Clonal chromosome abnormalites found in three non-neoplastic proliferative brain lesions

Clonal chromosome abnormalites found in three non-neoplastic proliferative brain lesions

Cláudia Aparecida Rainho¹, José Barbieri Neto², Lamartine Correa de Moraes³ and Silvia Regina Rogatto¹

¹Departamento de Genética, Instituto de Biociências, UNESP, 18618-000, Botucatu, SP, Brasil. Send correpondence to S.R.R. Fax: [phone]. E-mail: [e-mail]

²Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brasil.

³Departamento de Neurologia, Hospital Universitário Regional do Norte do Paraná, UEL, Londrina, PR, Brasil.

ABSTRACT

Chromosome analysis was made of brain lesions from three patients which, according to classical histopathological criteria, did not contain tumor cells. In addition to normal cells, we identified abnormal karyotypes with clonal numerical and structural chromosome alterations in at least two independently originated primary cultures from each lesion. Our data suggest that chromosomal aberrations can exist in vivo in non-neoplastic lesions. Other abnormalities may be due to genetic instability manifested only in vitro (culture artifacts) or may already have been present in brain tissue, reflecting previous chromosome damage (as a result of exposure to chemical treatment or enviromental clastogens).

INTRODUCTION

Glial cells are the most numerous cell type in the human brain and are subdivided into astrocytes, oligodendrocytes and ependymal cells. Gliosis represents a non-malignant reactive proliferation of astrocytes that occurs when these cells respond to central nervous system injury. Such reactive cells have been considered genetically normal astrocytes (Robbins et al., 1984).

We are aware of just two reports concerning cytogenetic analysis of non-neoplastic glial cells (Moertel et al., 1993; Dalrymple et al., 1994). In the first of these two studies (Moertel et al., 1993), 63 cases were analyzed, 57 of which presented at least one abnormal metaphase and 11 presented simple clonal karyotypes. The most common clonal alteration found was loss of chromosome Y. In a more complete study (Dalrymple et al., 1994), the results of cytogenetic analysis after short-term primary culture were compared to those obtained by the FISH technique using centromeric probes for chromosomes 7, 8, 9, 10, 12, 17, 18, X and Y in non-neoplastic, normal (11 cases) and gliotic brain tissues (10 cases). Interphase nucleus analysis revealed apparent monosomy of chromosomes 8 and 17, but this was not confirmed in the karyotype. A more careful analysis revealed that these monosomies were probably due to homologous centromere pairing in the interphase nucleus, as previously observed for chromosome 17 in normal human brain tissue (Arnoulds et al., 1991).

MATERIAL AND METHODS

Cases

Case 1 was a 29-year-old male with headache, fever and convulsive seizures. Carotid angiography revealed an expansive frontal lesion. Frontal craniotomy was later performed on the right side for excision of the lesion. Histological analysis revealed proliferating glial tissue together with a neutrophilic inflammatory infiltrate and a large number of foamy histiocytes, suggesting a chronic meningoencephalic inflammatory process with the formation of abscesses.

Case 2 was a 36-year-old female who for two years had complained of headache in the occipital region, anorexia, vomiting, and low visual acuity. Computer tomography revealed a thickening of the cerebellar tent on the right side compatible with an inflammatory process of indeterminate nature. She was treated with three daily doses of a combination of 800 mg sulfamethoxazole and 160 mg trimetoprim (Bactrim, Roche) for four months with no satisfactory response. Subsequently, she was submitted to craniotomy. Histopathological analysis revealed normal cerebellar tissue exhibiting signs of edema, acute vascular congestion and an apparent decrease in Purkinje cells. Glial proliferation with moderate anisocytosis and anisonucleosis were observed in a small area.

Case 3 was a 12-year-old female with headache and worsening consciousness level, rapidly progressing to coma. Computer tomography revealed a voluminous expansive right parietal process containing blood and surrounded by an extensive area of edema. The patient was submitted to surgery for full excision of the expansive process. Histopathological analysis revealed brain tissue with an extensive area of liquefying necrosis and the presence of foamy histiocytes and numerous microglial cells.

Cytogenetic analysis

Fresh tissue samples obtained under sterile conditions were promptly processed after surgery. Part of the sample, sent to the cytogenetics laboratory, was fixed and used for histopathological evaluation. The remainder of the sample was processed for in vitro cell culture in order to obtain chromosome preparations as previously described (Rogatto et al., 1993). Except for case 2, the patients did not receive presurgical radio and/or chemotherapy.

Metaphases were submitted to standard staining and GTG chromosome banding techniques (Scheres, 1972). The karyotypes were described according to the nomenclature proposed by ISCN 1991 and 1995. Clones were defined as two or more metaphases with the same additional chromosome or structural rearrangement or three or more metaphases with the same lost chromosome. Another criterion for classification as clones was individual analysis of each culture in order to determine if the same chromosome alteration was present in two or more independent primary cultures.

RESULTS

In addition to normal cells, we observed abnormal karyotypes in all three cases, with clonal numerical and structural alterations (Table I) as well as other nonclonal changes. Among the numerical alterations, monosomy of chromosome X (nonclonal in case 2 and clonal in case 3) was particularly frequent. Loss of chromosome Y was observed as a nonclonal phenomenon in case 1, and trisomy of chromosome 20 was detected in two cells in case 3. Structural clonal chromosome alterations involved chromosome 2 in case 1, chromosomes X and 8 in case 2, and chromosomes 4, 19 and 22 in case 3. Deletions del(1)(q12) and del(6)(q25) (case 1) as well as telomeric associations and chromosome breaks (case 2) were observed in sporadic cells.

