As per the study done by Collina et al.¹³, CD44 expression was detected in 143/160 breast cancer samples, i.e., 89.3 % of the specimens.

Applying the formula to calculate sample size for qualitative variables according to prevalence: 1.96²×p×q/d²

where:

p=prevalence (from previous studies)

q=1−p

d=marginal error (10%, <0.2 of prevalence), hence d=0.1×0.89=0.09;

sample size n=3.84×0.89×0.11/0.09×0.09

n=47

By using the above formula, the required sample size for the study was 47 participants. Thus, a total of 50 TNBC cases diagnosed on core needle biopsy or mastectomy specimens from July 2023 to November 2023 were enrolled in the study. Patient information was collected from the histopathology requisition forms, which were paper-based.

Histopathologically diagnosed cases of invasive breast cancer with TNBC subtype received in the Department of Pathology, VMMC, and Safdarjung Hospital were included in the study.

Non-invasive cancer, including in situ cases, Metastatic lesions, sarcomas and ER+, PR+ or HER2 Neu positive cases were excluded from the study. Histological parameters were determined from H&E-stained slides. Clinicopathological characteristics were evaluated for each tumor, including the patient's age at diagnosis, tumor laterality, tumor subtype, and histological grade using Modified Bloom-Richardson's grading system.¹⁴ pTNM stage was assessed in all the breast cancer surgery specimens, wherein T is tumor size, N is nodal status, and M is metastasis.¹⁵

Immunohistochemistry (IHC) was performed for the assessment of ER, PR, HER2neu, and Ki-67. Only the samples negative for ER, PR, Her2neu, i.e., TNBC subtype, were included in this study. CD44 IHC was performed using a mouse monoclonal antibody (BC8, Biocare Clone). Paraffin blocks of tumor sections were cut on poly-L-lysine-coated slides, which were followed by deparaffinization. The sections were subjected to descending concentrations of alcohol for hydration. Then, 3% Hydrogen peroxide was added, followed by antigen retrieval using the pressure cooker heating technique. Primary Mouse monoclonal antibody was applied for approximately 45 minutes to 1 hour, followed by secondary antibody for 45 minutes. The slides were washed using Tris buffer after every step. Chromogen-DAB was used to highlight the antibody expression, and hematoxylin was used as a counterstain.

The most representative areas were selected for IHC assessment by two pathologists, independently under a light microscope. CD44 expression in tumor cells was evaluated by considering the tumor percentage and intensity of immunopositivity in the cell membrane under low and high power (100× and 400×). There is no standardized scoring system for CD44 assessment; thus, we schematized our scoring system as follows:

Each specimen was evaluated using the mentioned scoring system and subcategorized into negative (score=0), low positive (1+), intermediate positive (2+), and high positive (3+). CD44 expression and scores were then correlated with histological grade, T and N stage, wherever applicable.

The presentation of the categorical variables was done in the form of numbers and percentages. Quantitative data were presented as means ± SD and as medians with 25th and 75th percentiles (interquartile range). The association between CD44 expression and prognostic parameters, such as tumor grade, T stage, and N stage, which were qualitative, was analyzed using Fisher's exact test, as at least one cell had an expected value of less than 5. The Spearman rank correlation coefficient was used to analyze the correlation of grade with CD44 score. Univariable logistic regression was used to find out significant risk factors for positive CD44. For statistical correlation, a P value of less than 0.05 was considered significant. Due to a lack of a statistically significant association in univariable regression analysis, multivariable regression model was not performed. The data entry was done in the Microsoft Excel spreadsheet, and the final analysis was done with the use of Statistical Package for Social Sciences (SPSS) software, IBM, Chicago, USA, version 25.0.

The study included fifty cases of TNBC, including 31 core needle biopsy specimens, 17 mastectomy specimens, and one of the WLE and BCS specimens. The age of the patients ranged from 32 to 74 years, with a mean (SD) of 49.4 (10.4) years. Tumors involved the right breast in 26 (52%) cases, 23 (46%) involved the left side, and one was bilateral. The majority of the cases were grade 2 tumors (24 cases, 48%), followed by grade 3 (23 cases, 46%), and 3 (6%) were grade 1. Cases belonging to higher tumor grades were predominant, perhaps because of their triple negative status (47/50). pTNM staging could be assessed in 19 of the total 50 specimens. Tumor sizes less than 2 cm (T1) and 2 to 5 cm (T2) were considered as low T stage and comprised 12 (63.16%) of the cases. Also, 7 cases (36.84%) belonged to a higher T stage, i.e., T3/T4 category. Lymph node involvement was found in 7 (36.84%) of the patients, whereas 12 (63.16%) had a negative nodal stage. Clinicopathological features are summarized in Table 1.

CD44 positive expression was observed in 20 (40%) of the total 50 cases (Figure 1).

Also, 13 samples (26%) showed intermediate (score 2+) to high (score 3+) expression for CD44. In 7 samples (14%), low expression (score 1+) was observed. The remaining 30 cases (60%) showed a negative expression (score 0). (Figures 2A–D and 3A–D).

The study found a significant association between positive CD44 expression and histological grade (P value using Fisher's exact test=0.002, Table 2).

Stronger CD44 expression was observed with increasing tumor grade. Of the total 20 cases with positive CD44 expression, the majority (15, 75%) were grade 3 tumors, followed by grade 2 (5, 25%), whereas none of the grade 1 (0%) cases showed positivity for CD44. On applying the univariable logistic regression model, the study failed to show a significant correlation between CD44 expression and tumor grade (P value using Fisher's exact test >0.05, Table 4).

The distribution of CD44 scores showed a similar pattern across different tumor grades. For cases with score 1+ i.e., low positive, the proportions were 0, 2 (28.5%), and 5 (71.5%) for cases at grades 1,2, and 3, respectively.

Similarly, in cases with score 2+, the proportions were 0, 2 (33.34%), and 4 (66.67%), respectively. Lastly, for cases exhibiting strong positivity, i.e., score 3+, the highest number of cases belonged to Grade 3, i.e., 6 (85.71%), followed by Grade 2, 1 (14.28%). The P value associated with this distribution was marginally significant (P=0.052 using Fisher's exact test, Table 3).

A stronger expression, both in intensity and tumor percentage, was noticed in the worst histological grade. Using the Spearman rank correlation coefficient, a moderately positive correlation was observed between CD44 score and increasing tumor grade (Correlation coefficient of 0.485, P=0.0004).

CD44 positivity was observed in 10/19 (52.63%) of the mastectomy specimens. The majority of the positive cases (5 cases, 50%) showed an intermediate immunoreactivity score (2+), followed by score 1+4 (40%). One case was stained high for CD44 with a score of 3+.

CD44 was additionally correlated with tumor size and lymph node spread in these cases.

No statistical significance was observed between CD44 immunoexpression and tumor stage (P value using Fisher's exact test=0.592). Logistic regression test was applied, and no significance was observed between CD44 expression and T stage (P value=0.494, Table 4). Ten out of the 19 surgery specimens showed a positive CD44 expression, with an equal number of cases belonging to the lower T1/T2 stage and the high T3/T4 stage.

The association between CD44 immuno-expression and nodal status did not show any statistical significance (P value using Fisher's exact test=1). Logistic regression analysis failed to show an association between CD44 positivity and N stage (P=0.620, Table 4). The majority of nodal negative cases (8 cases, 70%) displayed positive CD44 expression, compared to 2 (30%) cases of positive nodal involvement.