Comparison of Core Biopsy and Excision Materials in Terms of Histological Type, Histological Grade, Hormone Receptors, and Human Epidermal Growth Factor Receptor 2 Expression in Invasive Breast Carcinomas

IHC applied to CB is usually accepted accurate and it is not studied again in EM to reduce the cost.[] The question is, considering the tumor heterogeneity and the small amount of tissue obtained by CB, whether the tumor grade and immunohistochemical profile evaluated in CB reflect the entire tumor. The ASCO/CAP guideline supports to study of tumor markers to CB with sufficient number and size, but if the results are inconsistent with the histopathological characteristics or the tissue is not prepared as recommended in the guideline, IHC is recommended to be repeated in EM.[,]

In our series of 201 cases, in terms of histological type, the concordance between two methods was 89%. The concordance rate was 100% in ILC, MC, tubular carcinoma, and metaplastic carcinoma, 98% in IBC-NST, and 27% in mixed type carcinoma. In similar studies, it was reported that this concordance was highest in IBC-NST and metaplastic carcinomas; however, the rates are decreased to 14% in mixed type carcinomas.[,] In a study, the overall concordance was 73.6%, and the concordance rates were 82% in IBC-NST, 30% in ILC, 50% in MC, and 25% in mixed-type carcinoma.[] Greer et al.[] found the overall concordance as 81%, and the concordance rates were 96% in IBC-NST, 77% in ILC, 100% in metaplastic carcinoma, and 14% in mixed type carcinoma.

Park et al.[] investigated the accuracy of CB in breast carcinomas and found that the average number of cores required to ensure 100% concordance in tumor type was 5.1. The number of cores taken in our center varies between 1 and 6.

When we evaluated the cases with discordance of histological type, we found that 12 IBC-NST, 1 IMPC, and 2 MC cases according to the CB were diagnosed as mixed type carcinoma in EM. In one case which was diagnosed as mixed type carcinoma (IBC-NST+IMPC) by CB, MC component was also added in EM. Since it is not always possible to sample each component of heterogeneous tumors, in mixed type carcinomas, the diagnosis given by CB may be incomplete.[,]

Three cases diagnosed as mixed IBC-NST +ILC in CB were diagnosed as IBC-NST in EM. In revision, possibly depending on the artifact during taking biopsy, it was observed that the carcinoma cells were trapped in the stroma, formed single cell rows, and mimicked ILC in CB (Fig. 1). Similar diagnostic differences for mixed type carcinomas have been reported in the literature.[,]

In our study, the concordance rate in terms of grade between two methods was 75% and the concordance was moderate. The concordance rates were 80% for Grade I, 92% for Grade II, and 41% for Grade III tumors.

In total, 41 of 201 cases had higher tumor grade in EM. In these cases except one, the tumor grade increased from II to III. In one case, tumor grade changed to Grade III from Grade I. When it is revised, it was seen that the tumor was mixed type carcinoma consisting of MC and IBC-NST. Probably, due to the small tumor area sampling, only MC component was sampled by CB. Badoual et al.[] found 73.1% concordance rate between CB and EM in terms of grade, in a series of 110 cases. They observed that the concordance rates were 78.5% and 79.6% in terms of tubular formation and pleomorphism, respectively, and this rate decreased to 60.2% in terms of mitotic index. They suggested that the discordance in terms of tubular formation and pleomorphism was dependent on tumor heterogeneity and the tumor area in CB was too small to give mitotic index. Park et al.[] found that lower grade in CBs than EMs was associated with the lower calculation of mitotic index in CBs. In this study, when the average number of CBs was 5.1, the concordance rate between two methods was 80.8% for grade and 59.6% for mitotic index. As a result, they emphasized that five CBs were insufficient to accurately determine the grade. In other studies, the concordance rates between two methods in terms of grade varied between 63% and 77%.[,-] Based on these results, it is necessary to evaluate the grade in EM for optimal treatment and prognosis.[]

In our study, the concordance rate between two methods was 96% for ER and 89% for PR, similar to the literature. The reported rates ranged from 81% to 99%.[,,,,,] Compared to EM, ER staining percentage and intensity were higher in CB. One of the important factors explaining these findings is tissue fixation (cold ischemia time, type of fixative, and fixation time). The ASCO/CAP guideline recommends keeping cold ischemia time under 1 h for breast excisions.[]

Qiu et al.[] fixed breast cancer samples using different fixation times and performed IHC by using the same clones, they showed that the staining scores of ER started to decrease in 2-8 h and PR in 1-8 h. As shown in this and similar studies, it is important to standardize the cold ischemia time to prevent the changes in the level of target protein expressions to which ER, PR, and HER2 antibodies can bind.[,,] Formaldehyde permeates into the tissue at a rate of 1 mm/h, but tissue fixation is slower. Complete crosslink formation takes 24 h at room temperature and 18 h at 37oC. Tissue fixation time is the same, although the fixative permeates faster in smaller tissues.[,] ASCO/CAP recommends tissue fixation with 10% buffered formalin for at least 6 h.[26]

Alcohol, alcohol-based solutions (e.g. Carnoy's), acetone, and acidic fixatives (eg., Bouin"s, and B5) are also used for tissue fixation. Since ER is degraded in acidic fixatives, they are not recommended by CAP. [] Although alcohol-based fixatives and acetone are superior to formaldehyde in terms of preserving antigenicity, they lead to coagulation of tissue and are not recommended for routine usage.[]

Another factor affecting the IHC is tissue processing. Prolonged using of alcohol solutions may cause insufficient dehydration of the tissue. Insufficient dehydration may cause poor or no staining. Some authors recommend changing the solutions at least once a week. [] In our laboratory, automatic tissue processing device is used and solutions are renewed every week.

