 |
       |
|
|
ARGYROPHILIC NUCLEOLAR ORGANIZER REGIONS (AgNORs) IN PRECANCEROUS AND CANCEROUS LESIONS OF CERVIX
Ritu Singhal*, L K Pandey**
*Senior Resident, Dept. of Pathology; **Professor and Head, Dept. of Obst. and Gynaecology, IMS, BHU.
Argyrophilic nucleolar organizer regions (AgNORs) staining was employed in 112 female patients including 10 normal age matched controls by a method described by Crocker and Smith. The observations revealed significant increase of AgNOR count in cancerous lesions than in benign and pre cancerous lesions. AgNOR staining and scoring is a simple, inexpensive and useful adjunct to routine histopathology, though scores can not be standardized and fixed for a particular lesion as there are intra laboratory variations.INTRODUCTION
Nucleolar organizer regions (NOR) are components of ribosomal DNA that transcribe to ribosomal RNA. The structures studied in this technique are Argyrophilic non Histone Proteins whose silver stainability serves as an indicator for transcriptional activity of NORs.[1] Although their exact biological function is still under study, it has been suggested that the number of AgNOR per cell may be a marker of cellular activity including malignancy. In the present study AgNOR count in different cervical lesions was done and compared between benign, pre cancerous and cancerous lesions. The main objective of the present study is to evaluate the role of AgNOR in discriminating benign and precancerous lesions from cancerous lesions.
MATERIAL AND METHODS
Female patients clinically diagnosed as carcinoma cervix form the subject matter of the present study. Total 112 female patients were studied, out of them 102 were of cervical pathology and 10 were used as normal age matched controls. Detailed histological examination in haematoxylin and eosin was done along with the AgNOR studies which was evaluated in relation to clinical stage of the disease, histological type and histological grade.
Staining Procedure for nucleolar organizer regions (NORs)
This was carried out as per the method of Crocker et al [2]with little modification. After dewaxing and hydrating three micron thick paraffin sections to deionized water, sections were incubated in a silver colloidal solution for 40 minutes in the dark at room temperature. Silver colloidal solution was prepared by mixing one vol. of 2 gm/100 ml gelatin in 1% formic acid solution to two volumes of 50% aqueous silver nitrate solution. The stained slides were washed for three minutes in three changes of distill water. Counter staining was done with 0.01% safranin. Sections were dehydrated, cleaned and mounted in DPX. AgNOR stained as black dots within the nucleus. Their number was counted in one hundred adjacent cells and mean AgNOR count was calculated for each histological lesions.
RESULTS
Total of 112 cases were studied in detail as shown in Table 1. Carcinoma cervix accounted for majority of cases followed by cervical intra epithelial neoplasia. Age distribution is shown in Table 2. Majority of the cases of Ca cervix were in 41-50 years of age groups. CIN occurs nearly adecade earlier than infiltrating malignancy. Mean age of CIN being 31.5 years.
Cases No. (%) Infiltrating Ca Cervix of varying clinical stages 80 Cervical intra epithelial neoplasia of all grades 12 Chronic cervicitis 10 Normal age matched controls 10 Total 112
Age (in year) No. of cases Percentage 21-30 02 2.5 31-40 27 33.75 41-50 32 40 51-60 11 13.75 61-70 06 8 > 70 02 2.5
Fig. 1 Microphotograph of squamous cell carcinoma showing AgNOR dots(AgNOR x 1000x). AgNOR counts were done in normal age matched control, chronic endocervicitis, cervical intra-epithelial neoplasia and different stages and grades of carcinoma.
In normal squamous epithelium mean AgNOR counts were highest at parabasal level (2.1-2.9 with the mean of 2.3), less at the basal cell level (1.2-1.8). Reliable counts above this level were not possible due to nuclear maturation and pyknosis. In endocervical and glandular epithelium AgNOR count ranged from 1.0-1.3 with the mean of 1.2.
No significant difference in AgNOR counts was noted between normal age matched control and chronic cervicitis but significant increase of AgNOR counts was noted from grade I CIN to grade III CIN to Ca cervix.
AgNOR counts in different grades of cellularity, mitosis, necrosis and pleomorphism are shown in Table 4. Histological grade has been assessed on the basis of cellularity, mitosis, necrosis. Hyperchromatism and pleomorphism and tumour was graded from grade I to grade III. AgNOR in different histological grades are shown in Table 5. There is significant increase of AgNOR with increasing grade.
Type No. of patients Range Mean ± SD Chronic endocervicitis 10 0.81 ± 1.13 0.97 ± 0.10 Age matched controls 10 0.89 - 1.20 1.02 ± 0.09 CIN 12 — — Grade - I 04 0.76 - 0.98 0.9 ± 0.08 Grade - II 64 0.97 - 1.97 1.2 ± 0.42 Grade - III 04 1.83 - 4.27 2.69 ± 0.96 Ca Cervix 80 — — Squamous cell Ca 71 3.0 - 5.05 3.97 ± 0.69 Large cell nonkeratinizing Scc 27 3.0 - 4.19 3.33 ‘ 0.47 Keratinizing Scc 32 3.11 - 5.01 4.34 ± 0.49 Small cell Scc 12 3.26 - 5.05 4.38 ± 0.54 Adeno CA 9 2.63 - 4.26 3.10 ± 0.43 Ca Cervix IIa 4 2.98 - 4.44 3.59 ± 0.83 IIb 11 2.63 - 4.02 3.16 ± 0.55 IIIa 25 2.98 - 4.11 3.85 ± 0.58 IIIb 33 3.02 - 4.34 3.94 ± 0.68 IV 7 3.43 - 5.05 4.87 ± 0.16 CIN - Cervical intra epithelial neoplasia; Ca cervix - Carcinoma cervix; SCC- Squamous cell carcinoma
Fig. 2 Microphotograph of adenocarcinoma showing AgNOR dots and gland formation. ( AgNOR x 500x.) DISCUSSION
In this study we evaluate the significance of AgNOR in relation to clinical stage, morphological type and histological grade of carcinoma cervix and in different grades of cervical intra epithelial neoplasia.
