Japanese Journal of Clinical Oncology 33:463-469 (2003)
© 2003 Foundation for Promotion of Cancer Research
Multiple (Multicentric and Multifocal) Cancers in the Ipsilateral Breast with Different Histologies: Profiles of Chromosomal Numerical Abnormality
1 Department of Pathology and Surgery, Hamamatsu University School of Medicine and 2 Saiseikai Hospital, Hamamatsu, Shizuoka, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: Chromosomal numerical abnormality (CNA) is a characteristic of breast cancer as with other common solid cancers. Multiple mammary carcinomas are also sometimes encountered in the ipsilateral breast. Deciding whether they are multicentric (arising independently) or multifocal (of the same origin) has been a continued challenge to clinicians and pathologists.
Methods: We experienced two cases of macroscopically distinct double carcinomas in the ipsilateral breast, in which two cancers had a different histopathological morphology. Centromere probes identifying 17 different and specific centromeres were used to obtain the profile of CNA for each tumor, with a microwave-assisted FISH protocol. For comparison, a case of three lesions, two of which had the same and the other different histology was also studied.
Results: Contrary to our expectations, in spite of the particular difference in the histopathological picture of these tumors, the CNA profile was the same in one case and different in the other. The gain of chromosome 1 and loss of chromosome 15 are representative features shared by multifocal cases. On the other hand, the multiple cancers with the same histology had mostly the same CNA profile.
Conclusions: CNA profiling is useful to identify the lineage of multiple breast cancers. As shown here, regardless of the histological features, a certain common CNA profile can define the same origin of some multiple cancers.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synchronous multiple mammary carcinomas, especially when occurring in an ipsilateral breast, is still one of the challenges for both pathologists and clinicians in terms of identification of the origins and therapeutic management of the cancer (1,2). There are several ways to determine whether multiple tumors have arisen independently (multicentric) or originated from the primary lesion (multifocal) by, for example, the loss of heterozygosity profile (3–7). A general view over the chromosomal alterations is also a possible way to obtain information on cancer cell lineage. Karyotypic analysis based on short-term culture has been frequently applied to the analysis of bilateral breast cancer and multiple lesions in the same breast and has been shown to be able to successfully demonstrate the related karyotypes in multiple lesions (8). However, information from short-term culture and chromosomal karyotyping analysis is still not particularly accessible for ordinary pathology laboratories because of the associated technical problems, and thus development of molecular techniques is awaited (9–11). We recently developed a new FISH protocol to enable us to detect the chromosomal numerical abnormality (CNA) in the interphase nuclei of paraffin-embedded pathology archives (12) and successfully extended this technique to multiple early gastric cancers (13). In this report, two cases of apparently multiple mammary carcinoma, in which two lesions, located separately and appearing quite different microscopically, were analyzed by interphase FISH to disclose the CNA profile of each lesion.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Case 1
The formalin-fixed, paraffin-embedded specimens of surgically resected multiple breast cancers from the left breast of an 80-year-old woman were used for the following study. Multiple lesions had been revealed by computer-assisted tomography (CT) in 1998. Gross examination revealed two lesions which were located separately; no lesions were detected between them even after scrutinizing the pathology extremely carefully (Fig. 1a). The microscopic pathological features were discernibly different: one lesion was identified as the mucinous type (Ca#1), and the other lesion was identified as the scirrhous (invasive ductal) type (Ca#2), as shown in Fig. 2a and b, respectively.
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Case 2
Two lesions were detected in the C region of the right breast of a 45-year-old female (Fig. 1b). Histologically, one lesion, 1.0 cm in diameter, was the solid-tubular type of invasive ductal carcinoma and the other, 1.2 cm in diameter, had scirrhous features, as shown in Fig. 2c and d, respectively. As summarized in Table 1, both lesions were <2.5 cm in diameter and regional lymph node involvement was not detected. Histological classification was documented according to the Japanese criteria of breast cancer pathology.
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Comparative case
For comparison, we performed an additional survey on a third case, Case 3, in which multiple lesions shared histology of similar appearance. Two lesions were detected in the right breast of a 72-year-old female. One lesion, 2.2 cm in diameter, was in locus C and was immunohistologically ER(++), PgR(+/–) and HER-2/neu(–). The other was in locus D, 1.0 cm in size, ER(++), PgR(+++) and HER-2/neu(–). Both of these lesions had papillotubular carcinoma with scirrhous infiltration. Lymph node involvement was not detected (not included in Table 1).
