Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 64  |  Issue : 2  |  Page : 329--333

Isolation and morphology of circulating tumor cells by cell block technique in breast cancer


Nandini Agrawal1, Rajpal S Punia1, Uma Handa1, Ashok K Attri2,  
1 Department of Pathology, Government Medical College and Hospital, Chandigarh, India
2 Department of General Surgery, Government Medical College and Hospital, Chandigarh, India

Correspondence Address:
Nandini Agrawal
P-2, Police Officer's Colony, Sector-2, Panchkula, Haryana - 134 112, Chandigarh
India

Abstract

Background: Circulating tumor cells (CTCs) are cells present in the blood stream that are antigenically or genetically similar to a specific tumor type and are markers of tumor diagnosis, prognosis, residual disease and metastasis. The ever-increasing burden of breast cancer globally warrants the incorporation of this all-inclusive marker in the diagnostic repertoire using the simplest of techniques. Aims: To identify CTCs in peripheral blood by cell block (CB) technique in cases of breast cancer diagnosed on fine-needle aspiration (FNA) or core needle biopsy (CNB) and to correlate their presence with nodal metastasis. Material and Methods: This study was conducted in the Department of Pathology, at a tertiary care hospital. Peripheral blood samples from a total of 30 cases of primary breast carcinoma diagnosed on FNA or CNB without prior neoadjuvant chemotherapy were analyzed using the CB technique. Results: The age ranged between 29-74 years with the most common presenting complaint being a palpable, single, unilateral breast lump. CTCs were detected in 2 (6.7%) cases with a <5 cell cluster with both the cases being grade I breast carcinomas and also displaying nodal metastasis.



How to cite this article:
Agrawal N, Punia RS, Handa U, Attri AK. Isolation and morphology of circulating tumor cells by cell block technique in breast cancer.Indian J Pathol Microbiol 2021;64:329-333


How to cite this URL:
Agrawal N, Punia RS, Handa U, Attri AK. Isolation and morphology of circulating tumor cells by cell block technique in breast cancer. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jun 20 ];64:329-333
Available from: https://www.ijpmonline.org/text.asp?2021/64/2/329/313263


Full Text



 Introduction



Cancers prove to be the second leading cause of deaths globally with an estimated 18.1 million new cases and 9.6 million cancer deaths worldwide in 2018.[1] Out of all the carcinomas breast cancer is of paramount importance with Northern America, Western and Northern Europe and Australia having the highest incidence with a range of 85.8 to 96.0 per 100,000 women. Amongst the Indian population, the age adjusted incidence rate of breast cancer is 25.8 with a mortality of 12.7 per 100,000 women.[2] Diagnosis of breast cancer cases follows a multi-pronged approach with the amalgamation of clinical examination, radiological examination using mammography and BI-RADS scoring system, cytopathological and histopathological evaluation for utmost sensitivity and specificity. Cytopathological examination incorporates FNAC and nipple discharge smears with a sensitivity and specificity of 74% and 96% respectively. The histopathological examination can be carried out on CNB with sensitivity and specificity of 87% and 98%, respectively.[3] The most frightening prospect after diagnosis of breast carcinoma is the possibility of metastasis which can even develop several years after the diagnosis of primary tumor and herein lays the import of liquid biopsy in the form of CTCs.

CTCs are a heterogeneous group of cells with this diversification being attributed to two factors i.e., cancer stem cells and epithelial-to-mesenchymal transition.[4] An assortment of tests and techniques have been developed based on the physical, biological and immunomagnetic properties for the enumeration and enrichment of CTCs, where enumeration stands for the total number of cells and enrichment for the isolation and extraction of cells from the blood stream. However, these technologies require an exorbitant capital and substantial resources which hinders their widespread use especially in a resource poor country like India.