AgNOR studies were done in 112 patients. Our counts in normal squamous epithelium correlated well with the counts reported by Cullimore et al[3] and counts in endocervical epithelium was in accordance with the observations of Egan et al[4]. In AgNOR studies no significant difference of AgNOR counts was noted between normal cervix and non specific chronic endocervicitis.
Range Mean ± SD Amount of cellularity + (Sparse) 2.63 - 4.11 3.92 ± 0.76 ++ (Intermediate) 2.98 - 4.99 4.06 ± 0.77 +++ (Rich) 3.02 - 5.05 3.55 ± 0.63 Score of mitosis + (< 2/hpf) 2.63 - 4.44 3.54 ± 0.63 ++ (2-4/hpf) 3.12 - 4.69 4.07 ± 0.77 +++ (> 4/hpf) 3.54 - 5.05 4.27 ± 0.60 Degree of necrosis + 3.02 - 5.05 3.93 ± 0.71 ++ 2.98 - 5.01 3.86 ± 0.75 +++ 2.63 - 4.98 3.56 ± 0.70 Degree of pleomorphism + 2.63 - 4.08 3.34 ± 0.55 ++ 2.98 - 4.64 3.99 ± 0.67 +++ 3.11 - 5.02 4.19 ± 0.76
Histological grades Range Mean I 2.63 - 4.02 3.3 ‘ 0.45 II 3.11 - 5.02 4.1 ‘ 0.62 III 3.84 - 5.05 4.63 ‘ 0.63 In cervical intra epithelial neoplasia no significant increase of AgNOR count has been observed between CIN I and CIN II, but significant increase is found in CIN III and Ca insitu. These observations are in accordance with the observation of other workers.[4,5]
AgNOR counts were higher in carcinoma cervix, more in squamous cell Ca than in glandular carcinoma cervix. These findings correlate well with the findings of New bold et al[6]. In carcinoma cervix not only the number of NoRs increases, they become prominent and larger in size, many a times appearing as giant size and they are also seen in the nucleoli in the nucleus. This correlated well with the study by Murty et al[7].
Different morphological characters of carcinoma cervix were studied in detail. Depending upon the cellularity, Ca was divided into sparse, intermediate, and richly cellular Ca. However, mean AgNOR count did not vary significantly between three groups of cellularity, nor there is consistent pattern of NOR number from low to highly cellular tumours. There was also no correlation between tumour necrosis and AgNOR counts.
There was consistent rise of AgNOR counts from low grade mitotic cancer to the cancers showing large number of mitosis. Similarly AgNOR counts increases with increase in degree of pleomorphism.
There was also a significant increase in NOR from grade I to grade III Ca. This correlated well with the study by Murty et al[7]. So AgNORs position particularly ectopic AgNOR or abnormal AgNOR, distribution and number reflect degree of differentiation and transcriptional activity.[8]
Inspite of all limitations of two dimensional grading system, with the limited resources of developing countries particularly in India, histological grading appears to be most important morphological indicator of probable behaviour and it correlates with the AgNOR number, their topography within the nuclei and distribution pattern.
CONCLUSION
Quantitative study of AgNOR is valuable in field of oncologic histopathology and the size, number and position of NOR may provide useful morphometric data over and above morphological criteria. So it can be used as a marker of cellular activity as well as malignancy and therefore it can be utilized for discriminating benign, precancerous and cancerous lesions.
REFERENCES
- Howell WM. Selective staining of nucleolar organizer regions (NORs). In Busch H, Rothblum L, eds. The cell nucleus, New York : Academic Press. 1982; XI : 89-142.
- Crocker J. Nucleolar organizer regions. In : Underwood JCE, ed. Current topics in pathology : Pathology of the nucleus, Berlin : Springer Verlag. 1990; 91-149.
- Cullimore JE, Rollanson TP, et al. Nucleolar organizer regions in adenocarcinoma in situ of the endocervix. J Clin Pathol 1989; 42 : 1276-80.
- Egan M, Freeth M, et al. Relationship between cervical intra epithelial neoplasia and the size and number of AgNOR. Gynaecol Oncol 1990; 36 : 30-3.
- Rowlands DC. NOR in cervical intra epithelial neoplasia. J Clin Pathol 1988; 41 : 1200-2.
- New Bold KM, Rollason TP. Nucleolar organizer regions in glandular and squamous carcinoma of cervix. J Clin Pathol 1989; 441-2.
- Murty VVS, Mitra et al. Nucleolar organizer regions in patient with pre cancerous and cancerous lesions of uterine cervix. Cancer Genet and Cytogenet 1985; 18 : 275-9.
- Reeves BR, Casey G, Harris H. Variations in the activity of nucleolar organizers in different tissues, demonstrated by silver staining of human normal and leukemic cells cancer genet. Cytogenet 1982; 6 : 223-30.
 |