DNA Probes
A panel of 18 centromeric 
-satellite DNA probes (D1Z5, D2Z, D3Z1, D4Z1, D6Z1, D7Z1, D8Z2, D9Z1, D10Z1, D11Z1, D12Z3, D15Z1, D16Z1, D17Z1, D18Z1, D20Z1, DXZ1 and DYZ3) derived from chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 20 and X, respectively, were purchased from Vysis Inc (Downers Grove, IL). All probes were labeled with either Cy3 (orange) or FITC (green).
Modified FISH Analysis
The generalized procedure for FISH analysis consisted of sample preparation by denaturation, hybridization, rinsing, counterstaining and visualization according to the manufacturer’s instructions. We also employed a new FISH protocol, which we have recently developed and shown to be successful in identifying the sensitive and specific signals in pathology archives (12,13). This protocol includes a microwave treatment stage for better penetration of the probes. Briefly, in the sample preparation period the slides were subjected to microwave treatment and pepsin digestion. In the hybridization period, the slides were subjected to intermittent microwave treatment. In most of the experiments, two differently labeled probes were hybridized, counted and detected simultaneously.
Counting and Statistical Analysis
We observed the slides immediately through a fluorescence microscope equipped with epifluorescence filters and performed the evaluation of interphase nuclei. Photographs were taken from a CCD camera (Quantix 1400, Olympus Ltd, Tokyo, Japan). Every captured image was incorporated and stored in the image analysis program CytoVision (Applied Imaging, Tokyo, Japan). For each probe and each of the tumor areas, 100–200 intact and non-overlapping nuclei were counted, and the number of signals per nucleus was scored. We used the two indices, modal number (MN), defined as the most prevalent number of the chromosome in tumor cells, and variant fraction (VF), which is the percentage of tumor cells whose chromosome number differs from the mode (13–15), as is the case with the evaluation criterion of the CNA. Briefly, when 20% or more cancer cells had abnormal numbers of respective chromosomes, we defined gain or loss of that chromosome. Stereographs of the CNA profiles were depicted using Quick graph (developed by Tateo Fukui, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We obtained the CNA profile of four lesions with different, and two lesions with the same, histological features.
As shown in Table 2, there were common features in the CNA profile in Case 1. The results for the CNA expressed as MN and VF in each of the 16 autochromosomes and two sex chromosomes of the tumors are summarized in Table 2. Gains were noticed in the two tumors (Ca#1 and Ca#2), and there were no losses except in chromosome 15. Gains of chromosome 1, 2, 3, 6, 7, 8, 11, 17 and X were demonstrated in both lesions. Gains of chromosome 9, 12 and 16 were detected only in the specimens of the scirrhous type. A gain of chromosome 18 was detected only in the specimens of the mucinous type. A significant loss of chromosome 15 was observed in both lesions. The patterns of the other chromosomes including chromosome 4, 10 and 20 were confirmed as normal in both lesions. The common changes involving CEP 17, CEP 3, CEP 15 and CEP 18 are shown in Fig. 3a and d. In conclusion, in total 13 (72%) of the tested 17 CEP probes were concordant and 10 out of 11 altered chromosomes in each lesion were the same. Thus, we consider these two lesions to be related clonally. Fig. 4a and b shows the chromosome alterations in stereoscopic graphs in order to represent the similarity of these two lesions in a more obvious way.
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On the other hand, the two carcinomas in Case 2 had different CNA profiles (Table 3). The shared gains in the two lesions were only in chromosome 8. Only nine loci (50%) of 18 tested CEP probes were consistent between the two lesions and only one chromosomal change was concordant among the nine chromosomes altered in each lesion. Different profiles of CEP 1 and CEP 7 are shown in the two lesions in Fig. 3c and d. The stereographic graphs in Fig. 4c and d highlight the difference in profiles of these two lesions compared with those in Case 1 (Fig. 4a and b). In Case 2, we considered them to be different clones and it would be reasonable to think that they were of independent origin.
