Research ArticleExperimental Studies

Validation of 2-mm Tissue Microarray Technology in Gastric Cancer. Agreement of 2-mm TMAs and Full Sections for Glut-1 and Hif-1 Alpha

FELIX BERLTH, STEFAN P. MÖNIG, HANS A. SCHLÖSSER, MARTIN MAUS, CHRISTOPH T.H. BALTIN, ALEXANDER URBANSKI, UTA DREBBER, ELFRIEDE BOLLSCHWEILER, ARNULF H. HÖLSCHER and HAKAN ALAKUS
Anticancer Research July 2014, 34 (7) 3313-3320;

Abstract

Background/Aim: Tissue Microarray (TMA) is a widely used method to perform high-throughput immunohistochemical analyses on different tissues by arraying small sample cores from paraffin-fixed tissues into a single paraffin block. TMA-technology has been validated on numerous cancer tissues and also for gastric cancer studies, although it has not been validated for this tumor tissue so far. The objective of this study was to assess, whether the 2-mm TMA-technology is able to provide representative samples of gastric cancer tissue. Materials and Methods: TMA paraffin blocks were constructed by means of 220 formalin-fixed and paraffin-embedded gastric cancer samples with a sample diameter of 2 mm. The agreement of immunohistochemical stainings of Glut-1 and Hif-1 alpha in TMA sections and the original full sections was calculated using kappa statistics and direct adjustment. Results: The congruence was substantial for Glut-1 (kappa 0.64) and Hif-1 alpha (kappa 0.70), but with an agreement of only 71% and 52% within the marker-positive cases of the full-section slides. Conclusion: Due to tumor heterogeneity primarily, the TMA technology with a 2-mm sample core shows relevant limitations in gastric cancer tissue. Although being helpful for tissue screening purposes, the 2-mm TMA technology cannot be recommended as a method equal to full-section investigations in gastric cancer.

Tissue microarray (TMA) technology is used for tissue studies of multiple cancers (1-6). This technology was designed for high-throughput immunohistochemical studies on different tissues. By arraying core biopsies (0.6 to 2 mm in diameter) from formalin-fixed and paraffin-embedded donor specimens to a recipient block large numbers of tissue samples can be analyzed time and cost-efficient under identical conditions (7, 8). Tissue microarrays should allow an efficient and rapid analysis of large tissue numbers, whereby the representativity of the core for the corresponding full-section slide is crucial for this technique. In order to review the technique on different tissues, several validation studies with different types of cancers have been performed in recent years. These studies included cancer tissues of the esophagus, head and neck and endometrium (9-13). As there is an interest to investigate on the prognostic value of molecular markers in gastric cancer, it is inevitable to perform a TMA validation before using the TMA technology in immunohistochemical studies on gastric cancer tissues. The study of Guldman et al. investigated the validity of Tissue Microarrays taken out of biopsies of gastric cancer. This study with a number of thirty eight cases aimed to evaluate Tissue Microarray Technology for biopsies but still there is no TMA validation study for gastric cancer in a high number of patients and with use of full-section slides published (14). Besides the advantages cost-effectiveness, tissue saving and speed of analysis, a major disadvantage of TMA technology in gastric cancer might be disconcordances in downstream experiments between TMA cores and full sections as TMA cores are only a sample of a whole section (15). It is still discussed, if a tumor sample can be representative for the whole specimen regarding certain heterogeneity in the expression of molecular markers in tumor tissue (16). The aim of the present study was to evaluate the TMA technology on gastric cancer tissue by investigating the concordance of immunohistochemical staining patterns of a 2-mm biopsy core with the full-section slide. Regarding the fact that there are only few data concerning the 2-mm TMA technology available and the analyzed area in a single 2-mm TMA biopsy is three- to four-fold higher than three 0.6 mm biopsies (3.1 vs. 0.84 mm2) we decided to use single 2-mm biopsies.

Materials and Methods

Construction of TMA paraffin blocks. Disposable paraffin-embedded specimen materials of patients with a gastric cancer who underwent a gastrectomy with systematic lymphadenectomy at the Institute of General, Visceral and Cancer Surgery of the University of Cologne between 1996 and 2009 and were recruited from the archive of the Institute of Pathology. The population included patients who underwent neoadjuvant chemotherapy and cases of the intestinal and the diffuse-type regarding to the Laureén classification. Up to three representative tumor regions were marked in an H&E stained full section slide of each case by an experienced pathologist (U.D.). Of the three paraffin block counterparts, the best concerning tissue quality and amount of tissue was chosen to be the donor block for the TMA core biopsies. After taking 3-mm slides with a microtome for the full-section staining, a tissue cylinder of 2 mm diameter was stamped out of the donor block and in each case 23 cylinders were transferred to a recipient paraffin block, including one cylinder of a breast cancer donor block, for internal controls (Figure 1). As 220 cases of gastric cancer were available for this step, all in all ten recipient paraffin blocks with 22 cases each were constructed. Besides the breast cancer case, one specific spot on each of the recipient blocks was left empty to surely distinguish the ten recipient blocks. A manual tissue arrayer and particular recipient block paraffin was used for TMA construction.

