Howell’s cytological grading of breast carcinomas-comparison with histological grading and its association with lymph node metastasis

Background: Determining histological grade of breast carcinomas before mastectomy is necessary to decide about neoadjuvant chemotherapy. Grade obtained from fine needle aspiration cytology samples will help in such situations particularly when core needle biopsy is not done routinely as inresource-poor setups. Methods for doing such gradingare still under study. We performedgrading of breast carcinomas in Fine Needle Aspiration smearsby Howell’s method and correlatedthe resultswith modified Bloom-Richardson histological grade in mastectomy specimens. For the first time, we also studied the prognostic significance of Howell’s grade by studying its association with lymph node metastasis. Materials and methods: Fifty cases of Invasive carcinomano special type of breast in which both mastectomy and fine needle aspiration cytology were done between 2013 and 2015 were included in our study. Howell’s grading was done in Papanicolaou, Haematoxylin & eosin and May-Grunwald Giemsa stained cytology smears and correlated with modified Bloom Richardson histological grading in mastectomy specimens. Results: The two methods had same grade in 34 cases (68%) and a kappa agreement value of 0.505. They showed a good positive correlation (Spearman correlation coefficient 0.732) and significant association (chi-square test, p-0.0001). 12 cases were under-graded and four were over-graded. Lymph node metastasis significantly increased with increase in Howell’s cytological grade (p-0.018). Conclusion: Howell’s cytological grading is a simple method to grade breast carcinomas before mastectomy. This method has a good concordance with histological grading.The strong association with lymph node metastasis indicates the prognostic significance of this grading method. KeywordsBreast carcinoma, Histological grading, Fine needle aspiration cytology, cytological grading, lymph node metastasis ...................................................................................................................................................


Introduction
Breast cancer is the most common cancer among females globally as well as in India [1]. Histological grading is usually done in breast carcinomas by Nottingham modification of Bloom Richardson (NBR) grading method and is an important prognostic factor [2,3]. Apart from being a prognostic factor, grading also helps in choosing between the different treatment options available for breast carcinoma [4,5]. Hence it is essential to obtain the grade of breast carcinoma early at the time of diagnosis itself. Although core needle biopsy can be used for this purpose, be it is not done routinely in the resource-poor setup [5]. Hence this study aimed at grading invasive carcinomano special type of breast in FNAC samplesby Howell's method and compared it with NBR histological grading obtained from mastectomy specimens in the same cases.
We also studied the prognostic significance of Howell's grading method by studying its association with lymph node metastasis. To our knowledge, this aspect has not been studied in the past.

Materials and Methods
Place and Type of study-This study was done in the central laboratory of a tertiary health care hospital in South India by retrospectively studying cases of invasive carcinoma-no special type of breast diagnosed between 2013 and 2015.
Inclusion criteria-Cases of invasive carcinoma-no special type of breast diagnosed between 2013 and 2015 having both FNAC smears and mastectomy specimens were included in our study.
Exclusion criteria-Cases in which mastectomy were done after chemotherapy or radiotherapy were excluded from our study. This is because such therapies could have changed the grade of the tumor. Cases with inadequate material and those cases in which the slides/ blocks were not available were also excluded. Grading procedure-Since mitoses and tubules are difficult to identify in FNAC smears, Howell had modified the criteria to grade breast carcinomas in FNAC smears [7].

Sample collection and Sampling methods-
Both microacini as well as branching elongated three dimensional tubular structures were included as tubule formation in cytologicalgrading. Mitotic count threshold for each score was also reduced. Howell's modified criteria are given in Table 1. Criteria used in NBR histological grading is given in Table 2.
Metaphase, anaphase and telophase mitotic figures were included for mitotic count. Olympus CX21 microscope with field view number 18 and field diameter 0.45 mm for the high power view was used. Sections from axillary lymph node were examined for metastasis.
Statistical methods-Statistical analysis was done using SPSS software version 21.0. Association between the two grading systems and association of Howell's grade with lymph node metastasis wereassessed using Chisquare test.
Kappa value of agreement was used to measure the strength of agreement between the two grading systems.
Correlation between the two grading systems was assessed by Spearman rank correlation coefficient. p value less than 0.05 was considered statistically significant.

Results
Fifty cases were included in our study based on the inclusion and exclusion criteria. Grade II was the most common NBR histological grade in our study (21 cases, 42%) followed by grade III (15 cases, 30%) and grade I (14 cases, 28%).
Cytological grading was also done by assessing the three parameters tubule formation, pleomorphism and mitotic count (Figs 1-3). In cytological grading also grade II was the most common (25 cases, 50%). However grade I was the next most common grade in this method (16 cases, 32%) followed by grade III (9 cases, 18%).
12 of the 14 histological grade I cases (85.7%), 15 of the 21 histological grade II cases (71.4%) and 7 of the 15 histological grade III cases (46.7%) were graded similarly in the Howell's cytological method (Table 3). Thus the concordance was maximum in the grade I cases and least in the grade III cases.
The overall concordance was 68% with a kappa agreement value of 0.505. The two methods showed good positive correlation in Spearman rank correlation test with a rho value of 0.732.There was a strong association between these two grading methods in chi-square test with a p value of 0.0001. Overall 12 cases (24%) were under-graded and 4 cases (8%) were over-graded in the Howell's cytological grading method.
Thus under-grading was more common than over-grading in Howell's method. Among the total 50 cases, 24 cases (48%) had lymph node metastasis. Five out of the 16 Howell's grade I cases (31.3%), 11 out of the 25 Howell's grade II cases (44%) and eight out of the nine Howell's grade III cases (88.9%) had lymph node metastasis.
Thus the incidence of lymph node metastasis increased with increase in Howell's grade and this association was found to be statistically significant in chi-square test with a p value of 0.018.  Grade 1 -Total score 3 to 5, Grade 2 -Total score 6 to 7. Grade 3 -Total score 8 to 9   grading was common in their study as well [15]. In our study also, concordance was least for the histological grade III cases (46.7%).
Thus under-grading seems to be more common than over-grading in Howell's method and was more common in the high grade tumours in our study.
Although seen in a limited number of cases, this can be considered as a minor disadvantage of this method. We further observed that mitotic count score was decreased in most of these under-graded cases and was the major