To overcome this impediment, we have utilized the technique of CB, which is a routine practice in cytopathology wherein, the material obtained from cytological specimen, whether in the form of sediment, blood clot or tissue pieces, is processed similar to histopathology.[5]

 Materials and Methods



Samples selection

This prospective study was conducted at our tertiary care hospital from December 2017 to October 2019. It included 30 cases diagnosed as primary breast carcinoma, irrespective of age, on FNAC and CNB without prior neoadjuvant chemotherapy and about to undergo modified radical mastectomy (MRM). Ethical clearance was obtained from the Institutional Ethics Committee. This study was approved by the GMCH Chandigarh Institutional Ethics Committee on 8th December 2017. IEC Regd no. ECR/658/Inst/PB/2014. Complete clinico-radiological investigations and prior FNAC or CNB findings were recorded for each patient. 7.5-10 mL of venous blood was withdrawn, preferably from the median cubital vein, under aseptic conditions from the patient one day before she underwent MRM in sterile glass bottles which were kept in the biosafety cabinet.

Cell block processing

The blood was allowed to self-clot. After the clotting process was complete 10% neutral buffered formalin was poured in the bottle and it was made to stand overnight. The supernatant was poured off and the sediments were picked up by spatula, wrapped in tissue paper and placed in a tissue cassette. Rest of the processing was similar to routine paraffin embedding technique for any other tissue. Multiple sections were cut at 3 μm and the cell blocks were exhausted to search for CTCs and stained by hematoxylin and eosin (H & E). The CTCs were searched for under high power magnification (40×) and also under oil immersion (100×). The slides were examined by two pathologists blinded to each other's findings.

Criteria for a cell to qualify as a CTC on H and E stain: A cell size larger than the sizes of normal blood cellular components along with a visible nucleus. The slides were classified as being positive or negative for the presence of CTC and a cut-off value of ≥1 CTC was considered positive.

Processing of MRM specimen

After the MRM specimen had been received in histopathology in 10% neutral buffered formalin, it was grossed according to the standard protocols. The MRM specimen was kept for overnight fixation after making serial sections in the tissue perpendicular to the attached elliptical skin flap. The relevant measurements were noted and location of tumor was identified. Sections were taken from the tumor proper, fibrotic areas, surgical margins and nipple areola complex. Lymph nodes were searched for in the axillary tail and a minimum of 10 lymph nodes were isolated. The lymph nodes with size <0.5 cm were embedded as such, while those ≥0.5 cm were bisected and embedded. These sections were processed routinely with paraffin embedding technique. Sections of 3 μm thickness were cut and stained by H and E stain. Immnohistochemistry (IHC) staining with CK was applied in lymph nodes showing presence of metastatic tumor deposits wherever required.

Statistical analyses

Categorical variables were reported as counts and percentages. Group comparisons were made with the Chi-Square test or Fisher's exact test as appropriate. Continuous data were given as Mean/SD and range or median and inter-quartile range as appropriate. Spearman correlation coefficient was calculated to see relation between variables. A P value <0.05 was considered significant. Analysis was conducted using IBM SPSS STATISTICS (version 22.0).

 Results



All the patients were females with the average age range between 29 to 74 years and a mean of 54.03 ± 10.65 years. All the patients presented with palpable breast lumps which were single and unilateral. The lump was right sided in 19 (63.3%) patients and left sided in 11 (36.7%) patients. The duration of the lump varied from 1 month to 2 years with a mean of 7.03 ± 6.86 months. In 13 (43.3%) cases lump was located in central quadrant, 11 (36.7%) cases in upper outer quadrant, 4 (13.3%) cases in upper inner quadrant and 1 (3.3%) case each in lower outer and lower inner quadrants. The minimum lump size on mammography was 1 cm while the maximum size was 6 cm with a mean of 2.98 ± 1.2 cm and median of 3.05 cm. Maximum i.e., 12 (40%) cases had a BI-RADS score V in the involved breast while only 2 (6.7%) cases had a BI-RADS score III.

