The unknown biology of the unknown primary tumour: a literature review

A. J. van de Wouw1,+, R. L. H. Jansen2, E. J. M. Speel3 and H. F. P. Hillen2

1 Department of Internal Medicine, Slingeland Hospital Doetinchem, Doetinchem; 2 Department of Internal Medicine, University Hospital Maastricht, Maastricht; 3 Department of Molecular Cell Biology, Growth & Development Research Institute, University of Maastricht, Maastricht, The Netherlands

Received 12 June 2002; revised 4 September 2002; accepted 17 September 2002

Abstract

The unknown primary tumour (UPT) is an intriguing clinical phenomenon found in approximately 5% of all newly diagnosed patients with cancer. It is unclear whether UPT forms a distinct biological entity with specific genetic and phenotypic characteristics, or whether it is merely a clinical presentation of metastases in patients in whom the primary tumour cannot be detected and does not result in any visible clinical signs. Understanding the basic biology of UPT may shed light on this issue and, moreover, may have a direct impact on clinical care. A review of the literature revealed only a limited number of publications describing the genetic and phenotypic features of UPT, most of which focus only on the potential of these markers to predict prognosis. The question as to whether the biology of UPT is different from tumours of known primaries therefore remains unanswered. Further insight into the molecular mechanisms underlying the oncogenesis of UPT, e.g. by applying newly available DNA and gene profiling microarray techniques, will be necessary to understand its specific biology and to develop more effective treatments.

Key words: biology, review, unknown primary tumour

Introduction

The unknown primary tumour (UPT) is an intriguing clinical phenomenon found in approximately 5% of all newly diagnosed patients with cancer [13]. UPT, alternatively known as cancer of unknown primary (CUP), is defined as a biopsy-proven metastasis of a malignancy in the absence of an identifiable primary site after a complete history and physical examination have been carried out, along with basic laboratory studies, chest X-ray and additional directed studies, indicated by positive findings during the initial work-up [4]. Unknown primary tumours are predominantly classified as adenocarcinomas (50–60%) or poorly differentiated adenocarcinomas or carcinomas (30–40%). Only 5–8% of UPTs are squamous carcinomas and 2–5% undifferentiated malignancies [1, 2].

At present, several different explanations have been put forward to describe the heterogeneous UPT syndrome. One such explanation is that UPTs may be considered as metastases in patients in whom the primary tumour has not been found and which did not result in clinical signs of disease. On the other hand, they may represent a separate group of cancers harbouring genetic and phenotypic characteristics that underlie their unique clinical presentation, or alternatively, they may represent unusual primary tumours mimicking metastatic disease (in the case of one identified tumour site).

Advances in the understanding of the basic biology of UPT may have a direct impact on clinical care. If we regard UPTs as common metastases of an unrecognised primary, then diagnostic evaluation should concentrate on the identification of the primary origin of the tumour. Its identification would lead to a disease-directed treatment and a better-defined prognosis. Thus, if this is the case, research should focus on developing tools for better detection and/or classification of primary tumours, such as the use of magnetic resonance imaging (MRI) and positron emission tomography (PET) scanning, monoclonal antibody panels and molecular profiling techniques to detect the specific genomic and phenotypic characteristics of the malignant cells involved. In the concept of a specific biology of unknown primary tumours the search for a primary is of minor importance and diagnostic evaluation should focus on the identification of treatable subsets. Treatment should be UPT-specific and research should focus on the metastatic genotype and phenotype and on the detection of specific biochemical or molecular targets for the treatment of UPT.

Thus, it is of great importance to understand the relationship between genotype, phenotype and the biological behaviour of UPTs.

