic lesion, may thus provide fresh cytological material for an updated characterization of relevant predictive factors and a real-time HER-2/neu assessment for trastuzumabbased therapy. Furthermore, retesting of HER-2/neu status on metastatic lesions may be worthwhile when a negative score is obtained by IHC performed on the primary tumor sample resected many years before. In conclusion, our results underline the feasibility and advantages of two reliable, rapid, and informative techniques, such as FNAB and FISH. The FISH technique is suitable on unstained smears for patient candidates for preoperative chemotherapy, as well as on smears already stained for patients whose paraffin blocks of primary tumor are not available. Moreover, FISH, performed on cytologic smears obtained by FNAB from metastatic sites, as well as on cytologic preparation from pleural and ascitic fluids, allows not only a better definition of HER-2/neu status but also

4 HER-2/neu Amplification Detected by FISH in Cytological Samples from Breast Cancer aids in subsequent treatment decisions. Since the advent of trastuzumab, the characterization of the molecular profile in metastatic disease has been becoming increasingly important for targeted therapies selection (see also Part IV, Chapters 3 and 5).

Acknowledgment I am grateful to Dr. Giorgio Cocconi for his expert review of this manuscript.

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