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Primary or Secondary Malignancy? Fingerprint Evidence
Clinical Oncology (2003) 15: 408–411 doi:10.1016/S0936-6555(03)00062-1
Case Report Primary or Secondary Malignancy? Fingerprint Evidence W. Alazawi*, M. Gonzalez*,†, J. Abraham†, M. Arends‡, N. Coleman*,‡, C. Wilson† *MRC Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge, UK; †Department of Oncology, Addenbrookes Hospital, Hills Road,Cambridge, UK; ‡Department of Histopathology, Addenbrookes Hospital, Hills Road,Cambridge, UK Received: 7 August 2002
Revised: 9 January 2003
Introduction
It can be difficult to determine whether malignant neoplasms in certain anatomical locations represent primary or secondary lesions. Although morphological and immunophenotyping approaches can be used, relatively few discriminatory markers there are for common malignancies like adenocarcinomas. Genetic profiling may therefore be more effective in the differential diagnosis of such tumours. The tests that are likely to be of greatest value are those that detect changes across the entire genome, thereby generating a ‘fingerprint’ of abnormalities. One such test is comparative genomic hybridisation (CGH), a molecular cytogenetic technique that enables amplified or deleted chromosomal regions in tumours to be detected and mapped most commonly using metaphase chromosomes. Recent technological advances have enabled CGH to be applied to formalin-fixed, paraffin-embedded tissue, permitting the technique to be used in a number of clinical areas where retrospective tissue sampling has been performed. Here we report a case where a lung adenocarcinoma was detected in a man with a past history of rectal adenocarcinoma. It was not possible on clinical grounds to distinguish between primary and secondary lung neoplasia, but the demonstration of a CGH fingerprint informed the selection of a treatment strategy.
Case Report
A 77-year-old retired jockey presented acutely with worsening haemoptysis. Five years previously, he had been diagnosed with a Dukes A rectal carcinoma (pT2, pN0, pM0) that was treated with standard preoperative radiotherapy and low anterior resection with covering ileostomy. Clinical suspicion of left lower-lobe collapse Author for correspondence: William Alazawi, MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, U.K. Tel: +44 1223 763279; Fax: +44 1223 763284; E-mail: [email protected] 0936-6555/03/70408+4 $30.00/0
Accepted: 3 February 2003
was confirmed on his admission, and chest radiograph and fibre-optic bronchoscopy revealed a mass partially occluding the left main bronchus. A biopsy of the mass showed adenocarcinoma. Suboptimal pulmonary function tests (FEV1=0.9, FVC=2.1) deemed the lesion inoperable. Computed tomography raised the possibility that the pulmonary lesion could be a metastasis from the previous rectal adenocarcinoma, rather than representing a primary tumour, even though there was no radiological evidence of dissemination to the liver. The patient subsequently developed localised chest pain for which he received a single fraction of palliative radiotherapy. Despite substantial symptomatic relief, he developed further haemoptysis. A decision as to which chemotherapy regimen to instigate for further palliation was required. Standard cytotoxic regimens for primary lung adenocarcinomas are platinum-based, whereas metastatic colorectal malignancies respond to the less toxic 5-fluorouracil-based regimens. The clinical presentation was consistent with a primary lung adenocarcinoma whereas radiology could not rule out metastatic colorectal adenocarcinoma. Although metastases from colorectal cancer are usually associated with liver metastases, in 2–4% of patients with colorectal malignancy, pulmonary metastases do occur in isolation [1]. Rectal primaries, as in this patient, are more frequently associated with isolated pulmonary metastases than colonic primaries. The systemic venous drainage of the rectum via the middle and inferior rectal veins (which bypass the portal system) probably accounts for this. Only archival formalin-fixed, paraffinembedded tissue was available to resolve the dilemma. Recent development of immunohistochemical investigations using antibodies to different cytokeratins can (in certain, limited circumstances) help to distinguish the likely origin of a tumour. In this case, histological and immunohistochemical assessment was inconclusive, as both the pulmonary and rectal lesions were welldifferentiated adenocarcinomas and analysis of p53, bcl-2, MSH-2 and cytokeratin 7 expression revealed .