Immunohistochemistry. For each immunohistochemical marker and for the H&E staining, a 3-micrometer slide was taken from each of the TMA donor blocks and of the full section blocks. The details of the antibodies, the dilution, method and incubation time, that was used for this study is listed below.

The de-paraffinization process for the full-section slides was done with alternate xylene and acetone for 2×5 min followed by rehydration in distilled water. The TMA slides were deparaffinized in xylene for 15 h before this first step due to a different type of paraffin used for TMA constructing. Antigen retrieval was done in a pH 6.5 citrate buffer solution for 15 min. After a cooling down period of 10 min the slides were incubated in distilled water for another 5 min. For blocking the endogenous peroxidase, a solution of 0.3% hydrogen peroxide and methanol (1:100) was used for 20 min followed by a 2×5 min period in phosphate buffered saline solution (PBS: pH 7,4). The primary antibody was used as recommended in a solution of 1:200 for Glut-1 and 1:20 for Hif-1 alpha (Table V). The slides were incubated for 16 hours in a moist chamber at 4 degrees Celsius. Incubation time with the secondary antibody was 30 min. The staining itself was done by AEC solution, in which the slides were incubated for 30 min in a dark chamber at room temperature before counterstaining the slides with hematoxylin. In between, the slides were washed again in PBS buffer solution.

View this table:
Table I.

Overview of available cores in H&E staining for verifying the tumor content.

Immunohistochemical scoring. Slides of each TMA Block and the full section slides were used for immunohistochemical staining with Glut-1 and Hif-1 alpha antibodies. Slides of each TMA block underwent an H&E staining in order to monitor the tumor content of every single tissue core. Cores without tumor tissue and those with less than 50% of tissue shown in the H&E staining were not used for the validation. In the same manner the immunohistochemical cases were excluded from further analysis, if less than 50% of the tissue was presented in a TMA sample.

Glut-1 and Hif-1 alpha were marked negative (<10% of tumor cells stained) and positive (10-100% tumor cells stained)(17-19). Glut-1 staining appeared as a membranous and cytoplasmic staining pattern, whereas Hif-1 alpha presented a (peri-) nuclear staining pattern. Areas of necrosis were not counted as stained tumor tissue.

Statistical analysis. The statistical analysis was done with the help of SPSS (Windows-version 17.0, Chicago, IL, USA). All disposable cases were used for the statistical comparison of TMA and full section slides. For the statistical assessment of the agreement between TMA and full section slide, the Cohen‘s weighted kappa test was performed. The agreement was called poor if k<0.4, moderate if 0.4<k<0.6, substantial if 0.6<k<0.8 and almost perfect if k>0.8. In addition, the percentage of concordance of all TMA‘s and the full section slides was calculated (20).

Results

H&E staining. All 220 TMA cases were analyzed under H&E staining, which was chosen to be the reference for verifying the content of tumor tissue in every TMA core (Table I). In regards to H&E staining, 26 (11%) cases had to be excluded before TMA evaluation. 12 cores (5%) were missing in the TMA slides for H&E staining so that the tumor content could not be verified and 14 (6%) cores did not show tumor tissue under microscopic analysis (Figure 2).

Hif-1 alpha and Glut-1 staining. For Glut-1 and Hif-1 alpha staining, 216 and 218 cases respectively were available for TMA evaluation, as four and two cases of the full-section slides were missing due to tissue damage during the staining process (Table II). As this loss was not due to TMA technology, these cases were excluded from statistical analysis.

View this table:
Table II.

Number of available cores for Hif-1 alpha and Glut-1 staining

View this table:
Table III.

Agreement of TMA and full-section for Glut-1 staining.

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Table IV.

Agreement of TMA and full-section for Hif-1 alpha staining.

