Diagnostic Accuracy of Cell Block Using HistoGel Tube Method in Breast Cancer in Pakistan

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for detection and confirmation of breast cancer is the triple evaluation method which constitutes of clinical, radiological, and pathological evaluation of breast masses.Fine needle aspiration cytology (FNAC) is the preliminary pathological investigation except for cases where microcalcifications are found.It is relatively less painful, cost-effective, and yields high accuracy results in a shorter duration.Diagnostic accuracy of FNAC is as high as 98.9% in some settings 3 which can further be increased by cell block formation.Cell blocks prepared from the fine needle aspirate help increase the diagnostic accuracy by easy evaluation of the histological architecture of tumor cell clusters thus 4 avoiding the need for tissue biopsy.The cell blocks also offer the opportunity to get the special stains, and immunohistochemical markers such as Ki-67, Estrogen Receptor, Progesterone Receptor, Her 2 and p53 along with FISH for specific diagnostic and prognostic 5, 6 implications.The cell blocks can be prepared by the conventional Thrombo-plastin-Plasma Gell-block (TP-GB) method, Cell-Gel method, or Histogel-Tube method as compared to the older methods using 7,8 celloidin or agar.Among all, HistoGel-tube method is the most appropriate method as it yields a firm solidified cell gel button with embe-dded cells which can easily be maneuvered as a formalin fixed paraffin embedded tissue (FFPE).As stated in literature, FNAC and cell block together have combined diagnostic sensitivity and specificity of 96% and 100% 9 respectively.However, the gold standard is histopathological diagnosis made by core needle biopsy, incisional biopsy, excisional biopsy, or mastectomy with a pooled 10 sensitivity of 97% for the diagnosis of breast cancer .Lack of data in local settings and that too without the specification of cell block preparation technique raise the need to address this issue.Keeping in view the increasing global breast cancer burden, effective modalities should be employed for the evaluation and early diagnosis of breast cancer in resource-limited settings of Pakistan.The objective of this study was to determine the diagnostic accuracy of FNA cell blocks specifically made by the Histogel-tube method in the assessment and diagnosis of breast cancer taking histopathological diagnoses as gold standard.

Methods
This cross-sectional study was carried out at the Department of Pathology, King Edward Medical University/ Mayo Hospital, Lahore, six months after the approval from Institutional Review Board of KEMU (No. 430/RC/KEMU/).The data was collected using nonprobability convenient sampling.A sample size of 62 patients was calculated by using 95% confidence interval and 9% absolute precision.Participants included both male and female patients of all ages, having easily accessible breast lesion on palpation along with C III, C IV, and C V smears on initial cytological evaluation by rapid onsite evaluation (ROSE helps in the immediate categorization of 11 benign and malignant lesions).Participants excluded were the ones with deranged clotting profile, who had received neo-adjuvant chemotherapy or radiotherapy, hemorrhagic aspirate on fine needle aspiration, inadequate tissue on core needle biopsy, ultrasound-guided fine needle aspirations and benign lesions i.e., C I, C II smears on ROSE.After the aseptic measure, fine needle aspirate from the breast masses was taken using a 22-G disposable needle attached to a 10 ml syringe.They were quickly spread onto the slides, immersed in ethyl alcohol, air dried, and then stained 12 using Diff Quik.The cases were reported using the fivetier reporting system; C I -inadequate; C II-benign; C III-suspicious probably benign; C IV-suspicious, probably malignant; and C V-malignant breast lesions.The patients with lesions reported as C III, C IV, and C V on ROSE were then subjected to resampling in order to get a specimen for cell block formation.The cytology specimens from the selected cases were then transferred to a flat bottom glass tube and centrifuged at 3000rpm for 5 minutes.The supernatant was decanted after which an aliquot of 0.5ml melted Histogel was added.The tubes were centrifuged again to achieve uniform distribution of cells in the medium.Later, they were refrigerated for 15-20 minutes for solidification.The cell gel buttons were then squirted out from tubes, placed onto the filter papers and passed in properly labeled tissue 13 cassettes.Paraffin blocks were then prepared from the processed tissue by using reusable metallic moulds.The blocks were finally put in a refrigerator for 4-6 hours before microtomy after which they were cut in sections and stained by routine Hematoxylin and Eosin.Two pathologists reviewed them independently and the lesions were categorized according to 14 the WHO categorization of breast tumors.The grading was done according to the Modified Scarff Bloom Richardson system which consists of three attributes i.e., duct formation, nuclear pleomorphism, and 15 mitotic count.Data was entered in SPSS-version 26 and the analysis carried out was reported.