Clinical features

Although UPTs comprise a heterogeneous group of tumours with widely varying natural histories, the clinical picture of UPT demonstrates common typical characteristics. First, patients predominantly present with a short history of non-specific complaints (anorexia, weight loss, etc.). In most cases, the primary tumour remains unidentified during the patient’s lifetime, but if found during their lifetime or by autopsy, it is a small asymptomatic tumour often localised in the lung or pancreas [2, 5]. Even after thorough evaluation at autopsy, only 80% of the primaries have been found [6, 7]. Secondly, approximately 30% of patients with UPT present with three or more organs involved [8, 9]. This obviously differs from the percentage of patients with three or more involved organs in metastasised known primaries, which is below 15% [1012]. Thirdly, an unusual metastatic pattern was reported by Nystrom et al. [6] and Le Chevalier et al. [7]. A relatively high number of metastases in UPT are found in the kidneys, adrenal gland, skin and heart when compared with expected sites of metastases [10]. In addition, in patients where the primary tumour had been found during autopsy, differences in metastatic localisation were observed when the metastatic pattern of the UPT was compared with the common sites of tumour spread of known primary tumours. Fourth, the identification of the primary tumour by intensive radiological and/or endoscopic examination, by PET scan or by immunohistochemistry on tumour biopsy material did not seem to improve survival in the majority of UPT patients, mainly due to a lack of alternative therapeutic strategies [1319].

With the exception of some treatable subgroups, UPTs usually exhibit a relatively high resistance to available chemotherapy [1, 2, 2024]. Patients with UPT have a very poor prognosis of 3 to 4 months in an unselected population with <25% of patients alive 1 year after diagnosis [3, 25, 26]. However, new, more effective chemotherapeutic agents are available, and in more recent studies with selected patients from poor prognostic groups, median survival from 9 to 13 months is reached, with >40% of patients alive 1 year after diagnosis [2733].

Biological features of UPT

A PubMed search from 1970 until now revealed only a limited number of publications describing the genetic and phenotypic features of UPT, and most of them only described their potential as prognostic factors. An overview of the literature will be given below and is summarised in Table 1.


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Table 1. Biological features of unknown primary tumours (UPTs)
 
Aneuploidy
Aneuploidy (also referred to as chromosomal instability), which describes a chromosome complement that is not a simple multiple of the haploid set, is now a well-recognised phenomenon in 70–90% of solid tumours [3436]. There is increasing evidence that for many carcinomas, such as breast, prostate and colorectal cancer, a diploid DNA content is associated with better prognosis [37]. In order to determine favourable subgroups, Hedley et al. [38] measured the cellular DNA content of tumour biopsies of 152 patients with metastatic adenocarcinoma or undifferentiated carcinoma of unknown primary [38]. Aneuploidy was found in 70% of patients, evenly distributed between the two sexes, and there was no obvious relationship to the various patterns of metastatic involvement. Median survival of patients with diploid and aneuploid tumours was 4.2 months and 4.8 months, respectively. These results indicate that the incidence of aneuploidy in this heterogeneous group of patients is similar to that reported for carcinomas with overt primary. However, in contrast to many of these tumour types, in this single study, patients with metastatic adenocarcinomas of unknown primary that are diploid do not have a more favourable prognosis [38].

Chromosomal abnormalities
Until now, relatively few studies on the chromosomal abnormalities in UPT have been performed. In an attempt to define the unique characteristics of UPT, Abbruzzese et al. [39] and Bell et al. [40] developed a research program aimed at evaluating the karyotypic changes common to UPT. They were able to determine the karyotype of 13 out of 30 UPTs. In 12 of the analysed cell lines, there were, amongst other things, abnormalities identified in the short arm of chromosome 1. These abnormalities included deletion of 1p, translocations with a breakpoint at 1p, isochromosome 1q and evidence of gene amplification. These results are consistent with earlier descriptions of chromosome 1p abnormalities in advanced malignancy in general, as summarised by Atkin [41] and Mertens et al. [42].

Using karyotyping, Motzer et al. [43, 44] determined the frequency of specific abnormalities on chromosome 12 in patients with UPT. The hypothesis was that patients with undifferentiated carcinoma of unknown primary responding to cisplatin-based chemotherapy have unrecognised germ cell tumours, and that i(12p) is a specific chromosomal marker characterizing germ cell tumours. Twelve (30%) patients had increased 12p copy number or a deletion in the long arm of chromosome 12. This proved to be predictive for response. Complete responses to cisplatin-based therapy were achieved in patients with specific chromosomal aberrations associated with germ cell tumours and objective responses were achieved in 75% of these patients compared with 17% of patients without these aberrations. Summersgill et al. [45] and Ilson et al. [46] found a similar association. Thus, in undifferentiated carcinoma of unknown primary, i(12p) is correlated with a good response to cisplatin-based chemotherapy, although lack of i(12p) does not exclude response. Because i(12p) occurs in over 80% of germ cell tumours and only sparsely in a few other lesions (e.g. acute leukaemia, embryonal rhabdomyosarcoma and neuroepithelioma), determination of i(12p) is being used as a diagnostic tool for extragonadal germ cell tumours [47].

