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EPR17341 and A7H6R pan-TRK Immunohistochemistry Result in Highly Different Staining Patterns in a Series of Salivary Gland Tumors

Guibourg, Briac MD*; Cloarec, Emma BSc*; Conan-Charlet, Virginie MD*; Quintin-Roué, Isabelle MD*; Grippari, Jean-Luc MD*; Le Flahec, Glen MD*; Marcorelles, Pascale MD, PhD*; Uguen, Arnaud MD, PhD*,†

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Applied Immunohistochemistry & Molecular Morphology: October 2020 - Volume 28 - Issue 9 - p 719-724
doi: 10.1097/PAI.0000000000000825
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Abstract

Larotrectinib and entrectinib tyrosine kinase inhibitors have demonstrated encouraging efficacy in patients with unresectable or metastastic tumors of various subtypes with genes fusions involving NTRK1, NTRK2, or NTRK3.1,2NTRK rearrangements have been shown to be frequent in some rare subtypes of tumors as, for example, secretory carcinomas of the breast (about 95% of NTRK rearrangements) or of the salivary glands (anciently called mammary analog secretory carcinoma “MASC” with about 80% or NTRK rearrangements), but they are exceptional in more common cancer subtypes (eg, <1% of non–small cell lung carcinomas, colorectal cancers, or melanomas except Spitzoid ones and about 1% of gliomas in adults).3–5 In this manner, given on the one hand the rarity of these molecular alterations, and, in the other hand the need to accurately diagnose NTRK rearrangements for therapeutic purpose in patients with various advanced cancers, testing for NTRK rearrangement in daily practice could be challenging.

Ideally, a systematic molecular analysis of every cancer diagnosed at advanced stage using large cancer-dedicated gene panels comprising NTRK1, NTRK2, and NTRK3 may permit to identify and treat the patients with these rare NTRK rearrangements. Nevertheless, to date, because of workflow, technical, and economic reasons, such a systematic molecular analysis is not attainable for every cancer and targeted tests remain relevant. Examples of this relevancy of the targeted tests is provided by the diagnostic strategy used for ALK and ROS1 testing in non–small cell lung carcinomas with first lines ALK and ROS1 immunohistochemistry (IHC) analyses followed by fluorescent in situ hybridization (FISH) with ALK-dedicated or ROS1-dedicated break-apart FISH probes in case of ALK IHC low positivity (ie, scores 1+ or 2+) or any ROS1 IHC positive result (ie, scores 1+ to 3+), respectively. The diagnosis of NTRK1, NTRK2, and NTRK3 rearrangements is also possible using NTRK1, NTRK2, and NTRK3-specific break-apart FISH probes but performing 3 FISH tests for each patient with advanced cancer does not seem to consist in a time-effective and cost-effective approach. Pan-TRK IHC has been proposed as a premolecular test able to detect tumors with expression of any TRK-A, TRK-B, or TRK-C protein expression (coded by NTRK1, NTRK2, and NTRK3 genes, respectively). Two main pan-TRK IHC tests are used in loratrectinib-dedicated and entrectinib-dedicated trials: EPR17341 (Abcam, Cambridge, MA) and A7H6R (Cell Signaling Technology, Danvers, MA), respectively.5–10

To date, a single study has compared the 2 pan-TRK IHC tests in the literature. Indeed, in a previous melanoma-dedicated work, we have shown that the 2 pan-TRK IHC tests did not stain in a similar manner the same tumors but the few positive tumors in this field (3 with the A7H6R clone and none using EPR17341 clone) has prevented us to draw formal conclusions in this field.11 Recently, a study by Hung et al12 has reported a high rate of pan-TRK IHC positive tumors (with EPR17341 clone) in the field of salivary gland tumors with IHC positivity in 100% of pleomorphic adenomas, 83% of polymorphous adenocarcinomas, 20% of low-grade mucoepidermoid carcinomas, 64% of secretory carcinomas (but 0% of acinic cell carcinoma). For this reason, we decided to perform a method comparison study about A7H6R and EPR17341 pan-TRK IHC in a cases series of salivary gland tumors.

