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 »  Abstract
 »  Introduction
 »  Material and methods
 »  Results
 »  Discussion
 »  References

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Year : 2001  |  Volume : 49  |  Issue : 2  |  Page : 124-7

Flowcytometric and histopathological correlation of primary intracranial neoplasms.


Departments of Histopathology, Immunopathology and Neurosurgery, Postgraduate Institute of Medical Education and Research, Chandigarh, 160 012, India.

Correspondence Address:
Departments of Histopathology, Immunopathology and Neurosurgery, Postgraduate Institute of Medical Education and Research, Chandigarh, 160 012, India.

  »  Abstract

DNA ploidy and synthetic phase fraction (SPF) of 52 cases of primary intracranial neoplasms have been determined from fresh tissues and the data was correlated with histopathological typing and grading. Fresh tumour tissues from 52 random surgical biopsies (28 malignant and 24 benign) were obtained from neurosurgical operations during the period 1994-1996. The cells were analysed in Becton Dickinson flowcytometer fitted with Consort 30 programme and 'Sober' software. Percentage of diploid cells, proliferative cells and DNA aneuploidy were evaluated. The tumours were classified and graded according to WHO classification (1993). On histology, there were 28 malignant (grade II to IV) and 24 benign cases (grade I). All the histologically benign tumours in this study showed diploid DNA content with the exception of a pituitary adenoma which had a heterogeneous population of cells. The S phase fraction in all the benign cases was less than 10% except in the case of choroid plexus papilloma (S-phase 54%) and an atypical meningioma (S-phase 14%). Out of the 28 malignant tumours, 12 cases were aneuploid (43%) and the rest were diploid (57%). Among the 16 diploid tumours, SPF was more than 10% in eight cases. DNA aneuploidy and high SPF are more common in histologically malignant tumours than benign tumours. SPF is a reflection of proliferation potential of a tumour and may have some role in prognostication of brain tumours.

How to cite this article:
Lahiri M, Sehgal S, Kak V K, Banerjee A K. Flowcytometric and histopathological correlation of primary intracranial neoplasms. Neurol India 2001;49:124


How to cite this URL:
Lahiri M, Sehgal S, Kak V K, Banerjee A K. Flowcytometric and histopathological correlation of primary intracranial neoplasms. Neurol India [serial online] 2001 [cited 2021 Sep 25];49:124. Available from: https://www.neurologyindia.com/text.asp?2001/49/2/124/1281




   »   Introduction Top

The distinction between benign and malignant neoplasm is considerably less trenchant in tumours of central nervous system than in those arising in other systems. Histological grading systems attempt to predict the tumour behaviour from microscopic appearances. Recognition of limitations of histological grading has led to a search for other objective complementary parameters, such as assessment of DNA content, karyotype, proliferative capacity, etc.[1] Flowcytometry is one of the efficient techniques for estimation of DNA content, ploidy and synthetic phase fraction of tumour cells. DNA aneuploidy is considered as a marker of malignancy. Synthetic phase fraction (SPF) in a tumour represents its proliferative activity and aggressive behaviour and hence bears prognostic significance.[2] In the present study, DNA ploidy and synthetic phase fraction of 52 primary intracranial neoplasms have been determined from fresh tissue and correlated with histopathological typing and grading.