FINGERPRINTING EVIDENCE TO DETERMINE PRIMARY OR SECONDARY MALIGNANCIES
409
Fig. 1 – Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue from rectal tumour (a and c) and from lung tumour (b and d). Neither cytokeratin 7 expression (a and b) nor p53 expression (c and d) were significantly different between the two tissue samples.
no significant differences (Fig. 1). Cytokeratin 20 was expressed in over 75% of cells in the rectal tumour but absent in the pulmonary lesion, but this was not conclusive evidence that the lung mass was primary. CGH was therefore performed using DNA extracted from the rectal and lung tumours [2]. We reasoned that if the lung lesion was a metastasis, then it should share chromosomal copy number imbalances (CNIs) with the rectal tumour from which it arose. Conversely, a new primary pulmonary adenocarcinoma would appear cytogenetically distinct from the rectal lesion and would display a fingerprint of CNIs characteristic of lung adenocarcinomas. There were very few CNIs in the rectal lesion, in keeping with the findings for early-stage colorectal cancers [3]. There were many more CNIs in the lung tumour, mostly affecting different cytogenetic loci to those involved in the rectal tumour (Fig. 2). Furthermore, the lung tumour displayed CNIs typical of the ‘fingerprint’ of pulmonary adenocarcinomas, in particular the losses on 3p and 4q and the gains on 5p and 8q [4]. The cytogenetic evidence therefore suggests that these tumours were of different origins. These data were added to the clinical and radiological evidence
and the patient was given treatment for primary lung adenocarcinoma.
Methods
Comparative genomic hybridisation was performed on the paraffin-embedded archival material. Briefly, DNA from each of the tumours and DNA from normal peripheral blood lymphocytes were amplified and differentially labelled using degenerate oligonucleotideprimed polymerase chain reaction (PCR). Each labelled tumour DNA sample was then co-hybridised with labelled normal DNA to normal male metaphase chromosomes on a glass slide. Reference DNA was labelled with biotin and detected with avidin-Cy3 (a red fluorochrome, Fig. 2 (a1) and tumour DNA labelled with digoxigenin and detected with FITC (a green fluorochrome, Fig. 2 (a2). The green : red fluorescence intensity ratio along the length of each chromosome was calculated using QUIPS analysis software (Vysis, Downer’s Grove, IL, U.S.A.) and regions of DNA gain and loss in the tumour identified as those displaying increased green or red fluorescence intensity, respectively.
410
CLINICAL ONCOLOGY
Fig. 2 – Normal comparative genomic hybridisation target metaphase showing red fluorescence from normal reference DNA (a1), green fluorescence from lung tumour DNA (a2) and composite image from both, together with a blue counterstain (a3). Green : red fluorescence intensity ratios are calculated along the length of each chromosome from 10 metaphases and data presented in summary karyograms showing copy number imbalances from the rectal lesion (b) (six aberrant arms) and from the lung lesion (c) (14 aberrant arms). Bars in red to the left of each ideogram represent loss, bars in green to the right indicate gain. Boxed chromosomes are characteristically abnormal in primary lung adenocarcinomas [4], compared with rectal adenocarcinomas [3]. Discussion
Determining whether a malignant tumour is primary or secondary frequently has implications for clinical man-
agement. In this case study, we have applied a genomewide molecular cytogenetic ‘fingerprinting’ technique to identify the most probable site of origin of a new solid tumour, according to the chromosomal aberrations it
FINGERPRINTING EVIDENCE TO DETERMINE PRIMARY OR SECONDARY MALIGNANCIES
contains. CGH has been used widely as a research tool and both the number of CNI per case, and the presence of particular abnormalities has been associated with different prognoses [5]. CGH has been used to improve differential diagnosis of germ cell tumours [6], however we believe the case reported here to be the first in which a CGH fingerprint has been used in a clinical setting to assist in directing management decisions. The method was performed on standard formalin-fixed, paraffinembedded tissue of the type that is processed and stored in all histopathology departments. This case study illustrates the potential role of techniques such as CGH in resolving an important and common oncological dilemma. References 1 Turk PS, Wanebo HJ. Results of surgical treatment of nonhepatic recurrence of colorectal carcinoma. Cancer 1993;71:4267.
411
2 Zitzelsberger H, Lehmann L, Werner M, Bauchinger M. Comparative genomic hybridisation for the analysis of chromosomal imbalances in solid tumours and haematological malignancies. Histochem Cell Biol 1997;108:403. 3 De Angelis PM, Clausen OP, Schjolberg A, Stokke T. Chromosomal gains and losses in primary colorectal carcinomas detected by CGH and their associations with tumour DNA ploidy, genotypes and phenotypes. Br J Cancer 1999;80:526. 4 Goeze A, Schluns K, Wolf G, Thasler Z, Petersen S, Petersen I. Chromosomal imbalances of primary and metastatic lung adenocarcinomas. J Pathol 2002;196:8. 5 Kusano N, Okita K, Shirahashi H, et al. Chromosomal imbalances detected by comparative genomic hybridization are associated with outcome of patients with hepatocellular carcinoma. Cancer 2002;94:746. 6 Summersgill B, Goker H, Osin P, et al. Establishing germ cell origin of undifferentiated tumors by identifying gain of 12p material using comparative genomic hybridization analysis of paraffin-embedded samples. Diagn Mol Pathol 1998;7:260.