MATERIALS AND METHODS

Cases Studied

The cases included in this study were diagnosed at the Brest University Hospital. We selected 71 cases of various benign and malignant salivary glands tumors covering different histologic subtypes comprising notably some those studied by Hung and colleagues but also additional subtypes. Archival formalin-fixed and paraffin-embedded tumor samples were used to perform IHC and FISH analyses. The present study was conducted following our national and institutional guidelines. All samples were included in a registered tumor tissue collection and the present study was conducted in compliance with the Helsinki Declaration and after approval by our institutional review board (CHRU Brest; CPP no. DC—2008–214).

IHC

We used the 2 afford-mentioned pan-TRK monoclonal rabbit antibodies clones EPR17341 (6 µg/mL dilution) and A7H6R (1:50 dilution). IHC processes using Ventana Benchmark Ultra® automated slide preparation system (Roche Diagnostics, Meylan, France) comprised, after deparaffinization, a 64 minutes CC1 pretreatment step (Roche Diagnostics) followed by antibody incubation during 52 minutes, washing and OptiView DAB IHC Detection Kit (Roche Diagnostics) before hematoxylin counterstaining and mounting. We used 2 NTRK1-rearranged tumor (1 glioblastoma and 1 Spitz tumor) from previous studies as controls.11,13

Each IHC slide was read twice by 1 pathologist (A.U.). Nuclear, cytoplasmic, and/or membranous staining was considered for staining quantification. A “H score”-inspired interpretation method was used for interpretation: staining intensity was quoted as negative (0), weak (1+), moderate (2+), or strong (3+) in the different areas of the tumors and the percentage of tumor cells with different intensity within the tumor section staining was noted to calculate a staining scores of 0 to 300 as follows: [1×(% “cells 1+”)+2×(% “cells 2+”)+3×(% “cells 3+”)]. Given the H score values, tumors were classed as “IHC positive” (ie, corresponding to a H score ≥5 corresponding in at least a weak 1+ staining of 5% of tumor cells as previously chosen by Hung and colleagues) or “IHC negative” (ie, corresponding to a H score <5). The types and location of stained cells within tumor areas were also noted in positive samples and compared between the 2 IHC slides with the 2 clones.

Statistical Analyses

Statistical analyses were performed using MedCalc Statistical Software version 13.2.2 (MedCalc Software bvba; Ostend, Belgium; www.medcalc.org; 2014). The level of significance was set at P<0.05. The κ statistic test was used to quantify the inter-IHC method “IHC positive” versus “IHC negative” criteria. Intraclass correlations coefficients was used to calculate the inter-IHC method agreement for the H score values. The values of κ strength agreements and intraclass correlation were interpreted as follows: <0.20 poor, 0.21 to 0.40 fair, 0.41 to 0.6 moderate, 0.61 to 0.80 good, and 0.81 to 1.00 very good agreement.

RESULTS

Cases Studied

In total, 71 cases of salivary glands tumors from 33 men and 38 women with a mean age of 54 years (range, 19 to 87 y) and involving the parotid (59 cases, 83.1%), submandibular (9 cases, 12.7%), sublingual (1 case, 1,4%), or accessory salivary glands (2 cases, 2.8%) were selected for this study. Among the 71 cases of salivary gland tumors, 43 were benign tumors (60.6%) and consisted in 17 pleomorphic adenomas (23.9%), 15 cystadenolymphomas (21.1%), 6 basal cells adenomas (8.4%), 4 oncocytomas (5.6%), and 1 myoepithelioma (1.4%). Among the 28 malignant tumors (39.4%), 8 were carcinomas ex-pleomorphic adenomas (11.3%, with 4 adenocarcinomas, 1 epithelial-myoepithelial carcinoma, 1 myoepithelial carcinoma, 1 high-grade mucoepidermoid carcinoma, and 1 squamous cell carcinoma), 6 were mucoepidermoid carcinomas (8.4%, 4 low-grade, 1 high-grade, and 1 indermediate-grade), 5 were adenoid cystic carcinomas (7%), 2 were acinar cell carcinomas (2.8%), 6 were adenocarcinomas without other specification (8.4%), and 1 was a secretory carcinoma of the parotid (1.4%). More details about the cases included as well as the details about IHC results are listed in Table 1.

