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Original Article

Critical appraisal of cytological nuclear grading in carcinoma of the breast and its correlation with ER/PR expression
ABSTRACT Background: Cytological nuclear grading is one of the several key prognostic factors that should be addressed in cytological analysis of breast carcinomas. Aims: To evaluate different cytological nuclear grading methods on ne needle aspirates of breast carcinomas and its correlation with histopathological nuclear grading as well as with the immunocytochemical expression of estrogen receptor (ER) and progesterone receptor (PR). Materials and Methods: The smears from 30 cytologically proven cases of breast carcinoma were graded by - Nottinghams modication of Scarff Bloom Richardson, Fishers modication of Blacks nuclear grading and Robinsons cytological grading methods. 18 cases were available for correlation with histology grading. Results: Robbinsons cytological grading system was found to have the best correlation with histopathology grades (P < 0.001) as well as ER (P = 0.003) and PR (P = 0.001) expression on smears. Conclusions: The Richardsons cytology grading method is recommended for cytological nuclear grading along with ER / PR expression, without which the cytological diagnosis of breast carcinoma is incomplete. Key words: Breast carcinoma, nuclear grading; estrogen receptor; progesterone receptor; ne needle aspiration cytology.

Introduction
Breast cancer is the second most common cancer among Indian females, next only to cervical cancer. The cumulative incidence in females until 64 years of age is 1-2%.[1] In the past ten years, the importance of fine needle aspiration cytology (FNAC) has been well documented in the diagnosis of breast lesions. Fine needle aspiration has become a standard technique in evaluation and frequently, the source of primary diagnosis.[2] Noninvasive assessment of the aggressiveness of the cancer would therefore be valuable. It will avoid morbidity due to surgical intervention, particularly in low-grade tumors. However, very few studies have been conducted on cytological nuclear grading. Well-established prognostic factors include staging of the tumor, histological grade, lymph node status, and estrogen receptor (ER) and progesterone receptor (PR) status. Cytological nuclear grading is feasible and reproducible and may assume prime importance for patients who may receive chemotherapy prior to the resection of the tumors, and in those who present with metastases.

Materials and Methods

The present study was conducted in the Departments of Pathology and Surgery, Lady Hardinge Medical College and Smt. Sucheta Kriplani Hospital, New Delhi. Thirty cases of primary breast carcinomas were selected that had been diagnosed with the aid of FNAC. FNAC was performed using the 22-gauge needle. For nuclear grading, smears were stained for Papanicolaou (Pap) and Giemsa. At least four smears were prepared on poly-L-lysine-coated slides for immunocytochemistry (ER/PR) using avidin-biotin in the DAKO LSAB kit. For scoring of ER/PR, the simplified category formulated by Barnes and Millis in 1995 was used wherein negative and weak stainers were clubbed as negative, and moderate and strong stainers were grouped as positive.

Results
The patients ranged in age from 25-90 years; the mean age

VIDHI BHARGAVA, MANJULA JAIN, KIRAN AGARWAL, THOMAS S, SMITA SINGH

Department of Pathology, Lady Hardinge Medical College and Associated Hospitals, New Delhi, India
For correspondence: Dr. Manjula Jain, D-37, Hauz Khas, New Delhi - 110 016, India. E-mail: dr_manjulajain@rediffmail.com

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Bhargava, et al.: Cytological nuclear grading in carcinoma breast