Case Report Primary or Secondary Malignancy? Fingerprint Evidence W. Alazawi*, M. Gonzalez*,†, J. Abraham†, M. Arends‡, N. Coleman*,‡, C. Wilson† *MRC Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge, UK; †Department of Oncology, Addenbrookes Hospital, Hills Road,Cambridge, UK; ‡Department of Histopathology, Addenbrookes Hospital, Hills Road,Cambridge, UK Received: 7 August 2002
Revised: 9 January 2003
Introduction
It can be difficult to determine whether malignant neoplasms in certain anatomical locations represent primary or secondary lesions. Although morphological and immunophenotyping approaches can be used, relatively few discriminatory markers there are for common malignancies like adenocarcinomas. Genetic profiling may therefore be more effective in the differential diagnosis of such tumours. The tests that are likely to be of greatest value are those that detect changes across the entire genome, thereby generating a ‘fingerprint’ of abnormalities. One such test is comparative genomic hybridisation (CGH), a molecular cytogenetic technique that enables amplified or deleted chromosomal regions in tumours to be detected and mapped most commonly using metaphase chromosomes. Recent technological advances have enabled CGH to be applied to formalin-fixed, paraffin-embedded tissue, permitting the technique to be used in a number of clinical areas where retrospective tissue sampling has been performed. Here we report a case where a lung adenocarcinoma was detected in a man with a past history of rectal adenocarcinoma. It was not possible on clinical grounds to distinguish between primary and secondary lung neoplasia, but the demonstration of a CGH fingerprint informed the selection of a treatment strategy.
Case Report
A 77-year-old retired jockey presented acutely with worsening haemoptysis. Five years previously, he had been diagnosed with a Dukes A rectal carcinoma (pT2, pN0, pM0) that was treated with standard preoperative radiotherapy and low anterior resection with covering ileostomy. Clinical suspicion of left lower-lobe collapse Author for correspondence: William Alazawi, MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, U.K. Tel: +44 1223 763279; Fax: +44 1223 763284; E-mail: [email protected] 0936-6555/03/70408+4 $30.00/0
Accepted: 3 February 2003
was confirmed on his admission, and chest radiograph and fibre-optic bronchoscopy revealed a mass partially occluding the left main bronchus. A biopsy of the mass showed adenocarcinoma. Suboptimal pulmonary function tests (FEV1=0.9, FVC=2.1) deemed the lesion inoperable. Computed tomography raised the possibility that the pulmonary lesion could be a metastasis from the previous rectal adenocarcinoma, rather than representing a primary tumour, even though there was no radiological evidence of dissemination to the liver. The patient subsequently developed localised chest pain for which he received a single fraction of palliative radiotherapy. Despite substantial symptomatic relief, he developed further haemoptysis. A decision as to which chemotherapy regimen to instigate for further palliation was required. Standard cytotoxic regimens for primary lung adenocarcinomas are platinum-based, whereas metastatic colorectal malignancies respond to the less toxic 5-fluorouracil-based regimens. The clinical presentation was consistent with a primary lung adenocarcinoma whereas radiology could not rule out metastatic colorectal adenocarcinoma. Although metastases from colorectal cancer are usually associated with liver metastases, in 2–4% of patients with colorectal malignancy, pulmonary metastases do occur in isolation [1]. Rectal primaries, as in this patient, are more frequently associated with isolated pulmonary metastases than colonic primaries. The systemic venous drainage of the rectum via the middle and inferior rectal veins (which bypass the portal system) probably accounts for this. Only archival formalin-fixed, paraffinembedded tissue was available to resolve the dilemma. Recent development of immunohistochemical investigations using antibodies to different cytokeratins can (in certain, limited circumstances) help to distinguish the likely origin of a tumour. In this case, histological and immunohistochemical assessment was inconclusive, as both the pulmonary and rectal lesions were welldifferentiated adenocarcinomas and analysis of p53, bcl-2, MSH-2 and cytokeratin 7 expression revealed .