TMA validation. Out of all tumor tissue cores that were transferred into the ten paraffin blocks, an average of 87% (Tables I and II) was evaluable in immunohistochemical analysis. In addition to the 26 cases that were excluded after the H&E staining, due to tissue loss within the Hif-1 alpha and Glut-1 staining, respectively 9 (4%) and 6 (3%) additional cases were not evaluable. Allowing for the intersections, cases that were excluded in terms of the H&E staining and at the same time missing for the corresponding immunohistochemical analysis due to tissue loss, 188 cases were evaluable both for Glut-1 and Hif-1 alpha (Table II). Concerning the Glut-1 staining the total agreement was 82%, with an accordance of 71% within the positive scored full-section slide cases. Interestingly 7 cases (4%) showed a positive staining pattern in the TMA slides, as these cases did not present any tumor tissue positive for Glut-1 in the full-section slide. The weighted kappa score for Glut-1 was 0.64, which is counted as substantial (Table III).

View this table:
Table V.

Materials used.

The total agreement of the full-section slides and the TMA slides for Hif-1 alpha was 85%, whereas the agreement for the positive cases referring to the full section slides was only 52% (Table IV). The agreement within the negative full section cases was almost perfect (99%). The weighted kappa score was 0.70, which is also counted as substantial (Table IV).

Discussion

Since the modern TMA technology has been introduced in 1998 (7), many studies have used it for immunohistochemical analysis on different malignant tissues (1, 21, 22). As many validation studies showed almost perfect concordance rates, other investigations revealed significant differences between TMA and full section slides (23). One major problem of taking samples of full-section tumor blocks is the heterogeneity of marker expression of the tumor cells, which is predicted to show a unique expression pattern for each marker. Many evaluation studies have used a tissue core diameter of 0.6 mm (13, 24-26). In order to take samples from different parts of the tumor, two to six samples were obtained. In this study, one core of 2 mm in diameter was taken from each donor block. The circumstance that only one sample was taken was countered by the fact, that one 2-mm core is almost two-times bigger than six 0,6-mm cores, or in other words, the 2-mm core is equipped with about eleven-times more tissue than a single 0.6-mm core. Furthermore, the content of tumor and the quantity of staining is assessable better in one bigger piece of tissue, and a core of bigger diameter is more stable when processing the paraffin block and the TMA slide. Prior studies have shown that a needle size of 2 mm leads to a more efficient target selection and a more successful target arraying (27, 28). On the other hand many 0,6-mm cores can be taken out of different localizations of the tumor in the paraffin block, which could lead to higher representativity regarding the expression of antigens. As there is a lack of data concerning the 2-mm TMA technology, therefore this technique was evaluated.

Figure 1.
Figure 1.

Recipient paraffin block with 23 embedded tissue cylinders.

Nevertheless, it is essential to evaluate the TMA technology for every cancer entity individually and to our knowledge this has not been done in gastric cancer with gastrectomy specimens so far.

As another disadvantage of TMA technology the tissue loss during constructing and preparing is recorded (29). Our TMA blocks were constructed to deliver 220 different tumor spots. The overall percentage of assessable cases was 86 and 87% for Hif-1 alpha and Glut-1 and is comparable to results reported in other studies (13, 30, 31). In fact the tissue loss of the TMA slides at each immunohistochemical staining process was even relevantly lower, but in reference to the preceding H&E staining, 26 (12%) cases had to be eliminated (Table I). Both, tissue loss itself and insufficient tumor content were equally contributing factors for this reduction of evaluable cases. Tissue loss can be caused by tissue treatment with more fragile slides, containing many, sometimes-loose cores. The fact that some cores did not include tumor tissue might be contributed to improper selection of the tumor area, incorrect punching of these areas out of the donor block or a minor quality of the donor block itself. The tissue loss of following slides taken from these TMA blocks will be listed as well and compared with the resent loss in order to elucidate, if a deeper layer of the TMA blocks is delivering more, less or the same number of evaluable cores.

The decrease of total cases to 218 and 216 in Hif-1 alpha and Glut-1 staining was caused by missing full section slides and was consequently not counted as a TMA dependent tissue loss (Table II).

Figure 2.
Figure 2.

Immunhistochemical and H&E staining of the Tissue Microarrays of gastric cancer specimens. (A, B) Glut-1-positive immunhistochemical staining (4× and 10× objective). (C, D) Hif-1 alpha-positive immunohistochemical staining (4× and 10× objective). (E) H&E staining (4× objective). (F) Missed array on a H&E-stained microarray slide (4× objective).

An overall tissue loss under 15% seems acceptable in respect of the expense that TMA technology is able to save in the staining process and the number of cases that are processed simultaneously.

