J Cancer Res Clin Oncol (1996) 122:127 131 �9 Springer-Verlag 1996

Robert Kiss �9 Sandrine Rorive Isabelle Camby �9 Jean-Lambert Pasteels Jacques Brotchi �9 Isabelle Salmon

DNA Ploidy level assessments in 83 human brain metastases Relationship to the survival of 35 patients

Received: 30 January1995/Accepted: 13 August 1995

Abstract The nuclear DNA content (DNA ploidy) level was determined in a series of 83 human brain meta- stases, for which 35 complete clinical follow-ups were available. The DNA ploidy level determination was carried out by means of DNA histogram types. The results show that certain brain metastases were diploid, while others exhibited aneuploidy levels ranging from low to very high. The present study also shows that a significant proportion, i.e. 18%, of the 83 brain meta- stases, exhibited very high levels of aneuploidy, i.e. hypertetraploidy, hyperpentaploidy and octoploidy. We had previously observed that this feature appeared only rarely, i.e. in less than 2% of primary nervous turnouts. Furthermore, the present study shows that DNA ploidy level in brain metastases is related signifi- cantly (P < 0.001) to patient survival. Indeed, while 9/13 (69%) patients with diploid brain metastases sur- vived longer than 9 months, none (0%) of the 22

The present work was carried out on the basis of grants awarded by the Fonds de la Recherche Scientifique M+dicale (FRSM~ Belgium).

R. Kiss t (EN) �9 S. Rorive a . I. Camby 3 - J.-L. Pasteels Laboratory of Histology, Faculty of Medicine, Free University of Brussels, 808 route de Lennik, 1070 Brussels, Belgium

J. Brotchi Department of Neurosurgery, Hospital Erasme, Free University of Brussels, Belgium

I. Salmon Department of Pathology, Hospital Erasme, Free University of Brussels, Belgium

1 Research Associate with the Fonds National de la Recherche Scientifique (FNRS, Belgium) 2 Physician-in-Training at the Faculty of Medicine of the Universit6 Libre de Brnxelles 3 Research Assistant with the FNRS.

patients with aneuploid brain metastases survived lon- ger than the 9 months following the diagnosis of their brain metastases.

Key words Brain metastases �9 Primary nervous tumours �9 DNA ploidy �9 Image cytometry - Prognosis �9 Feulgen staining

Introduction

As stated by Patchell et al. (1990), metastases to the brain occur in 20-30% of patients with systemic cancer (Cairncross and Posner 1983) and constitute the most common type of intracranial tumour (Silverberg and Lubera 1988; Walker et al. 1985). Mehta et al. (1992) report that it has been estimated that 10%-20% of all cancer patients undergoing post-mortem examination are found to have brain metastases (Posner 1977), and that the annual number of cases in the United States probably exceeds 100000. Mehta et al. (1992) further state that very few modern prospective studies have evaluated the natural history of untreated cerebral metastases, adding that data from the 1970s suggest that the median survival time for patients with un- treated brain metastases is approximately 1 month. This opinion is also shared by Engenhart et al. (1993).

Like several other authors (Engenhart et al. 1993; Mehta et al. 1992), Smalley et al. (1992) agree with the fact that surgery plus radiotherapy is more effective for solitary brain metastasis treatment than radiotherapy alone. Patchell et al. (1990) report that the patients with brain metastases who are most likely to benefit from surgical resection are those with a single surgically accessible lesion, no remaining systemic cancer limited to the primary site, and a life expectancy of at least 2 months. This concerns approximately 25% of all patients with brain metastases. Engenhart et al. (1993) report that, while this relatively favourable subset of patients has a median survival time of 6-12 months, it

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also includes some remarkable long-term survival rates. In contrast, the median survival time of patients with multiple metastases is increased from 1 to only 3-6 months by whole-brain irradiation.

The aim of the present study is to characterize the nuclear DNA content (DNA ploidy) level in a series of 83 human brain metastases stemming from various histopathological tumour types. The determination of the DNA ploidy level was carried out by means of DNA histogram typing (Salmon et al. 1992a, 1993c, 1994). This ploidy level was studied as a function of patients' survival.