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In Case 3, the two carcinomas had the same histopathological morphology and we detected gains of chromosomes 3, 8, 9, 17 and 20 in one lesion (Fig. 4e) and chromosome 3, 8, 9, 17 and X in the other lesion (Fig. 4f). We considered the CNA profiles of these lesions to be mostly the same in this case.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent applications of allelotype analysis (3,16–18) to common cancers have made it feasible to identify clones of multicentric tumors, but the markers claimed as evidence for their identity are sometimes problematic. Middleton et al. (1) claimed that most of the apparently multicentric tumors are from the same clone based on the histological features and the local invasiveness. On the other hand, Tsuda and Hirohashi (19) reported that concordance in the pattern of the loss of heterozygosity indicated the monocentric origin in some cases. Teixeira et al., carefully making karyotypic analyses, concluded that three cases of different karyotypes were from multicentric loci (9). Previous procedures often had the problem that they could deal only with somewhat mature tumors. They also required DNA analyses dissected from the lesions, which often resulted in difficulties of qualification. FISH techniques are more feasible because they can show the changes in situ compared with karyotype analysis, but several problems have persisted. For formalin-fixed, paraffin-embedded samples, the naive application of this technique was not successful in generating clear signals for the DNA. Although a commercially-available paraffin pretreatment kit (Vysis) has been developed to raise the contrast, there is still a limitation with this modification, especially for specimens kept in fixation for longer than 28 h (data not shown). We applied the microwave-assisted FISH technique (Kitayama) for gastric cancer in previous work (12–15), and for breast cancer for the first time in this report. In both multiple breast cancer cases, gains of chromosome 1, 2, 3, 6, 7, 8, 11, 17 and X were observed (Table 2). These changes, at least in some part, are consistent with other reports about chromosomal abnormalities of breast cancers (20).
In our analysis of the four lesions, a minimal change was found in Ca#2 in Case 2, that is, a gain of chromosome 8 was the only alteration. Recently, Rummukainen et al. (21) used CGH, FISH with arm- and locus-specific probes and the spectral karyotyping (SKY) method and confirmed that chromosome 8q was structurally abnormal in breast cancer cell lines (13 of 16 cell lines). Thus, involvement of chromosome 8 would be the most consistent change in human breast cancer. Takami et al. (22) had investigated chromosomes 1, 2, 6, 7, 10, 11, 17 and 18 with the FISH method using chromosome-specific DNA probes. They claimed that the instability profile of the chromosome number in breast cancer showed a significant correlation with lymph node metastases. Our cases had extended CNA but had no lymph node involvement, and thus it seems that marked abnormality in the chromosome numbers of breast cancer occurs before its spread to the lymph nodes.
What are the lessons to be learned from the CNA profiles in multiple lesions in the ipsilateral breast? We believe the most important point is that the clonality, which has been delineated from the information of chromosomal abnormality, sometimes cannot be predicted from the histological features including tubular structure, mucin production or desmoplastic reactions. From the data provided by the CNA profiling, it would be convincing to reason that the two lesions in Case 1 are of the same clonal origin and those in Case 2 are not. We cannot of course exclude the possibility that a drastic clonal progression occurred during the course in Case 2. In any event, however, our report is the first to provide valid procedures to delineate multiple cancer lesions in the ipsilateral breast, validating the use of the microwave-assisted FISH protocol in the diagnostic pathology laboratory.
To further validate our observation and interpretation of the histological pictures and CNA profiles in the present Cases 1 and 2, an additional case of double ipsilateral breast cancer with the lesions having the same histological picture was investigated. As expected, the morphologically identical phenotype had the common CNA profile, though there may be an exception in extending this study in future. At the moment we think that CNA profiling is particularly useful when the histological picture varies among multiple lesions in ipsilateral breast.
This method (modified FISH analysis protocol) is very useful for analysis of chromosomal numerical change of solid tumors with various pathological features, including multicentric, heterogeneous and recurrent. We have already succeeded in applying this to gastric cancer series (23). Reports on further cases will be awaited.
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ACKNOWLEDGEMENTS |
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This work was supported by, in part, Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, the Ministry of Health, Labor and Welfare, of Japan, and the Foundation for Promotion of Cancer Research, and the Smoking Research Foundation.
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FOOTNOTES |
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+ For reprints and all correspondence: Haruhiko Sugimura, 1-20-1, Handayama, Hamamatsu 431-3192, Japan. E-mail: hsugimur{at}hama-med.ac.jp
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Received May 23, 2003; accepted August 21, 2003
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