FINGERPRINTING EVIDENCE TO DETERMINE PRIMARY OR SECONDARY MALIGNANCIES
409
Fig. 1 – Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue from rectal tumour (a and c) and from lung tumour (b and d). Neither cytokeratin 7 expression (a and b) nor p53 expression (c and d) were significantly different between the two tissue samples.
no significant differences (Fig. 1). Cytokeratin 20 was expressed in over 75% of cells in the rectal tumour but absent in the pulmonary lesion, but this was not conclusive evidence that the lung mass was primary. CGH was therefore performed using DNA extracted from the rectal and lung tumours [2]. We reasoned that if the lung lesion was a metastasis, then it should share chromosomal copy number imbalances (CNIs) with the rectal tumour from which it arose. Conversely, a new primary pulmonary adenocarcinoma would appear cytogenetically distinct from the rectal lesion and would display a fingerprint of CNIs characteristic of lung adenocarcinomas. There were very few CNIs in the rectal lesion, in keeping with the findings for early-stage colorectal cancers [3]. There were many more CNIs in the lung tumour, mostly affecting different cytogenetic loci to those involved in the rectal tumour (Fig. 2). Furthermore, the lung tumour displayed CNIs typical of the ‘fingerprint’ of pulmonary adenocarcinomas, in particular the losses on 3p and 4q and the gains on 5p and 8q [4]. The cytogenetic evidence therefore suggests that these tumours were of different origins. These data were added to the clinical and radiological evidence
and the patient was given treatment for primary lung adenocarcinoma.
Methods
Comparative genomic hybridisation was performed on the paraffin-embedded archival material. Briefly, DNA from each of the tumours and DNA from normal peripheral blood lymphocytes were amplified and differentially labelled using degenerate oligonucleotideprimed polymerase chain reaction (PCR). Each labelled tumour DNA sample was then co-hybridised with labelled normal DNA to normal male metaphase chromosomes on a glass slide. Reference DNA was labelled with biotin and detected with avidin-Cy3 (a red fluorochrome, Fig. 2 (a1) and tumour DNA labelled with digoxigenin and detected with FITC (a green fluorochrome, Fig. 2 (a2). The green : red fluorescence intensity ratio along the length of each chromosome was calculated using QUIPS analysis software (Vysis, Downer’s Grove, IL, U.S.A.) and regions of DNA gain and loss in the tumour identified as those displaying increased green or red fluorescence intensity, respectively.
410
CLINICAL ONCOLOGY
Fig. 2 – Normal comparative genomic hybridisation target metaphase showing red fluorescence from normal reference DNA (a1), green fluorescence from lung tumour DNA (a2) and composite image from both, together with a blue counterstain (a3). Green : red fluorescence intensity ratios are calculated along the length of each chromosome from 10 metaphases and data presented in summary karyograms showing copy number imbalances from the rectal lesion (b) (six aberrant arms) and from the lung lesion (c) (14 aberrant arms). Bars in red to the left of each ideogram represent loss, bars in green to the right indicate gain. Boxed chromosomes are characteristically abnormal in primary lung adenocarcinomas [4], compared with rectal adenocarcinomas [3]. Discussion
Determining whether a malignant tumour is primary or secondary frequently has implications for clinical man-
agement. In this case study, we have applied a genomewide molecular cytogenetic ‘fingerprinting’ technique to identify the most probable site of origin of a new solid tumour, according to the chromosomal aberrations it
FINGERPRINTING EVIDENCE TO DETERMINE PRIMARY OR SECONDARY MALIGNANCIES
contains. CGH has been used widely as a research tool and both the number of CNI per case, and the presence of particular abnormalities has been associated with different prognoses [5]. CGH has been used to improve differential diagnosis of germ cell tumours [6], however we believe the case reported here to be the first in which a CGH fingerprint has been used in a clinical setting to assist in directing management decisions. The method was performed on standard formalin-fixed, paraffinembedded tissue of the type that is processed and stored in all histopathology departments. This case study illustrates the potential role of techniques such as CGH in resolving an important and common oncological dilemma. References 1 Turk PS, Wanebo HJ. Results of surgical treatment of nonhepatic recurrence of colorectal carcinoma. Cancer 1993;71:4267.
411
2 Zitzelsberger H, Lehmann L, Werner M, Bauchinger M. Comparative genomic hybridisation for the analysis of chromosomal imbalances in solid tumours and haematological malignancies. Histochem Cell Biol 1997;108:403. 3 De Angelis PM, Clausen OP, Schjolberg A, Stokke T. Chromosomal gains and losses in primary colorectal carcinomas detected by CGH and their associations with tumour DNA ploidy, genotypes and phenotypes. Br J Cancer 1999;80:526. 4 Goeze A, Schluns K, Wolf G, Thasler Z, Petersen S, Petersen I. Chromosomal imbalances of primary and metastatic lung adenocarcinomas. J Pathol 2002;196:8. 5 Kusano N, Okita K, Shirahashi H, et al. Chromosomal imbalances detected by comparative genomic hybridization are associated with outcome of patients with hepatocellular carcinoma. Cancer 2002;94:746. 6 Summersgill B, Goker H, Osin P, et al. Establishing germ cell origin of undifferentiated tumors by identifying gain of 12p material using comparative genomic hybridization analysis of paraffin-embedded samples. Diagn Mol Pathol 1998;7:260.