   »   Material and methods Top

Fresh tumour tissue from 52 random surgical biopsies (28 malignant and 24 benign) was obtained from neurosurgical operations at Postgraduate Institute of Medical Education and Research, Chandigarh, during the period 1994-1996. The tumour tissue was minced using scalpel followed by pipetting and syringing. The minced tissue was incubated in Tween 20 and citric acid at room temperature. After washing, the cell pellet was resuspended and modified Krishan's buffer (sodium citrate - 0.25gm, RNAse - 0.005gm, Nonidet P40-0.75, propidium iodide - 0.125gm and distilled water 250 ml, cover buffer with alluminium foil and store in dark at 4oC) was added to 1x106 cells and was incubated for 30 minutes.[3] The suspension was centrifuged at 1,000 rpm for 5 minutes and the supernatant was removed. Fresh 1 ml of modified Krishan's buffer was added and each sample was syringed through a 100 micron nylon mesh. The cells were analysed in Becton Dickinson flowcytometer fitted with Consort 30 programme and 'Sober' software. Normal peripheral blood lymphocytes were used as the reference 2N peak. Percentage of diploid cells, proliferative cells (S,G2 and M) and DNA aneuploidy were evaluated. Histopathological study was performed on formalin fixed paraffin embedded routinely processed heamatoxylin and eosin stained sections. Special staining including immunostaining for glial fibrillary acid protein (GFAP), neurone specific enolase (NSE) and neurofibrillary protein (NFP), were applied in some cases. The tumours were classified and graded according to WHO classification.[4]


   »   Results Top

Detailed histological classification and DNA flowcytometry findings of all the cases is shown in [Table I]. There were 28 malignant (grade II to IV) and 24 benign (grade I) tumours. Out of the 28 malignant tumours, 12 were aneuploid (43%) and the rest were diploid (57%). Out of 16 diploid tumours, SPF was more than 10% in eight [Figure - 1].
All the histologically benign tumours in this study showed a diploid DNA content, with the exception of a pituitary adenoma which had a heterogenous population of cells. The SPF in all the benign cases was less than 10%, except in the single case of choroid plexus papilloma (S-phase 54%) and an atypical meningioma (S-phase 14%). All three pilocytic astrocytoma were diploid.
There were 20 cases of astrocytic tumours. Out of the two cases of anaplastic astrocytoma (WHO grade III), one was diploid and the other aneuploid. There was no difference in the histological features in these two cases. There were five cases of diffuse astrocytoma (WHO grade II) and two of these were aneuploid and three diploid cases [Figure - 2]. There were total 13(56%) cases of glioblastoma multiforme (GBM). Seven of them were diploid and the rest aneuploid [Figure - 3], with SPF ranging from 4.5 to 24%. Three cases of medulloblastomas were diploid with a high SPF and one aneuploid. One out of three oligodendrogliomas also showed DNA aneuploidy.
One case of a malignant tumour, which could not be classified further despite immunostaining, showed diploid DNA content with SPF of 4.97%. The association of DNA aneuploidy in the malignant tumour group was statistically significant when compared to the benign group of tumours (p<.0001). The SPF was also significantly higher in malignant tumours as compared to benign tumours (Mann Whitney U Test, p<.01).


   »   Discussion Top

DNA ploidy and SPF determinations have been proven to have a definitive role in prognostistication of urinary bladder, prostate and ovarian carcinomas.[2] The role of DNA FCM in brain tumours, however, still remains doubtful inspite of several studies.[5],[6],[7] Most of these studies were performed on archival materials and this may explain the contradictory results. The present study was prospective and was done on fresh tumour tissues.
In this study, all the benign tumours were diploid, except for a case of pituitary adenoma which showed heterogeneous population of cells. The presence of such two cycling cell lines has been previously described by other workers as well.[8] SPF was consistently low (<10%) in benign tumours as compared to malignant tumours, except in one case of meningioma which showed 14% SPF. This case showed high mitotic activity on histological section also and was labelled as an atypical meningioma. Other authors have also observed that DNA aneuploidy and SPF were significantly higher in atypical, anaplastic and recurrent meningioma,[9] and correlated well with histological features. However, a study from the Mayo clinic described that flowcytometry does not provide any prognostic information in meningiomas.[10]
One case of choroid plexus papilloma included in this study had a high SPF that could be due to the large number of S phase arrested cells.[11] There were 23 astrocytic tumours in this study. With increasing grade, the number of aneuploid cases were higher and the SPF was as high as 24% in one case of GBM. Other authors have also observed that anaplastic astrocytomas and GBM are more frequently aneuploid than low grade astrocytomas.[12] Association of high SPF with clinical aggressiveness has also been reported.[12]
There were three oligodendrogliomas, out of which two were diploid. Both these cases had high SPF (>10%). However, there was no histological difference between the diploid and aneuploid oligodendrogliomas. Kros et al concluded that flowcytometry had no value in predicting the biologic behaviour of these tumours.[13]
Three of the four cases of medulloblastoma were diploid with a high SPF. A similar observation had been made earlier by Mork et al,[14] who commented that the apparent ability to proliferate rapidly and malignant behaviours are perfectly compatible with diploid DNA content of tumour cells.
In conclusion, DNA aneuploidy and high SPF are more common in malignant tumours than histologically benign tumours. SPF is a reflection of the proliferation potential of a tumour and thus it may have some role in prognostication in cases of brain tumours.