Results
The descriptive statistics of findings revealed the mean age of diagnosed patients to be 47 ± 13 years with an interquartile range (IQR) of 31 years.Out of 62 selected cases, the majority were females i.e., 61 cases (98.4%), and only 1 (1.6%) patient was a 78 year old male.The ultrasonographic findings indicated that 53.2 % of patients were directly opted for mammography because of their age (more than 40 years).46.7 % showed suspicious-looking masses with irregular margins, 4 patients (4.8%) had suspicious-looking masses with internal vascularity and microcalcifications while 16.1% were reported to have axillary lymphadenopathy along with the suspicious breast swellings.55 patients (88.7%) had their mammography reports while 7 (       old patients which shows to be consistent with the present research finding i.e., mean age as 47±13 years and among females which conforms to the incidence of breast cancer among males being 1 out of every 100 20 cases.Though the only male included in the present study was a 78-year-old suggesting the fact that increasing age is a risk factor for breast malignancy in men and requires future research. Further findings showed that the FFPE sections archived from the cell blocks revealed 47(75.80%)true positive and 9(14.15%)true negative cases.Four(6.45%)cases were false positives i.e., over diagnosed on cell as "Positive for atypical/malignant cells".Three cases were confirmed as "Adenosis" while 1 as "Granulomatous inflammation" which were due to complex architecture of ducts and reactive atypia, thus, justified in 21 research by Guirguis MS et al.In this study, the researchers took a detailed account of various inflammatory and proliferative breast diseases which radiologically and pathologically were considered among the benign mimickers of breast carcinoma.Two (3.22%) cases were false negative and under diagnosed as "Stromal fragment only" and "Atypical ductal hyperplasia" which later came out to be "Spindle Cell lesion" and "IDC grade II" respectively on core biopsy.Thus, loss of diagnostic material or alteration in cellular morphology owing to fixation and processing were responsible for the limited diagnostic utility of cell blocks in such cases.
The diagnostic indices of Histogel cell blocks came out to be 96%, 69%, 92%, and 81% respectively.The diagnostic accuracy was calculated to be 90%.To the best of our knowledge, no specific study has been performed to determine the diagnostic accuracy of Histogel cell blocks in suspicious breast lesions.However, these 22 findings were supported by Kawatra et al. who concluded the sensitivity of conventional cell blocks as 100%, specificity 81.8% and diagnostic accuracy as 86.8%.

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Moreover, according to the study by Methew et al sensitivity, specificity and diagnostic accuracy of cell blocks came out to be 71.11%,100% and 71.73% respectively, proving the findings to be reliable and accurate in form of diagnostic utility of cell blocks in conjunction with cytopathology.However, scarcity of the diagnostic material and inability to determine the absolute efficacy of HistoGel were the limitations encoun-tered in the present study.Therefore, use of 24G needle and comparison of various methods of cell block preparation can improvise better outcome.

Conclusion
Cellblock technique should be employed in all cases along with FNAC to help in the accurate diagnosis of breast cancer.Cell block provide an alternative to invasive techniques i.e., tissue biopsy.Moreover, the sections of cell block can be used for IHC and ancillary studies.
In the present study, Histogel proved to be an effective medium for cell block preparation and the diagnostic accuracy of HistoGel cell blocks was comparable to that of the gold standard i.e., histopathology.

Figure 3 :
Figure 3: (a) Histogel cell block of a 78-year-old male patient showing papillary architecture (10X magnification) (b) Section of the Core Needle biopsy from the same patient.Both the cell block and biopsy were reported as Papillary Neoplasm.

Table 1 :
Correlation of findings on HistoGel cell block and Histopathology

Table 2 :
2×2 Contingency table for HistoGel cell block and histopathological diagnoses April -June 2024 | Volume 30 | Issue 02 | Page 200 Approval: The Institutional Review Board of KEMU approved the study vide letter No. 430/RC/KEMU/.Analysis & interpretation of data, drafting of article, critical revision for important intellectual content, final approval RS: Analysis & interpretation of data, critical revision for important intellectual content, final approval