Out of all the 30 cases, both FNAC and CNB had been performed in 4 (13.3%) cases, only FNAC in 17 (56.7%) cases and only CNB in rest of the 9 (30%) cases. Out of the 21 (70%) cases on whom FNAC was performed, a maximum of 16 (76.1%) cases were diagnosed as IDC, 2 (9.5%) cases as suggestive of IDC and 1 (4.8%) case each as suspicious of IDC, poorly differentiated malignant tumor and mucinous carcinoma. FNAC displayed a 100% sensitivity and specificity in our study. Out of the 13 (43.3%) cases on whom CNB was performed, a maximum of 9 (69.2%) cases were diagnosed as IDC, 1 (7.7%) case each of IDC with medullary features and infiltrating carcinoma likely lobular while in 2 (15.4%) cases no definitive diagnosis was possible. CNB displayed a sensitivity of 84.6% and a specificity of 100%.

Multiple sections were obtained from the CB prepared from peripheral blood and a total of 2 (6.7%) cases out of all the 30 cases included in this study showed presence of CTCs. In both the cases the CTCs were present in clusters of <5 cells. The cells displayed moderate pleomorphism with vesicular nuclear chromatin, prominent nucleoli and mild to moderate amount of cytoplasm [Figure 1]a and [Figure 1]b.{Figure 1}

In the MRM specimens out of all the cases, 27 (90%) cases were diagnosed as IDC, NOS, and 1 (3.3%) case each of IDC with medullary features, IDC with mucinous features and medullary carcinoma. Using the Modified Scarff-Bloom Richardson Grading out of all the 27 cases of IDC, NOS 21 (77.8%) cases were classified as grade II. IDC with medullary features and medullary carcinoma were classified as grade III while IDC with mucinous features was classified as grade I. A minimum of 10 lymph nodes were isolated in all the cases with a maximum of 27 lymph nodes in 2 (6.7%) cases. The mean number of lymph nodes isolated was found to be 16 ± 5.32. 17 (56.7%) cases showed presence of nodal metastasis.

In the present study, both the cases with positive CTCs had a histopathological tumor grade I with one case being IDC, NOS, and other being IDC with mucinous features [Table 1] and [Table 2]. On statistical analysis using Chi square test, it was found to be significant with a P value of <0.001. Lymph node metastasis was noted in both the cases showing presence of CTCs. In addition, lymph node metastasis [Table 3], percentage of metastatic deposits and LVI were compared with the CTCs in peripheral blood however, no association was found between them as the P value was >0.05 in each of these correlations.{Table 1}{Table 2}{Table 3}

 Discussion



Breast cancer is one of the most commonly encountered tumors and is well known for its notorious clinical behavior leading to substantial mortality and morbidity globally. This in large part can be allocated to the phenomenon of metastasis. The clinical diagnosis of breast cancer, in most of the developing countries, is still dependent on the triple testing which includes palpation of breast lump, radiological findings and tumor detection using histopathological techniques like FNAC or CNB. The mean age of patients with breast cancer in this study was 54.03 ± 10.65 years which are in concordance with the results of survey carried out by Indian Council of Medical Research (ICMR) published by Malvia et al.[2] It was found that breast cancer occurs one decade earlier in Indian women as compared to western population with a peak between 40-50 years. All the patients presented with a palpable unilateral breast lump which in most cases i.e., 19 (63.3%) were right sided. These findings are mostly similar to those obtained by Takalkar el al.[6] however; left-sided breast lump (50.7%) were more frequent in their study.

In our study 23 (76.7%) cases had a BIRADS score of IV-V with only 2 (6.7%) cases having a BIRADS score III. BN et al.[7] have reported similar findings with most of the malignant breast lesions belonging to BIRADS categories IV and V. A sensitivity and specificity of 100% for FNAC was noted in our study which is equivalent to the results obtained by BN et al.[7] A sensitivity of 84.6% and specificity of 100% was displayed by CNB in the present study which is in concordance with Saha et al.[8] who found the sensitivity and specificity of CNB to be 88.3% and 100%, respectively.

