Introduction

Breast cancer has been ranked as number one cancer among Indian females with a mortality of 12.7 per 100,000 women and age adjusted rate as high as 25.8 per 100,000 women. [1] Five year survival rate after primary diagnosis is about 80- 90 %, even though 4.5 million succumb annually to breast cancer.[2] Prognosis in breast cancer determines the survival. Lymph node metastasis, lymphovascular invasion, oestrogen receptor alpha, progesterone receptor, proliferation rate, Her2Neu, size of the tumour, histological grade, intrinsic subtypes and tumour buds (both intra and inter tumoral buds) are important established prognostic factors in breast cancer. One of the new prognostic factor emerging in breast cancer prognosis is adipose tissue invasion in the tumour margin.

Breast tissue is composed by 90% of adipose tissue (AT) with permanent interactions between epithelial cells and adipose cells. [3] AT is a loose connective tissue characterized by marked cellular heterogeneity. It is made up of about one-third of adipocytes and two-thirds of stromal-vascular fraction cells, a combination of mesenchymal stem cells, endothelial precursor cells, fibroblasts, smooth muscle cells, pericytes, macrophages and preadipocytes in various stages of development. [4] In the mammary gland, adipose cells are characterized by high plasticity and support the growth and function of the mammary epithelium. [5] Adipose cells communicate with cancer cells within the breast, and this may contribute to cancer progression, through different mechanisms of mechanical support and energy supply.

Invasion of tumour cells into the marginal adipose tissue (ATI) leads to larger area of contact between peritumoral lymphatics and tumour cells. Functional lymphatics at the peritumoral site is mainly responsible for lymphovascular invasion. Once tumour cells come in contact with peritumoral lymphatics, it leads to increased chances of lymphovascular invasion (LVI) which is a bad prognostic indicator ultimately leading to lymph node metastasis (LNM).

Few authors had previously studied the association of adipose tissue invasion by tumour cells with other clinic- histological prognostic indicators. [6-8] The present study was conducted with the aim to evaluate whether ATI of cancer cells at the tumour margin influenced lymph node status and prognosis in patients with invasive ductal carcinoma of the breast.

Materials and Methods:

This was a cross-sectional study conducted over a duration of three years from January 2019 up to December 2022. Ethical approval for this study (IEC/AIMS-13/2018) was provided by institutional ethical committee, AIMS, on 12^th December 2018.

Study population and demographics:

Study populationincluded43 cases of invasive ductal carcinoma diagnosed at histopathology in the department of Pathology during the study period. Written informed consent from the patients was obtained. Convenient sampling technique was followed. Cases with special histologic type such as mucinous, lobular, medullary, or squamous cell carcinoma were excluded from the study. Patients with bilat­eral breast cancer, multifocal or multicentric tumours in the unilateral breast, skin or striated muscle invasion, inflammatory carcinoma, distant metastasis, or malignancy at another site and for patients who had received preoperative neoadjuvant chemotherapy were also excluded from the study.

Study population was divided in two groups, one with adipocyte tissue invasion (ATI +) which included 33 cases and the other group without adipocyte tissue invasion (ATI-) comprising of 10 cases.

Processing of samples:

The resected breast lesions or core biopsy specimens were fixed using 10% neutral buffered formalin. Representative bits were processed by routine paraffin embedding. Subsequently, 4 micron thick sections were prepared and stained with haematoxylin and eosin stain. Lymph nodes were extracted from modified radical mastectomy specimens and examined histopathologic ally for presence of absence of metastasis.

Definition of variables:

Adipocyte tissue:

Adipose tissue was defined as a pure aggregate consisting of more than 20 fat cells without intervening fibrous tissue in the breast. The adipose tissue included tissue surrounding the mammary ducts or lobules and those in the subcutaneous layers.

Marginal adipocyte tissue invasion:

Marginal ATI was defined as the presence of more than 20 cancer cells in direct contact with the adipose tissue or the location of cancer cells in the adipose tissue.[6] Only cases with unequivocal ATI were considered positive (ATI+) and doubtful cases were considered negative (ATI–).

Lymphatic vessel invasion:

Lymphatic vessel invasion was defined as the presence of neoplastic emboli within the endothelium-lined spaces in areas adjacent to but outside the margins of the carcinoma. [8] Modified Bloom Richardson histologic grading system was used for grading of the tumours at histopathology.