was 46.7 years. Most of the cases were in stage T4 (50%) and T2 categories (33.3%); no case of T1 was seen. The size of the tumor varied from 2 to 15 cm; the maximum number of cases (36.6%) were in the range of 2.6-5 cm. Of the 30 cases, 20 (66.6%) had ipsilateral axillary lymph nodes, two had contralateral axillary nodes, and two cases had enlarged supraclavicular nodes. None of the cases showed distant metastasis. Cytological nuclear grading was done by the following three methods [Table 1]: 1. Nottingham modification of the Scarff Bloom Richardson (SBR)s method:[3] This grading system is based on three features: tubule formation, nuclear pleomorphism, and mitosis/10 HPF. Tubule formation in histopathology sections is considered equivalent to the presence of clusters in cytology smears. 2. Fishers modification of Blacks nuclear grading (NG):[4] This grading system is based on four nuclear features which include nuclear size, nuclear membrane, chromatin, and the presence/ absence of nucleoli. 3. Cytological grading by Robinsons method:[2] This method has exclusively been used for cytology; six features are assessed, i.e., cell dissociation, cell size, cell uniformity, nucleoli, nuclear margin, and chromatin [Figures 1-3]. The SBR method indicated that nine cases (30%) belonged to nuclear grade I (NG1), 18 cases (60%) belonged to grade II (NG2), and three cases (10%) belonged to grade III (NG3) [Table 1]. By using Fishers modification of Blacks NG, two cases (6.6%) were NG1, 13 cases (43.4%) were NG2, and 15 cases (50%) NG3 [Table 1]. According to the results of Robinsons method, the maximum number of cases belonged to NG3 (46.6%). There were ten cases (33.3%) in NG2 II and six (20%) in NG1 [Table 1]. Hence, 46.6% of the cases were NG III according to Robinsons classification, 50% cases NG3 according to Fishers modification of Blacks NG, and only 10% as NG3 according to the SBR method. This discrepancy could be due to the
Table 1: Cytological nuclear grading by Scarff-Bloom Richardson, Fishers modification of Blacks NG and Robinsons method.
Nottingham modification of SBR
Grade I Grade II Grade III 9 (30) 18 (60) 3 (10)

difficulty in assessing mitoses using cytology, which is one of the features of the SBR grading system. In low-grade tumors, i.e., NG1, the SBR method showed more cases (30%) compared to Fishers modification of Blacks NG and Robinsons methods, which showed 6.6 and 20% respectively. This could be because the SBR method uses very few criteria to assess nuclear features, i.e., only variations in size and shape. Nuclear margin, chromatin, and nucleoli, which are important for grading, are not assessed in this method. All 14 cases of NG3 tumors showed pleomorphic cells with marked variations in shape, prominent nucleoli, and clumped chromatin. In NG3 cases, mixtures of single and cell clusters were observed in eight out of 14 cases, and only single cells were observed in five out of 14 cases. Mastectomy specimens were received in 18 out of 30 cytologically proven cases of primary breast cancer. Tumor grading by the Nottingham modification of SBR was subsequently done on the sections. Of 18 cases, nine (50%) belonged to NG3, seven (38.8%) to NG2 and two cases (11.1%) to NG1. All cases (100%) that had been judged histopathologically to be NG1 by the SBR method were also found to be NG1 by SBR and Robinsons methods by cytological examination. In contrast, only 66.6% cases histopathologically judged to be NG1 by the SBR method were NG1 according to Fishers modification upon cytological investigation. Correlation was seen between NG2 tumors in 66.6% cases cytologically classified by the SBR method and 83.3% of the cases histopathologically classified as such by Fishers modification and Robinsons methods. In NG3 tumors, cytological results of the SBR method of only 22.2% of the cases correlated with the histopathological results of Fishers modification in 77.7% of the cases. The best correlation was observed with Robinsons method where cytology of 88.8% cases correlated with histopathology by SBRs method [Table 2]. Therefore, histopathological results of Robinsons method correlated best with those of the SBR method in all the three nuclear grades and this correlation was found to be statistically highly significant (P < 0.001) and therefore, we used this method only for further correlations. On immunocytochemistry of the 30 cases, six cases (20%) were found to be ER-positive and five cases (16.6%) were PR-positive. A significant positive correlation was seen with nuclear grade and tumor size, however no correlation was seen either with
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Fishers modification of Blacks NG

2 (6.6) 13 (43.4) 15 (50)

Cytological grading by Robinsons method

6 (20) 10 (33.3) 14 (46.6)

Figures in paranthesis indicate percentage

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Bhargava, et al.: Cytological nuclear grading in carcinoma breast