The agreement of the immunohistochemical scoring between the full Section slides and the TMA slides can be called substantial for Glut-1 (k=0.64) and for Hif-1 alpha (k=0.70), which implicates the congruence of all cases, marker-positive and marker-negative (Table III, IV). Although this adjustment is used for many TMA evaluation studies one weak spot is the equal allowance of immunohistochemical-positive and -negative cases. In studies, that used the immunohistochemical results of Hif-1 alpha or Glut-1 staining, the positive marker expression was correlated with the clinicopathological parameters of the patients (32-35). For these study purposes, the cases of interest are the ones showing a positive expression of the molecular marker. When regarding to the cases that show an expression of Glut-1 and Hif-1 alpha in the full section, the agreement with the TMA is only 71% and 52% (Table III, IV). This deviation can be explained by tumor heterogeneity or the scoring system used.

Tumor heterogeneity is a major concern when using TMA technology and therefore some other studies recommended at least two or three 0.6-mm core biopsies to be most efficient (36, 37). A TMA validation study for prostate cancer tissue showed that five to ten 0.6-mm core biopsies are needed to assess focal expression (38). In this study the idea was to take one large biopsy of 2 mm in diameter to have a larger area of tissue to assess. In some cases the biopsy was hardly smaller than the tumor area in the paraffin block, which made it very likely to get a representative TMA core. On the other hand when dealing with a large area of tumor, three samples could have been taken from different locations. But when thinking of tumor heterogeneity, it is expected to lead to an individual expression of every marker, so TMA can logically never meet the requirements for every marker as the samples are taken only once and before the staining. Regarding tumor heterogeneity TMA technology, irrespective of the core diameter taken, is always a compromise, so the center of interest should be the utility when correlating the staining results with clinicopatholigical parameters, which does not necessarily require a full agreement between full section and TMA.

Therefore a two-class system was used to interpret the immunohistochemical staining, as it was also planned to do so when only using the full section slides result for correlation with clinicopathological parameters. Preceding studies showed a higher agreement for the two-class system compared to a three class system in endometrial cancer (12). Beyond that, the cut off values affect the level of agreement, which could be seen when comparing the Glut-1 and Hif-1 alpha staining results (data not shown). Although for both stainings the same cut off was used, the agreement within the positive full section slides differentiated (71% vs. 52%). An explanation is that the Glut-1 staining presented relevantly more cases with a positive staining result (93 vs. 56) and showed a higher number of cases with a highly positive staining pattern (data not shown). If a tumor area expresses a marker in a high proportion it is more likely to hit that area with the TMA punching machine. Consequently a higher cut off value should produce a higher agreement between full section slide and TMA.

In conclusion, the TMA technology using a 2-mm sample core delivers a substantial agreement with the full section slides in gastric cancer, but a minor agreement within the cases of positive expression. In respect of the limitation of the TMA technology in gastric cancer TMA slides could be recommended to be used with markers of a high expression and a more homogenous staining pattern. Therefore, before using a marker with the TMA slides, a lower number of full section slides should be stained in order to check the criteria. For markers with a low expression grade or those, which show a focal and inhomogeneous staining pattern, the full section slides is the superior method. It has to be investigated, if the 0,6-mm TMA technology shows a higher concordance for those markers with such heterogeneous expression. Being evaluated for other tissues, a 0,6 mm or 1 mm TMA validation study for a large number of gastric cancer cases is still to be done. Besides that the TMA slides can be used for high throughput marker trial in order to save tissue, time and effort and to review many markers on a large number of cases to select the markers with the most promising results to use them on the full sections for more differentiated results.

In summary, the 2-mm TMA technology is a helpful method for tissue investigation in gastric cancer but should be used only after conducting a validation study for the TMA blocks and in respect to the limitations of this technology so far.

Footnotes

  • Conflicts of Interest

    The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.

  • Received March 8, 2014.
  • Revision received May 9, 2014.
  • Accepted May 12, 2014.

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Validation of 2-mm Tissue Microarray Technology in Gastric Cancer. Agreement of 2-mm TMAs and Full Sections for Glut-1 and Hif-1 Alpha
FELIX BERLTH, STEFAN P. MÖNIG, HANS A. SCHLÖSSER, MARTIN MAUS, CHRISTOPH T.H. BALTIN, ALEXANDER URBANSKI, UTA DREBBER, ELFRIEDE BOLLSCHWEILER, ARNULF H. HÖLSCHER, HAKAN ALAKUS
Anticancer Research Jul 2014, 34 (7) 3313-3320;
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