Materials and methods

Patient data

Of the 83 brain metastases, 55 were supratentorial, and 28 infi'aten- torial. A total of 49 metastatic patients underwent surgical resection after the discovery of a primary tumour, while for 23 of them the primary tumour was discovered after the surgical resection of the metastasis. For the remaining 11 patients, the primary turnout was never discovered. Of these 83 patients with brain metastases, 65 exhibited a single metastatic lesion while the remaining 18 had multiple ones.

Table 1 gives the phenotypic and histogenic classifications of the 83 brain metastases under study. We obtained complete clinical follow-ups for 35 patients. The explanation of why the present study is based on the assessment of DNA ploidy level in 83 patients, while clinical follow-ups were available for only 35, is as follows. We unfortunately lost track of a large number of patients because after their operations they left hospital and we did not see them again. They were thus lost as far as clinical follow-up was concerned. In addition, other patients had been operated upon too recently to be included in the present study under the heading of clinical follow-up.

from each block. The first, third and fifth (5 btm thickness) were subjected to histopathologicai checking (after haematoxylin/eosin) for the presence of tumour tissue on the block submitted to analysis. The second and fourth (8 btm thickness) were subjected to a method described in detail elsewhere (Kiss et al. 1993). This method makes it possible to obtain single-cell nuclei suspensions (after pronase diges- tion) that are centrifuged onto glass slides and then stained by the Feulgen reaction (Salmon et al. 1992a, b; 1993a d).

Ploidy level determinations

Ploidy level determination was carried out by means of a SAMBA 200 microscope image processor (Alcatel-TITN, Grenoble, France) with a 100 x magnification lens (numerical aperture: 1.30). After a rapid low-resolution analysis of the images to memorize the nuclei addressed at the motorized stage, each nucleus (200400 nuclei selected/slide, i.e. 400-1600 nuclei/case) was analyzed in high-resolu- tion mode (Kiss et al. 1992, 1993).

The nuclear integrated absorbance was computed on 256 den- sitometric levels on each pixel (0.16 ~tm2). The nuclear DNA content of each nucleus was thus calculated from its integrated absorbance parameter value, which corresponded to the sum of the absorbance values of each pixel of the nucleus.

The nuclear images of the Feulgen-stained nuclei were digitized at 540 nm wavelength, evaluated by eye, and accepted by the observers on the same basis as those previously described (Salmon et al. 1992a, b; 1993a-d).

The DNA histogram typing was carried out as described pre- viously for primary and metastatic tumours of the nervous system (Salmon and Kiss 1993; Salmon et al. 1992a, 1993c). In these pre- vious studies, six DNA histogram types were defined on the basis of this method; these included diploid (type I), byperdiploid (type IV), triploid (type II), hypertriploid (type V), tetraploid (type III) and polymorphic (type VI) patterns. Three additional histogram types are defined in the present study. They include the hypertetraploid (type VII), the octoploid (type VIII) and the hyperpentaploid (type IX) types. The definition of these nine DNA histogram types is given in Table 2.

Cytological samples

The series of 83 brain metastases were all formalin-fixed paraffin- embedded tissues. Depending on tumour size, between one and four paraffin blocks were available for each case. Five sections were cut

Table l Description of phenotypic and histogenic classifications

Phenotypic classification n Histogenic classification ~ n

Glandular carcinoma 40 Lung 47 Epidermoid carcinoma 20 Skin 5 Poorly-differentiated Colon 4

carcinoma 6 Breast 3 Small-cell carcinoma 8 Prostate 3 Melanoma 6 Esophagus 2 Ewing sarcoma 1 Kidney 2 Grawitz tumour 2 Ovary 2

Nodes 1 Bone 1 Pancreas 1 Salivary gland

aWith respect to 11 patients, the origin of the primary cancer remained unknown

Table 2 Definition of the nine DNA histogram types identified in the present series of 83 brain metastases. The DNA index corres- ponds to the ratio between the DNA content of the aneuploid G0-G1 peak and the DNA content of the diploid G0-G1 peak. In the case of a diploid tissue, the DNA index is 1.00

Type Definition DNA index (DI) range of the major G0-G1 DNA peak

I Diploid ~ 0.90 < DI < 1.15 II Triploid 1.40 < DI < 1.60 II1 Tetraploid 1.90 < DI < 2.20 IV Hyperdiploid 1.16 < DI < 1.39 V Hypertriploid 1.61 < DI < 1.89 VI Polymorphic b VII Hypertetraploid 2.21 < DI < 2.50 VI[I Octoploid 3.90 < DI < 4.10 IX Hyperpentaploid 2.61 < DI < 2.90

a A true diploid tumour must show a DNA histogram type with a G0-G1 DNA peak containing at least 70% of its cell nuclei population (Salmon et al. 1993c, 1994) bA polymorphic DNA histogram type must contain at least two of the four following DNA histogram types: hyperdiploid, triploid, hypertriploid or tetraploid (Salmon et al. 1993c, 1994)

All the parameters described in the present section were calculated in ignorance of the histopathological and clinical data.