Table I
- Clonal chromosome abnormalities in non-neoplastic proliferative brain lesions.

Case No.	Age/Sex	Days in vitro	Karyotype
1	29/M	11	46,XY,add(2)(q36)[14]/
			46,XY[7]
2	36/F	25	46,X,inv(X)(q11.2p22.3),del(8)(q21.2)[3]/
			46,XX[14]
3	12/F	20	46,XX,-X[6],add(4)(q3?5)[4],del(19)(p13.1)[4],del(22)(q13.1)[6][cp20]/
			46,XX[10]

Clone definition required that the abnormalities were found in at least two primary cultures set up independently.

Figure 1 shows a cell from case 3 with a 46,XX,add(4) (q3?5),del(22)(q13.1) karyotype.

Figure 1 -
GTG-banded cell from a non-neoplastic proliferative brain lesion (case 3) showing the clonal chromosome abnormalities add(4)(q3?5) and del(22)(q13.1)(arrows).

DISCUSSION

The pathogenetic significance of sex chromosome aneusomy and chromosome 7 trisomy in neoplastic glial cells is currently a matter of strong debate (Heim et al., 1989; Logan et al., 1990; Lindström et al., 1991; Hecht et al., 1994; Rogatto et al., 1994; Yamada et al., 1994). This is due to the fact that these abnormalities have also been observed in cultures of apparently normal brain tissue (Heim et al., 1989). These changes have also been reported to occur in renal, pulmonary and gastric neoplasias (Limon et al., 1990; Castedo et al., 1992), sometimes being present as a single abnormality. Some investigators have suggested that they play a relevant biological role in the brain because of the nonrandom nature of these alterations (Hecht et al., 1995). The presence of these aneuploidies in non-neoplastic cells is a controversial new topic in the study of human tumors, and it would be of great importance to resolve whether they are representative of parenchymal tumor cells or whether they indicate non-neoplastic in vivo mosaicism and/or in vitro artifacts occurring during cell culture (Lee et al., 1987; Heim et al., 1989; Kovaks and Brusa, 1989; Elfving et al., 1990; Lindström et al., 1991; Castedo et al., 1992).

Sporadic cells presenting 20 trisomy have been detected in five other cases of gliosis (Moertel et al, 1993). Chromosome X inversion and chromosome 1 deletions have also been reported as nonclonal alterations in gliosis (Moertel et al., 1993; Dalrymple et al., 1994).

The presence of these nonclonal abnormalities in our cases, taken together with other factors such as duration of in vitro culture (11 to 25 days) and presence of breaks and telomeric associations in sporadic cells (data not shown), support the hypothesis that most of these abnormalities are due to genetic instability manifested under in vitro conditions, or that they represent technical artifacts. On the other hand, some abnormalities may appear in normal, though reactive, tissue and may be absent in truly normal brain tissue. We detected clonal structural abnormalities in at least two independently established primary cultures in all three cases. This fact suggests that these abnormalities are representative of in vivo tissue, even though their meaning is not fully understood. The presence of these chromosome abnormalities may be related to intrinsic characteristics of the expansive process itself since dividing cells are more prone to various types of genetic errors.

Another possibility is that some cells in the gliotic tissue suffered previous chromosome damage (through exposure to clastogens, drug treatment, etc.). This may be the case for patient 2 who was submitted to treatment with sulfamethoxazole and trimetoprim. Trimetoprim is known to induce the expression of fragile sites (Lejeune et al., 1982). However, the breakpoints involved in the rearrangements observed in this case did not coincide with the location of fragile sites, although breaks or acentric fragments were observed in 4% of the cells (data not shown).

The pathogenetic meaning of an overwhelming majority of chromosome abnormalities in terms of cell proliferation and/or transformation remains unclear. Interpretation of cytogenetic findings in solid tumors should be quite cautious (Heim et al., 1989). The presence of abnormal karyotypes in benign tumors has dispelled the notion that chromosome aberrations are associated solely with malignancy (Sandberg, 1990). More recent data have indicated that acquired alterations may be present in lesions that by all classic pathological criteria are considered to be non-neoplastic (Ray et al., 1990; Rubin et al., 1992; Johanson et al., 1993; Aly et al., 1994). Existence of constitutional tissue-specific mosaicism should be considered (Pandis et al., 1994).

ACKNOWLEDGMENTS

This work was supported by grants from CNPq, CAPES and CPG-UEL. Publication supported by FAPESP.

RESUMO

Foi realizada análise cromossômica em lesões de três pacientes que, de acordo com critérios histopatológicos clássicos, não continham células tumorais. Além de células normais, nós identificamos cariótipos anormais com alterações cromossômicas clonais numéricas e estruturais em pelo menos duas culturas primárias originadas independentemente de cada lesão. Nossos dados sugerem que aberrações cromossômicas podem existir in vivo em lesões não neoplásicas. Outras anormalidades podem ser devidas a instabilidade genética manifestada apenas in vitro (artefatos de cultura) ou podem já estar presentes no tecido cerebral, refletindo danos cromossômicos prévios (como resultado de exposição a clastógenos químicos ou ambientais).

(Received April 6, 1998)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  06 Apr 1998