Breast cancer is the most common cancer and a leading cause of death in women. Locally invasive breast cancers are often treated with neoadjuvant chemotherapy (NAC). Pathological complete response (pCR) is defined as the absence of invasive breast cancer (in breast as well as in nodes) after the completion of chemotherapy. Disease-free and overall survival depend on the response to standard NAC in invasive breast cancer.¹ Invasive breast cancer shows different responses to NAC, ranging from complete absence of disease to extensive residual disease. More than 15 grading systems, including the Miller-Payne system, AJCC staging system, residual cancer burden (RCB) system, Neo-Bioscore, etc., have been proposed to evaluate the pathological response. Among them, the RCB index developed by the MD Anderson Cancer Center is most commonly used.^2-5 RCB can be calculated by a web-based calculator as a score or category. It has four categories, which include RCB 0 showing pathological complete response, RCB 1 showing minimal residual disease, RCB 2 showing moderate residual disease, and RCB 3 showing extensive residual disease. The index is based on several histopathological parameters, such as two-dimensional size and cellularity of the tumor bed, percentage of in situ cancer, number of involved nodes, and diameter of the largest metastasis. It provides more quantitative information about the response, unlike other systems, which are more descriptive.² However, there is concern regarding the subjectivity of this grading system, especially in assessing cellularity.³ To date, only a few studies have verified the feasibility of the RCB index in stratifying the prognosis of patients treated with NAC, with no study on the Indian population.^4,5

The aim of the present study was to assess the inter-observer agreement of the RCB index when slides were examined by four pathologists with different levels of expertise in breast pathology. Several clinical and pathological parameters were also evaluated.

In the present study, 49 patients were included (mean age of 52 years), out of whom 39 (80%) were above 45 years. There was no statistically significant association between age and NAC response (P=0.38). The mean tumor bed diameter was 4.8 cm. The size of the tumor bed dimension was divided into two groups: those ≤5 cm (26 cases, 53%) and those >5 cm (23, 47%). No statistical significance was found with the size of the tumor bed and response to NAC (P-value=0.8). There was no statistically significant correlation between neoadjuvant chemotherapy cycles (fewer than or more than 4 cycles) with RCB categories (P-value=0.49). We could retrieve the IHC data of 36 cases, among whom 20 (56%) cases were of Luminal subtype, 8 (22%) were HER2-neu positive, and 8 (22%) were triple-negative (Table 1). The pairwise observer agreement between the pathologists ranged from 57.1% to 83.67%, and the pairwise kappa value ranged from 0.43 to 0.78 (Table 2).

The cases were reviewed independently by four pathologists without knowing the original diagnosis. Individual pathologists’ diagnosis (RCB score), as a percentage of the total number of cases, is depicted in Figure 1. In the analysis, the four pathologists were treated similarly and none of them was considered to provide a reference score. The RCB index of all the cases as assigned by the four pathologists was statistically calculated by Fleiss’ kappa statistics, and the overall agreement was calculated to be 82.6%. The free marginal kappa was 0.76, and the fixed marginal kappa was 0.71 (Figure 1).

Many grading systems have been proposed to report post-neoadjuvant chemotherapy-treated surgically excised specimens of breast cancer, such as the RCB index, Miller-Payne system, Residual Disease in Breast and Nodes, etc.^2,4,5 Among them, the RCB index formulated by the MD Anderson Cancer Center is the most relevant and commonly used.² Sahoo et al. have argued that almost all available post-NACT assessment methods at present are similar to each other, except for RCB, which is a web-based system⁷. Sejben et al. recommend the use of the RCB index in histopathology, as this classification makes the best distinction in outcomes.⁸ It has been thoroughly explained with directions on how to use this grading system, and a freely accessible online calculator is available.²

Out of all our cases, 10 belonged to the age group less than 45 years, and the rest were above this age. Twenty cases belonged to the luminal subtype, 8 to the HER2-neu positive subtype, and 8 to the triple positive subtype. Also, 26 patients had received four cycles of neoadjuvant chemotherapy, and the rest of them had received 5 to 8 cycles of the same chemotherapy. The largest tumor dimension was less than or equal to 50mm for 26 cases. All these parameters have shown that they have no significant impact on the response to chemotherapy as graded using the RCB index. However, Lv et al. showed that high histological grade, negative HER2 status, and lymph node metastasis, positive HER2 status, and taxane-based regimens were significantly associated with the achievement of pCR with NAC.⁹ Tang et al. have demonstrated that among 84 patients aged less than 45 years and 189 patients aged ≥45 years, 12 and 26 patients achieved pCR, respectively. The P-value was not statistically significant. Additionally, among 230 patients with a tumor size less than 5 cm and 43 patients with a tumor size more than 5cm, 32 and 6 patients achieved pCR, respectively. The P-value was not statistically significant. Thus, similar to our study, they also concluded that age and tumor size have no effect on NAC response.¹⁰ Our sample size was small, and a larger cohort is needed to establish a statistical correlation between clinical parameters affecting the response to NAC.

Peintinger et al. found that accuracy and overall concordance for the agreement in the RCB score among pathologists are significant and highly reproducible.² In our study, the Fleiss’ kappa statistics have shown that the percentage of overall agreement among the pathologists was 82.31%, where the free marginal kappa was 0.76 and the fixed marginal kappa was 0.71. Thus, it can be inferred from the results that RCB has good inter-pathologist reproducibility for reporting post-neoadjuvant chemotherapy-treated breast cancer specimens, and can be reliably used for the same purpose.

For proper use of the RCB grading system, a training program is highly recommended for a better understanding of fixation to microscopy. It is also recommended that the tumor bed area be marked with a pin during trucut biopsy, as it is not often discernible during the grossing of specimens after chemotherapy. Radiography is required for the identification of the tumor bed area as per the protocol, with photography being available at the grossing station. It is often very difficult to reconstruct and measure the tumor bed area by microscopy alone. Hence, proper orientation of the area by drawing pictures and meticulous grossing are necessary steps that were followed in this study. The entire tumor bed area embedding had been followed as per protocol, and lymphovascular invasion was not included in the calculation. It is also recommended that cellularity percentage images be displayed in the reporting room to reduce inter-observer variation in reporting. In this study, we observed that out of 8 cases that demonstrated triple negative status, 2 patients (25%) achieved pCR with NAC. In other molecular subtypes, none of the patients showed pCR. Jeon et al. found that pCR rates were significantly lower in hormonal receptor-positive patients, i.e., 55.4%, compared to pCR rates in hormonal receptor-negative patients, which were 77.5%.¹¹ Jin et al. reported that hormone receptor-positive and HER2 low/negative cases have a pCR rate ≤8%, whereas in cases of hormone receptor-negative with HER2 low and HER2 negative have pCR rates of 21.95% and 23.64%, respectively.¹²

Apart from the small sample size, another major limitation of this retrospective study was that the pathologists were uninvolved in the grossing, which could have been a potential cause of variability among them (i.e., grossing of specimens and identification of the tumor bed might be different).

It has been observed that there is no significant association between RCB indices and age of the patient, the largest tumor dimension, and the number of chemotherapy cycles. It can also be concluded that RCB can be reliably used to report specimens of breast cancer patients who have received prior chemotherapy.

Ethical clearance was taken from the Institutional Ethics Committee, and approval was given by the West Bengal University of Health Sciences, India.