Table 2: Correlation of Scarff-Bloom Richardson on histopathology with cytological nuclear grading

Nuclear grading on histopathology Scarff-Bloom Richardson
I II III

No. of cases (n = 18)

Nuclear grading on cytology Scarff-Bloom Richardson

I II 2 4 (66.6) III 7 2 (22.2) I 2 (66.6) 1 3 (100) -

Fishers modification
II 5 (83.3) 1 III 2 7 (77.7) I 3 (100) -

Robinsons
II 5 (83.3) 1 III 1 8 (88.8)

3 6 9

Figures in paranthesis indicate percentage

Figure 1: Robinsons grade I - monomorphic cells with smooth nuclear membrane (score 1) and indistinct nucleoli (PAP, 1000).

Figure 2: Robinsons grade II - moderate variation in shape of cells (score 2), coarse chromatin with single cell showing nucleolus (Giemsa, 1000).

ipsilateral axillary nodes or with metastasis. On correlating the Robinsons nuclear grading with ER and PR status, 66.6% of NG1 lesions and 20% of NG2 lesions were found to be ER-positive (P = 0.003), whereas 66.6% of NG1 and 10% of Grade II lesions were PR-positive (P = 0.001). All NG3 lesions were negative for both ER and PR. Hence, there was a significant correlation between ER and PR positivity and the lower nuclear grades.

Discussion
As neoadjuvant therapy including preoperative chemotherapy, herceptin, and tamoxifen, is becoming increasingly common for the treatment of early breast cancer, it is desirable to grade tumors before surgery so that the most appropriate medical regimen can be selected. Hence, cytological grading may assume prime importance for patients who may receive chemotherapy prior to resection of the tumor and in those who present with metastases. In the present study, our findings were compatible with the study of Howell et al,[5] who used the cytology-based SBR method and found significant correlation with histopathological results. They used the SBR grading system for 35 cases of breast carcinomas for cytological as well as
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Figure 3: Robinsons grade III - pleomorphic cells with irregularly clumped chromatin (score 3) and nuclear budding (PAP, 1000).

histological investigations. They assigned the same SBR score to 74.3% of biopsy tissue and 65.7% of fine needle aspirates. The cytological grade could be used to predict the histological grade in 57.1% of the cases as compared to the present study, where the cytological grade could predict the histological grade in 50% of the cases. However, a difficulty in identifying tubule formation and mitoses was observed on FNAC, which has been explained by the fragility of cells and the fewer number of cells.[6] There are limitations with SBR grading as it has few criteria to assess nuclear features.

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Bhargava, et al.: Cytological nuclear grading in carcinoma breast

Nuclear margin, chromatin, and nucleoli are not evaluated. There is an inability to identify a few high-grade and lowgrade carcinomas in aspirates. Fishers modification of Blacks NG was used more accurately in cytology than SBR, since it emphasizes nuclear features which are highlighted to greater extents in Pap and Giemsa stained smears. No studies are available which correlate the two cytological methods. Dabbs et al.[7] and Zoppi et al.[8] have used Fishers modification. Dabbs et al.[7] observed that grading of tissue specimens and aspirates revealed concurrence of nuclear grades in 95% of the cases using Fishers modification of Blacks NG. Zoppi et al.[8] found an agreement between cytological and histological nuclear grading in 70.37% of tumors. The lack of correlation in 29.63% of the tumors was accounted for by tumor heterogeneity and observer subjectivity when assigning the nuclear grade. These studies concluded that this method can be easily established on fine needle aspirates. Ohri et al.[9] analyzed 50 cases of breast carcinoma. They found that agreement between simplified Black cytological grade and the histological grade was 95%. In the present study, the best correlation was observed with Robinsons method where 100% cases of NG1, 83.3% cases of NG2 and 88.8% cases of NG3 correlated with the SBR methods histopathological results (P < 0.001). This high correlation between these two methods could be due to the fact that mitosis, a feature that is difficult to assess by cytological examination, can be assessed well by histopathological examination. So, the grading by SBRs method can be more accurately assessed on histopathology. Robinson et al.[2] also observed that the cytological grade corresponded well with histological grade in 281 cases. On histopathological grading, 32% tumors were found to be NG1, 43% NG2, and 25% NG3. On cytological grading, 34% tumors were NG1, 44% grade 2, and 22% were grade 3. On regression analysis, every cytological feature was found to have a significant correlation to the histological score. Meena et al.[10] compared 100 cases of breast carcinoma that were graded cytologically by using Robinsons grading system with 71 cases which were graded histologically by the modified Bloom Richardson system. They found that the sensitivity and specificity of the cytological grading system were 90.77 and 84.42%, respectively. The results showed that both Robinsons cytological grading system and Fishers modification of Blacks NG had a statistically significant correlation with histopathology but Robinsons grading system correlated better (P < 0.001) than Fishers modification of Blacks NG (P = 0.001). This is because Robinsons method has two more criteria, i.e., cell dissociation and uniformity (variation in cell shape and size) which are not present in Fishers modification of Blacks NG. No studies are available to correlate the immunocytochemical