Preliminary data from our own group revealed that three out of five tumours of different UPT patients, analysed by comparative genomic hybridisation (CGH) to compare DNA copy numbers between tumour DNA and a normal reference sample, could be further diagnosed as having most probably metastases of the colon (two cases) and lung (one case) [48, 49]. Furthermore, these results indicate that genomic characterization in combination with phenotypic analysis may improve the classification of UPTs, which may have implications for therapy. Application of CGH to microarrays of genomic DNA clones harbouring, for example, known cancer genes could have even a more important role in this respect, because genes predictive of response to therapy or of known prognostic significance, such as c-erB-B2, could be directly analysed for amplification or deletion [5052].

Oncogenes
The oncogenes ras, c-myc, bcl-2 and Her2/neu, also known as c-erB-B2, are overexpressed in many solid tumours. Determination of the levels of these gene products are considered to be useful prognostic factors, although reports on c-myc and bcl-2 are often conflicting on this matter [5360].

Pavlidis et al. [61] found high levels of overexpression of c-myc, ras and c-erB-B2 in 26 UPT patients by immunohistochemical staining (96%, 92% and 65%, respectively) [61]. They found neither further relationship with histological or clinical parameters nor diagnostic or prognostic value of this overexpression.

Hainsworth et al. [62] evaluated 100 tumour species of poorly differentiated adenocarcinoma (PDA) or poorly differentiated carcinoma (PDC) of unknown primary site for Her-2 protein [62]. Ten (11%) tumour species overexpressed Her-2. No major differences in overall response rate to chemotherapy were observed between the patients who overexpressed Her-2 and those who did not. Evaluation of the efficacy of trastuzumab in UPT patients with Her-2 overexpression is indicated.

Briasoulis et al. [63] studied bcl-2 expression in 40 UPTs (8% squamous, 36% adenocarcinoma, 55.5% PDC). Staining was evaluated by intensity (+1 to +3) and the percentage of positive cells (1–100%). Only staining of >5% of tumour cells was interpreted as positive tumour staining. Bcl-2 was expressed in almost half of the tumours; this was not expected, because in most studies bcl-2 has been found to be up-regulated in premalignant lesions rather than in advanced malignancies, and has also been associated with a less aggressive phenotype [64, 65]. In this study, bcl-2 expression by itself had no prognostic value. When combined with a high expression of p53, there was a trend towards a higher response to cisplatin-based chemotherapy.

Taxanes could be used in an anti-bcl-2 approach. By prevention of polymerisation or depolarisation of microtubules taxanes cause phosphorylation of bcl-2, leading to apoptotic cell death [66]. In recent studies, paclitaxel has been used in combination therapy in UPTs with promising results (response rates over 40% and median survival of 9–13 months) in a phase II study [32, 33].

Tumour suppressor genes
It is now known that a high number of gene inactivations occur in a wide variety of human cancers, and a number of additional putative tumour suppressor gene loci have been mapped on specific chromosomes. At present, p53 is the best-known tumour suppressor gene. It can suppress tumour development by arresting the cell cycle or initiating apoptosis. Mutations in p53 are a common occurrence in human cancers and immunohistochemistry has shown that approximately 55% of all human cancers express p53 [6769].

Using immunohistochemistry, Briasoulis et al. [63] studied p53 expression in 47 UPTs. Staining was evaluated by intensity (+1 to +3) and the percentage of positive cells (1–100). p53 was expressed in over 70% (33/47) of tumours. Assessment using the immunoreactivity index showed 25 tumours (53%) expressing a high immunoreactivity index and 22 (47%) a low immunoreactivity index. In this study, p53 expression by itself had no prognostic value. When combined with a high expression level of bcl-2, there was a trend towards a higher response to cisplatin-based chemotherapy.