Tumour size:

Tumour size was measured as the largest dimension of the microscopic invasive component on the pathologic specimen and lymph node involvement was recorded histologically.

Clinicopathologic prognostic parameters of 33 cases of invasive ductal carcinoma with marginal ATI were compared with that of 10 cases without ATI. Value of the combination of ATI and peritumoral lymphatic vessel invasion (LVI) was also assessed.

Statistics:

Statistical analyses were performed using IBM Statistical Package for the Social sciences (SPSS) Statistics for Windows, version 24.0 (IBM Corp., Armonk, NY) and the results were expressed as mean, standard deviation and percentage. Chi square test was applied for comparison of categorical variables in the two groups. The statistical significance was set at p<0.05.

Results: A total of 43 cases of infiltrating ductal carcinoma in whom modified radical mastectomy was performed were considered for the study.

Demographic details: (Table 1)

Out of 43 cases 14 (32.5%) cases were in the age group of 41 to 50 years, followed by 13 (30.2 %) cases in 51 to 60 years age group. Majority of the cases were seen in the right side of the breast, 24/43, (55.8%) and all were unifocal and unilateral tumours. Maximum number of the cases 34/43 (79%) presented with lump in the breast, followed by lump in the breast with nipple discharge 6/43 (13.4%) and lump in the breast with nipple retraction 3/43 (6.9%). Quadrant wise distribution of the cases showed maximum number of cases 11/43 (25.83%) in the upper outer quadrant followed by lower outer quadrant 10/43 (22.08%).

Figure 1: Section shows marginal adipose tissue which was defined as a pure aggregate consisting of more than 20 fat cells without intervening fibrous tissues in the breast. (Haematoxylin and Eosin, x 400)	Figure 2: Section shows marginal adipose tissue invasion which was defined as the presence of more than 20 cancer cells in direct contact with the adipose tissue or the location of cancer cells in the adipose tissue. (Haematoxylin and Eosin, x 400)

Among total 43 cases of infiltrating ductal carcinoma examined, adipocyte tissue invasion was noted in 33 (76.74%). Table 1 shows demographic profile of cases with and without adipocyte tissue invasion.

Association of Adipose tissue invasion with various clinicopathologic prognostic parameters: (Table 2)

The frequency of axillary lymph node metastases was higher in patients with ATI (20/33, 63.7%) as compared to that without ATI (0/10,0%) and this difference was found to be statistically significant (p < .001). The frequency of peritumoral lymph vascular invasion was higher in patients with ATI (24/27, 88.88%) as compared to that without ATI (3/27, 11.1%) and this difference was also found to be statistically significant (p < .001). In addition, patients without ATI or LVI had no lymph node metastasis (n =-7).

Mean tumour size was significantly higher ATI+ cases as compared to that without ATI ((2.2 +0.3 cm vs 1.1+0.1 cm, p<0.001)

Among total 33 cases with ATI, maximum number of cases were histopathologically grade 3 tumours (13/33,39.3%), followed by grade 2 (11/33,33.3%) and grade 1 (9/33,27.2%) tumours. In contrast to this, among total 10 cases without ATI, maximum cases (6/10,60%) were grade 1, whereas two cases each (20%) were grade 2 and 3. Surgical margin invasion was seen in only 8/33 (24.2%) cases of ATI, whilst none of the cases without ATI showed surgical margin invasion. Perineural invasion was present in 9.09% (3/33) of cases with ATI and 20% (2/10) of cases without ATI. There was no statistically significant association between presence of absence of ATI and tumour grade, surgical margin invasion and perineural invasion (p- 0.162, p-0.014 and p-0.375) respectively.

Discussion:

Adipocyte tissue next to breast cancer cells show modulation of gene expression profile in the form of down-regulation of the adipogenesis-related genes Homeobox C Cluster (HOXC)8, HOXC9, fatty acid binding protein 4 (FABP4) and hormone sensitive lipase (HSL) and up-regulation of inflammatory cytokines, like TNF-α, monocyte chemoattractant protein 1 (MCP-1), leptin, with a decrease of adiponectin levels.[9] Compared to normal adipocytes, the so called “cancer- associated adipocytes” (CAA) are smaller cells, with a reduction in the number and size of lipid droplets and modification of basement membrane proteins. [10] The reduction of lipid droplets takes place with the metabolic reprogramming that adipocytes undergo in contact with breast cancer cells and with the acquisition of a brown-fat like phenotype. Indeed, cancer cells induce the lipolysis in CAAs via HSL and adipose triglyceride lipase (ATGL). [10] Despite lower adiposity, Xbp1s overexpression in these cells promote tumour progression.[11] These modifications in the AT close to tumour cells are responsible for promoting tumour progression and influencing the biologic behaviour of the breast cancer. This adipocyte tumour cell cycle may be a potential target for therapeutic interventions in future.