expression of ER/PR with cytological grades, but studies by Ruibal et al.[11] and Thike et al.[12] showed an association of the histological tumor grade with the ER/PR content. A transition from lower to higher histological grades was accomplished by a decrease in the ER/PR content. The same result was obtained with nuclear grades upon cytology and immunocytochemical expression of ER/PR in this study. ER positivity was seen in 66.6% of NG1 lesions and 20% of NG2 (P = 0.003) whereas 66.6% of NG1 and 10% of NG2 lesions were PR-positive. All NG3 lesions were negative for both ER and PR.

Conclusions
In this study, we compared Robinsons method, Fishers modification of Blacks nuclear grading, and the SBR method for cytological nuclear grading in breast carcinomas with SBR method on histology. The Richardsons cytology grading method showed the best correlation with the histology grading. Therefore, we recommend this method for cytological nuclear grading along with ER / PR expression without which the cytological diagnosis of breast carcinoma is incomplete.

References
1. 2. National Cancer Registry Programme. ICMR; July 1992. Robinson I, McKee G, Nicholson A, DAcy J. Prognostic value of cytological grading of ne needle aspirates from breast carcinoma. Lancet 1994;343:947-9. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer.I. The value of histological grade in breast cancer: experience from a large study with long term follow up. Histopathology 1991;19:403-10. Fisher ER, Redmond C, Fisher B. Histologic grading of breast cancer. Pathol Annu 1980;15:239-51. Howell L, Gandou-Edwards R, OSullivan D. Application of SBR tumor grading system to ne needle aspirates of breast. Am J Clin Pathol 1994;101:262-5. Wallgren A, Zajicek J. The prognostic value of aspiration biopsy smear in mammary carcinoma. Acta Cytol 1976;20:479-85. Dabbs D. Role of nuclear grading of breast carcinomas in ne-needle aspiration specimens. Acta Cytol 1993;37:361-6. Zoppi JA, Pellicer EM, Sundblad AS. Cytohistological correlation of nuclear grade in breast carcinoma. Acta Cytol 1997;41:701-4. Ohri A, Jetly D, Kaushambi S, Bansal R. Cytological grading of breast neoplasia and its correlation with histological grading. Indian J Pathol Microbiol 2006;49:208-13. Meena SP, Hemrajani DK, Joshi N. A comparative and evaluative study of cytological and histological grading system prole in malignant neoplasm of breast: An important prognostic factor. Indian J Pathol Microbiol 2006;49:199-202. Ruibal A, Arias JI. Histological grade in breast cancer: Association with clinical and biological features in a series of 229 patients. Int J Biol Markers 2001;16:56-61. Thike AA, Chng MJ. Immunohistochemical expression of hormone receptors in invasive breast carcinoma: Correlation of results of H-score with pathological parameters. Pathology 2001;33:21-5.

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Source of Support: Nil, Conict of Interest: None declared.

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