Statistical analyses

The results are given as the breakdown of DNA histogram type percentages as opposed to various histopatho-clinical subgroups. Statistical comparisons of the data were performed by means of the z2-test. All the statistical analyses were carried out using the Statistica/Dos software (StatSoft, Tulsa, Okla.).

Results

Determination of the ploidy level in the 83 brain metastases

The nine DNA histogram types indentified in the pres- ent study are defined in Table 2. Of the 83 brain metastases, there were 13 diploid (type I), 4 hyperdip- loid (type IV), 6 triploid (type II), 9 hypertriploid (type V), 6 tetraploid (type III), 30 polymorphic (type VI), 9 hypertetraploid (type VII), 2 hyperpentaploid (type IX) and 4 octoploid (type VIII) cases.

This series of 83 cases therefore included 23% euploid (16% diploid and 7% tetraploid), 5% weakly aneuploid (hyperdiploid) and 72% highly aneuploid tumours. Of the 72% of highly aneuploid tumours, 18% exhibited very high levels of aneuploidy, i.e. hypertetraploidy (11%), octoploidy (5%) and hyper- pentaploidy (2%).

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Relationship between the ploidy level of the brain metastases and patient survival time

Figure 1 shows that a statistically significant (P < 0.001/)~2-test) relationship exists between the ploidy level of the brain metastases and patients' correspond- ing survival time. Indeed, while 9/13 (69%) patients with diploid brain metastases survived more than 9 months (area A in Fig. 1), none (area C in Fig. 1C) of the 22 patients with aneuploid (DNA index more than 1.20) brain metastases (area D in Fig. 1D) survived longer than the 9 months following histopathological diagnosis. When the cut-off value was raised from 9 to 12 months, 6/13 (46%) of the patients with diploid brain metastases still survived as opposed to the 0/22 (0%) patients with aneuploid brain metastases (P = 0.002/z2-test).

Of these 35 patients, 3 and 4 exhibited multiple metastatic lesions in the diploid and aneuploid groups respectively. This did not significantly (P > 0.05) influ- ence the results.

Discussion

The results that we obtained in previous studies with respect to ploidy level determination in tumours of the human central and peripheral nervous systems show that the diagnostic and prognostic fields of application of this determination must be kept completely separate. Indeed, we tried using this ploidy level determination to distinguish between benign and malignant nervous

Fig, 1 Relationship between the ploidy level in a given brain metastasis (x axis) and the corresponding survival period of the patient (y axis). The vertical black line separates the diploid (DNA index < 1.20) from the aneuploid (DNA index > 1.20) cases. The horizontal black line separates the patients who survived more than 9 months from those who survived less than 9 months following the diagnosis of their metastases

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1,60 1,80 2,00 2,20 2,40 2,60 2,80

DNA INDEX ( arbitrary units )

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tumours. The results indicated clearly that definitely benign meningiomas (Salmon et al. 1993b) and nerve sheath tumours (Salmon et al. 1992b) can be highly aneuploid, while highly malignant meningiomas (Salmon et al. 1993b) and primary neuro-ectodermal tumours (Dangou et al. 1993) can be diploid, at least within the limits of resolution of the image cytometry methodology.

With respect to the prognostic field, in studying a series of 206 adult astrocytic tumours we observed that the hypertriploid subgroup of aneuploid tumours gives more hope of survival to the patient than the group associated with any other kind of tumour (Salmon et al. 1992a, 1994). This might be due to the lower proliferation rate inside hypertriploid tumours as compared to observations made on other types of tu- mours (Salmon and Kiss 1993).

All the results reported above seem to point to an apparent paradox. Indeed, while ploidy level deter- ruination is not associated with any diagnostic value, it is associated with a strong prognostic value in low- grade and high-grade astrocytic tumours.