Earlier studies have envisaged the significance of dispersed adipocyte invasion and invasive length of adipose tissue invasion as a marker of prognosis in breast cancer. Very few studies have evaluated the role of ATI at the tumour margin in prognostication in breast cancer.

The present study showed significant positive association of ATI with lymphovascular invasion. This was in concordance with the findings reported by Moriuchi H et al.[7] Though no prognostic significance was noted between ATI + cases and LVI +, ATI - and LVI-cases did not show evidence of any recurrence or metastasis among the study population reported by Yamaguchi et al.[6] This finding further portrays combined utility of ATI and LVI in determining the need for axillary dissection or chemoradiotherapy post-surgery. Predictive value of peritumoral LVI in lymph node metastasis has been well established and is considered as an adverse prognostic indicator. Similarly, tumour cells in contact with peritumoral lymphatics not only leads to nodal but also distant metastasis. Our recent work highlights the utility of D2-40 stain in determining lymphatic vessel density in invasive breast carcinoma Amita et al reported significant positive correlation between intratumoural and peritumoral lymphovascular density and tumour size and stage in breast cancer patients. Similarly, higher peritumoral lymphovascular density was associated with LVI.[12] Further studies exploring molecular markers directed towards development for newer therapeutic targets are essential.

The current study also showed that the ATI had significant association with lymph node metastasis. The frequency of axillary lymph node metastases was significantly higher in patients with ATI as compared to that without ATI.

Yamaguchi et al, studied 735 early invasive breast cancer cases of both luminal type and triple negative breast cancer. Authors observed that ATI was strongly associated with poor survival in triple negative breast cancer. Their findings recognized the invasion of cancer cells in adipocytes as the earliest step in cancer progression.[6] One of the major limitation of the present study was the inability to assess the prognostic significance of ATI and LVI with the molecular subtypes of breast cancer.

In the present study, there was no significant association of tumour size with ATI. Small sample size and less number of cases with large tumour size, in the present study, limits the generalization of this finding. These findings were contrary to that reported by Yamaguchi et al and Moriuchi et al, who observed that larger tumours had higher rate of adipose tissue invasion.[6,7]

In the present study, tumours with ATI had higher grades, whereas tumours without ATI showed more cases with grade 1 morphology on histopathology. Nevertheless, grade of the tumour did not show any significant association with the ATI. Similar findings were reported by Moriuchi et al.[7] However in contrast to this, Yamaguchi et al reported grade 3 tumours to be associated with adipose tissue invasion.[6] This indicates that more than aggressiveness of the tumour cells there may be other factors that may paly role in the interaction between tumour cells and adipocytes which needs further investigation.

Age did not have any impact on ATI in the present study. Similar findings have been reported in the literature. However, with regard to LVI, studies have reported LVI to associated with younger age group. This phenomenon has been attributed to lesser fat content and aggressive nature of cancer cells in younger patients in contrast to the older population.

In the present study, tumour size was significantly higher in ATI+ cases as compared to ATI- cases. The significance of tumour size in lymph node metastasis is a well-established fact.[13] Larger size tumours have greater opportunity to interact closely with the adipocytes, thereby paving way for altering the tumour microenvironment in favour of intravasation and metastasis.

Adipocyte tumour invasion (ATI) of cancer cells at the tumour margin is one of the biologic indicators of tumour aggressiveness in invasive breast cancer. The present study showed that ATI had significant association with well-established prognostic parameters like nodal metastasis and LVI. The present study emphasized that ATI was an independent factor influencing nodal metastasis.

Conclusion:

Presence of adipocyte tissue invasion was associated with LVI and lymph node metastasis. ATI and peritumoral LVI can serve as novel biomarker for prognosis, risk stratification and planning treatment. However, further studies involving large sample size must be conducted for generalization of the results. If proved as proposed, ATI could be incorporated in synoptic cancer reporting protocol of infiltrating ductal carcinoma of the breast.

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