 

  »   References Top

1.Barlogie B, Raber MN, Schuman J et al : Flowcytometry in clinical cancer research. Cancer Res 1983; 43 : 3982-3997.   Back to cited text no. 1    
2.Koss LG : FIowcytometry. ln : Diagnostic cytopathology and its histological bases.4th Edition, J.B. Lippincott Company, Philadelphia, 1992; 1613-1656.   Back to cited text no. 2    
3.Wresto RP, Greenebaum E, Deitch D et al : Deoxyribonucleic acid ploidy and cell cycle events in benign colonic epithelium peripheral to carcinoma. Lab Invest 1988; 58 : 218-225.   Back to cited text no. 3    
4.Kleihues P, Berger PC, Scheithauer BW : Histological typing of tumors of the central nervous system. In : International histological classification of tumors. World Health Organization. 2nd Edition, Springer-Verlag, Berlin, 1993; 5-10.   Back to cited text no. 4    
5.Dwaranath BS, Manogram PS, Das S et al : Heterogeneity in DNA content and proliferative status of human brain tumors. Indian J Med Res1994; 100 : 127-134.   Back to cited text no. 5    
6.Muller W : DNA estimations in cerebral tumors of man. Neuropath Pol 1972; 10 : 121-128.   Back to cited text no. 6    
7.Cho KG, Nagashima T, Barnwell S et al : Flowcytometric determination of modal DNA population in relation to proliferative potential of human intracranial neoplasms. J Neurosurg1988; 69 : 588-592.   Back to cited text no. 7    
8.Amiko M, Tribukait B, Wersall J : DNA ploidy and cell phase in human pituitary tumors. Cancer 1984, 53 : 1708-1713.   Back to cited text no. 8    
9.ZeIlner A, Meixensberger J, Roggendrof W et al : DNA ploidy and cell cycle analysis in intracranial meningiomas and haemangiopericytomas : a study with high resolution DNA flow cytometry. Int J Cancer 1978; 79 : 116-120.   Back to cited text no. 9    
10.Perry A, Stafford SL, Scheithauer BW et al : The prognostic significance of MIB-1, p53, and DNA flowcytometry in completely resected primary meningiomas. Cancer 1988; 82 : 2262-2269.   Back to cited text no. 10    
11.Coons SW, Johnson PC, Haskett D et al : Flowcytometric analysis of deoxyribonucleic acid ploidy and proliferation in choroid plexus tumors. Neurosurgery 1992; 31 : 850-856.   Back to cited text no. 11    
12.Danova M, Giaretti W, Merlo F et al : A prognostic significance of nuclear DNA content in human neuroepithelial tumours. Int J Cancer 1991; 48 : 663-667.   Back to cited text no. 12    
13.Kros JM, Van Eden CG, Vissers CJ et al :Prognostic relevance of DNA flowcytometry in oligodendroglioma. Cancer1992; 69 : 1791-1798.   Back to cited text no. 13    
14.Mork SJ, Laerum OD : Modal DNA content of human intracranial neoplasms studied by flowcytometry. J Neurosurg 1980; 53 : 198-204.   Back to cited text no. 14    

 

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