We hypothesized that high levels of aneuploidy in a given tumour, whether benign or malignant, could reflect a degeneration phenomenon linked to tumour ageing (Salmon et al. 1993c). In other words, highly aneuploid tumours are in fact tumours that have accu- mulated a large number of chromosomal accidents during the large number of cell cycles characteristic of their great age. Of these highly aneuploid tumours, hypertriploid ones might be the most degenerate, as is suggested by their low proliferative activity which, in turn, might handicap their spontaneous growth. This feature was observed not only in tumours of the central and peripheral nervous systems (Salmon and Kiss 1993) but also in those of the thyroid gland (Salmon et al. 1993a) and bladder (van Velthoven et al. 1995). Diploid tumours, whether benign or malignant, might therefore correspond to young "healthy" tumours that have not yet had time to accumulate such a large number of chromosomal aberrations because their cells have passed through a much smaller number of cell cycles than in the case of aneuploid ones.

We have partly validated our hypothesis by demon- strating that certain human colorectal cancers, which are mostly diploid or slightly aneuploid at the time of grafting onto nude mice, become more and more aneuploid as they pass through their successive animal transplants (Jannot et al. 1993).

This hypothesis fits in with the present results. In fact, in the present series of 35 patients for whom we obtained complete clinical follow-ups, we observed that a very highly significant (P < 0.001) proportion of those with diploid brain metastases survived longer (more than 9 and even 12 months) than those with aneuploid metastases. Therefore, according to the above-mentioned hypothesis, the potential biological explanation of how ploidy difference might produce

prognostic differences in brain metastasis patients is as follows. According to our hypothesis, diploid tumours are biologically younger than aneuploid ones. The pa- tients with diploid brain metastases survived longer than those with aneuploid ones because, fortunately for them, the brain metastases were detected at an earlier clinical stage than in the case of the patients with aneuploid brain metastases. In other words, the diploid metastases might have been developing for a shorter period of time than the aneuploid ones.

Another explanation of aneuploidy occurrence in human tumours relates to their genomic instability. Indeed, several studies have shown that, in embryo fibroblasts (Harvey et al. 1993) and in various types of benign (Blount et al. 1994) and malignant (Carder et al. 1993; Eyfj/Srd et al. 1995; Offerhaus et al. 1992) tumours, mutant p53 destabilises the genome and facil- itates the development of aneuploidy.

The survival figures in the present study may also lead to the hypothesis that diploid metastases exhibit a lower degree of activity than aneuploid ones, thus explaining the longer survival periods of patients with diploid metastases. This hypothesis cannot be retained since we observed that diploid and aneuploid meta- stases exhibited similar (P > 0.05) proliferation indices (data not shown).

We also checked that the cohort of lost patients was similar to the cohort of follow-up patients included in the analysis of DNA ploidy levels and patient survival, thus the lost patients had no influence on the survival statistics relating to the follow-up patients.

Lastly, neither histogenic (P > 0.05) nor phenotypic (P > 0.05) classifications of brain metastases were asso- ciated with significant results in regard to survival times (data not shown).

The present study also shows that about 20% of the 83 metastases under sthdy exhibited very high levels of aneuploidy, i.e. hypertetraploidy, hyperpentaploidy and octoploidy. This is in sharp contrast with the fact that fewer than 2% of the primary nervous tumours exhibited such very high aneuploidy levels (Salmon et al. 1993c).

In our laboratory we are currently setting up various experimental models in an attempt to understand why such high levels of aneuploidy occur in brain metastases when they are only rarely observed in pri- mary tumours of the central and peripheral nervous system. We are investigating whether a relationship might exist between the ploidy levels in a primary tumour and its metastasis. Preliminary results seem to indicate that such a relationship does not exist (data not shown).

In conclusion, the present study confirms previous data demonstrating that ploidy level determination cannot be used as a diagnostic marker since certain brain metastases, i.e. highly malignant tumours, may be diploid. The present study also shows that a significant proportion, i.e. 18%, of the 83 brain metastases

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exhibited very high levels of aneuploidy, i.e. hypertetra- ploidy, hyperpentaploidy and octoploidy. This feature only rarely appeared, i.e. less than 2% in primary nervous tumours (Salmon et al. 1993c). The present study further shows that a clear-cut relation (P < 0.001) exists between the ploidy levels in brain metastases and patients' corresponding survival times.

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