Bar-Eli et al. [70] investigated the frequency of p53 mutations in a series of 15 UPT biopsies and eight cell lines established from UPTs. Mutations in the conserved regions of the p53 gene were analysed by single-strand conformation polymorphism analysis of exons 5–9 and were verified by direct DNA sequencing of polymerase chain reaction (PCR) products. The p53 gene was mutated in six out of 23 (26%) patients with UPT. Thus, although UPTs represent bad prognostic tumours which are often aneuploid, the frequency of p53 mutations is relatively low in this study. Therefore, they suggest that p53 mutations may not play a major role in the development and progression of UPT. These data are in contrast with the immunohistochemical data mentioned above as well as with our preliminary data on microsatellite analysis of UPTs. Besides aneuploidy, we identified in 90% of the metastases an allelic imbalance/loss of heterozygosity of the p53 locus at 17p13, indicating a relationship between p53 mutation and UPT [65].

The difference in the results of p53 studies may probably be explained by discordance between immunohistochemical and molecular genetic methods, which may occur in 25% of tumours [62]. Two reasons are mentioned. First, 20% of the mutations occur outside the hot spot region (exon 5–9) [66]. On the other hand, the fact that the same monoclonal antibodies can detect both wild and ‘mutated’ protein makes it difficult to distinguish the proportion of each protein [67]. Secondly, alterations in the proteins regulating p53 expression, such as p14ARF and MDM2, as well as the p53 substrate p21, may be responsible for the low number of p53 mutations found by Bar-Eli et al. [70].

No association was detected between positive versus negative immunodetection of p53 and any of the clinicopathological parameters studied. However, patients with a high immunoreactivity index for p53 and bcl-2 tended to respond better to cisplatin-based chemotherapy.

No research has been performed yet on metastasis-suppressor genes, which seem to play an important role in regulating the growth of disseminated cancer cells at secondary sites [71].

Microvessel density (MVD)
There is strong evidence that angiogenesis, as measured by MVD, correlates with the incidence of metastases in several solid tumours [72]. In order to investigate whether angiogenesis has a specific biological role in the metastatic phenotype of UPTs, our laboratory compared the MVD in liver metastases of UPT with the MVD in liver metastases of colon and breast tumours [73]. We found no difference between the MVD in liver metastases of UPT and known primaries: both showed a high degree of visualisation. As in other solid tumours, a high MVD was correlated with short survival in univariate as well as in multivariate analysis.

Discussion

Mutual clinical features of widespread metastases in uncommon sites and a primary tumour that remains asymptomatic support the paradigm of a specific clinical entity. Whether UPT demonstrates a specific biology with mutual genetic characteristics is still unclear.

Only a few studies have tried to identify the unique biological characteristics of UPTs. Except for karyotypic abnormalities of the short arm of chromosome 1, as found in many metastatic solid tumours, no further consistent similarities have been found. The finding of aneuploidy in 70% of UPTs and the frequency of c-myc, HER2/neu, bcl-2 expression and p53 mutation is probably not different from known primary tumours. Most of these items have the same prognostic power in UPT as in metastatic tumours with an overt primary.

It is hypothesised that in UPT the primary acquires a metastatic phenotype soon after transformation and stays small either by inborn errors within the primary, leading to involution or an extremely slow growth rate, or by metastases inhibiting the growth of the primary tumour [14, 40, 74]. There are also suggestions that aggressive tumour cells can leave a relatively mild tumour early and circulate through the blood and form autotrophic, excessive metastases in other organs [75]. But none of the biological hypotheses is confirmed by results from the literature.

New biological approaches to the dormancy problem in patients may reveal an explanation for the behaviour of UPT [75].

In conclusion, the question as to whether UPTs are really different from tumours of known primaries remains unanswered. Further research on the biology of UPT should concern the determination of whether this group of tumours share unique genetic, chromosome and/or phenotypic anomalies. It is postulated that new molecular techniques, such as DNA and gene expression profiling as well as proteomics, are going to play an important role in this search.

Footnotes

+ Correspondence to: A. J. van de Wouw MD, Department of Internal Medicine, Slingeland Hospital Doetinchem, PO Box 169, 7000 AD, Doetinchem, The Netherlands. Tel: +31-314-329751; Fax: +31-314-329068; E-mail: y.van.de.wouw{at}slingeland.nl Back

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