In our center, CBs are placed into formalin immediately, so fixation begins at the 0th min. Tissue processing is also performed the same day. EMs sent for frozen process are taken into formalin as soon as the process is over. For the others that kept in the operating room or refrigerated the time without fixation can sometimes extend up to 6 h. In case of delay, EMs are taken into formalin without sectioning and reaches to our laboratory the next day. In this case, formaldehyde penetration into the tissue center is insufficient and these areas are not fixed. In our department, EMs are divided into slices of 0.5-1 cm thickness to ensure formaldehyde penetration to the whole tissue, kept in formalin for 24 h, and sampled the next day and taken to the tissue processing device. Yıldız-Aktaş et al.[] showed that ER and PR staining percentage and intensity of EMs significantly decreased when EMs kept more than 2 h at room temperature and 4 h refrigerated.

Another reason of higher ER expression in CBs is peripheral sampling. Douglas-Jones et al.[] reported that ER expression is higher in the tumor periphery than in the center in breast tumors. They found that ER staining intensity decreased by approximately 2% per millimeter from the periphery to the center. It was emphasized that may be due increased biological activity and mitosis, as well as due to better fixation at periphery. Greer et al.[] also compared the HR status between CB and EM and found stronger and higher positivity in CBs.

CAP recommends to use internal control (normal breast tissue) during ER evaluation to avoid false negative results. In CBs without normal cells, if ER is negative, the procedure must be repeated with another block or EM. Staining of the external control as expected indicates that IHC procedure is correct.

The histological type and grade are also important in the evaluation of ER; if ER is negative in low-grade tumors such as mucinous and tubular carcinoma, where it is expected to be positive, the study must be repeated.[]

In our study, the concordance rate between two methods for PR was 89%, and it was lower than ER as in the literature. It is thought that this was related to the tumor heterogeneity and PR had more heterogeneous distribution than ER within the tumor.[,,] Although there was no significant difference in terms of mean PR staining percentage, intensity of staining was higher in EMs. Greer et al.[] showed that the concordance between CB and EM in terms of ER, PR, and HER2 decreased in heterogeneous tumors, and the concordance increased with the increased number of CB samples. In the study, PR positivity was detected in more cases in CBs compared to EMs. In many studies, a higher rate of PR positivity in CBs was found associated with rapid fixation and better formaldehyde permeation into the tissue.[,,]

Immunohistochemical HER2 results may vary between laboratories and this discordance rate can increase up to 18-26%.[,] In our center, for CBs, we can successfully provide fixation conditions recommended in ASCO/CAP guideline. However, cold ischemia time for EMs may sometimes exceed 1 h.[]

In our study, the concordance rate of immunohistochemical HER2 expression between CB and EM was 73%, moderately concordant and significant. The concordance rates were 97%, 24%, and 92% in positive, suspicious, and negative cases, respectively. HER2 was positive in 20% of CBs and 15% of EMs. We detected a higher rate of immunohistochemical HER2 positivity in CBs compared to EMs. Park et al.,[] in a series of 104 cases, showed positive HER 2 staining in 22 CBs and 20 EMs but HER2 amplification was not searched. In our study, SISH method was applied to all patients with score 2+. When IHC and SISH results were evaluated together, concordance rate between two methods reached to 96%. In the previous studies, high concordance rates ranging from 86.5% to 100% in terms of HER2 positivity have been reported.[,-]

The general opinion is that CB represents a very small part of the tumor. Especially, at multiple tumors and at the tumors larger than 4.5 cm, heterogeneity is higher, and CB does not reflect the entire tumor.[,-] Greer et al.[] showed that the concordance between CB and EM in terms of HER2 expression was worse than ER and PR in heterogeneous tumors, so they emphasized that HER2 study should be repeated in EM.

In our study, since the SISH method was applied only to cases with suspicious HER2 positive, the concordance between two methods could not be examined in terms of SISH. Shousha et al.[] found 89% concordance between two methods in terms of SISH results, and suggested that 11% discordance was associated with tumor heterogeneity.

Limitations of the Study
In the current approach, Ki67 is routinely studied together with HR and HER2 in patients with breast cancer. However, since our study was retrospective and there were no Ki67 results for most cases in the pathology reports, core biopsies and EM could not be compared in terms of Ki67.