CTCs are referred to whole tumor cells which are either present singly or in clusters in the blood stream and are released directly by the main tumor or from the metastatic sites. They can also be defined as non-leucocytic cells which are usually 20-30 μm in size with variable morphological aspects depending on the primary tumor.[9],[10] The growing emphasis on CTCs stems from the fact that it is a valuable prognostic marker at every stage of breast cancer including prevention in an at-risk population, early detection, minimum residual disease after a resected tumor, harbingers of metastasis and monitoring for cancer recurrence in a clinically treated patient. It also pitches in as a predictive marker by guiding treatment therapies in resistant cancers.[11] Cristofanilli et al.[12] have demonstrated a direct correlation between the absolute number of CTCs in 7.5 mL of peripheral blood with overall survival and progression free survival. Owing to its import as an all-inclusive marker emphasis has been on evolution and elaboration of CTC detection techniques. The various distinct biophysical and immunological properties are utilized for this purpose. The differences in cell size and density are exploited in physical filter, density gradient, photoacoustic and microfluidic devices along with morphology-based imaging cytometry. Techniques based on antibody capture incorporate CellSearch and Magsweeper with functional characteristics being utilized in Epispot assay and invasion assay. Nanotechnology is the latest methodology to join this long list of existing techniques.[9],[10] All of them show variable sensitivity and specificity for the detection of CTCs, however, CellSearch is the only FDA cleared technology for the detection of CTCs currently wherein the CTCs are captured using ferrofluids tagged with EpCAM antibody. In a study conducted by Pierga et al.[13] out of the total 97 patients without neoadjuvant chemotherapy 22 (23%) cases showed presence of at least 1 CTC. Several studies have contrasted the efficacy of different assays in isolation of CTCs with that of CellSearch; however, the CB method has not been utilized for this purpose. A study was conducted by Nam et al.[14] to detect CTCs in patients of liver cancer using CB method. Out of the 29 patients included in their study CTCs were detected in 14 (48.3%) cases whereas, in our study, using the CB technique, CTCs were isolated in 2 (6.7%) cases out of the total 30 cases. CB is a long-established universal technique in cytopathology having better results in terms of cellular yield as compared to conventional smears with a sensitivity and specificity of around 92% and 99%, respectively.[15] Herein, the material obtained whether in the form of sediment, blood clot, or tissue pieces is processed similar to histopathology with creation of paraffin blocks. However, CTC being a scarce and rare event in the peripheral blood with a half-life of few minutes to few hours could conceivably be difficult to detect using this methodology. CB although being the least technically challenging of all and offering the advantage of several sections for the examination of CTCs may lead to loss of single tumor cells during section cutting.

Also, in the present study nodal metastasis was demonstrated in a total of 17 (56.7%) cases. These findings are consistent with those of Pathak et al.[16] with 66.1% cases demonstrating nodal metastasis. In our study, CTCs were detected in both the histopathological grade I tumors and this finding was statistically significant. However, none of the cases in grade II or III showed presence of CTCs in the CB preparation. These findings are at variance with those of Bansal et al.[17] where the highest CTC positivity was seen in Grade III tumors (31.25%) followed by grade II (1.31%) and then grade I (0%). Both the CTC positive cases, in our study, displayed presence of metastatic deposits in the axillary lymph nodes; however, this finding was not significant statistically. On the contrary, Nakagawa et al.[18] have demonstrated a statistically significant correlation between these variables with a directly proportional relationship between number of metastatic lymph nodes and CTC detection. The findings of the above-mentioned study is reproduced by Bansal et al.[17] with 81.82% CTC positive cases having nodal metastasis. An association between presence of CTCs and LVI was also elucidated in our study but it was non-significant. Similar findings have been described by Biggers et al.[19] with 41.4% cases displaying positive CTCs in the absence of LVI.

A few shortcomings of our study include identification of only a small cluster of CTCs which was not present on the subsequent sections, thwarting the use of IHC. Similarly, we may have missed single tumor cells during technical processing and in the absence of IHC leading to lack of identification of tumor cells in grade II and III tumors.

Take home message

CTCs have proved their mettle as a stellar prognostic and predictive marker with a variety of exemplary studies authenticating its import. Cytopathological techniques primarily cell block can be availed for the purpose of detecting these CTCs, our study being a small step in that direction. However, further studies are recommended for the corroboration of our findings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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