TABLE 1 - Details of IHC Analyses for the 71 Tumors
EPR17341 IHC A7H6R IHC
Sex Age (y) Salivary Gland Tumor Type ± H Score ± H Score
M 30 Parotid Secretory carcinoma + 160 + 160
F 71 Parotid Ex AP carc. + 10 + 25
M 51 Parotid Ex AP carc. 0 0
M 66 Parotid Ex AP carc. 0 0
F 76 Parotid Ex AP carc. 0 5
M 80 Parotid Ex AP carc. 0 0
F 51 Parotid Ex AP carc. 0 0
M 68 Parotid Ex AP carc. 0 0
F 71 Submandibular Ex AP carc. 0 0
F 69 Submandibular Adenoid cystic carc. + 160 0
M 61 Accessory Adenoid cystic carc. + 25 0
F 25 Parotid Adenoid cystic carc. 0 0
F 60 Parotid Adenoid cystic carc. 0 0
F 32 Submandibular Adenoid cystic carc. 0 0
F 44 Parotid Mucoepidermoid carc. 0 0
F 57 Parotid Mucoepidermoid carc. 0 0
M 35 Parotid Mucoepidermoid carc. 0 0
F 41 Parotid Mucoepidermoid carc. 0 0
F 75 Parotid Mucoepidermoid carc. 0 + 10
M 76 Submandibular Mucoepidermoid carc. 0 0
M 33 Parotid Acinar cells carc. 0 0
F 20 Parotid Acinar cells carc. 0 0
M 43 Parotid ADK 0 0
M 64 Parotid ADK 0 0
F 81 Parotid ADK 0 0
F 71 Parotid ADK 0 + 40
M 67 Parotid ADK 0 0
F 51 Parotid ADK 0 0
F 50 Sublingual Pleomorphic ad. + 60 + 40
F 31 Submandibular Pleomorphic ad. + 75 + 10
M 45 Submandibular Pleomorphic ad. + 10 + 45
M 48 Submandibular Pleomorphic ad. + 20 + 90
M 45 Parotid Pleomorphic ad. + 30 + 30
F 55 Parotid Pleomorphic ad. + 15 0
F 50 Parotid Pleomorphic ad. + 5 0
M 30 Parotid Pleomorphic ad. + 45 0
F 38 Parotid Pleomorphic ad. + 40 + 25
F 87 Parotid Pleomorphic ad. + 10 + 25
M 38 Parotid Pleomorphic ad. + 40 + 60
M 28 Parotid Pleomorphic ad. + 180 0
F 61 Parotid Pleomorphic ad. + 30 + 5
M 79 Parotid Pleomorphic ad. + 30 + 75
M 53 Parotid Pleomorphic ad. + 35 + 20
M 57 Submandibular Pleomorphic ad. 0 + 15
F 40 Parotid Pleomorphic ad. 0 0
F 41 Parotid Cystadenolymphoma 0 0
F 42 Parotid Cystadenolymphoma 0 0
F 76 Parotid Cystadenolymphoma 0 0
M 53 Submandibular Cystadenolymphoma 0 0
F 59 Parotid Cystadenolymphoma 0 0
F 56 Parotid Cystadenolymphoma 0 0
M 51 Parotid Cystadenolymphoma 0 0
M 74 Parotid Cystadenolymphoma 0 0
M 64 Parotid Cystadenolymphoma 0 0
M 60 Parotid Cystadenolymphoma 0 0
M 47 Parotid Cystadenolymphoma 0 0
M 57 Parotid Cystadenolymphoma 0 0
F 66 Parotid Cystadenolymphoma 0 0
F 83 Parotid Cystadenolymphoma 0 0
M 53 Parotid Cystadenolymphoma 0 0
M 54 Parotid Basal cells ad. + 15 + 150
F 19 Parotid Basal cells ad. + 100 + 150
F 21 Parotid Basal cells ad. + 135 + 150
F 44 Parotid Basal cells ad. 0 0
M 58 Parotid Basal cells ad. 0 0
M 66 Accessory Basal cells ad. 0 0
F 38 Parotid Oncocytoma 0 0
F 70 Parotid Oncocytoma 0 0
M 50 Parotid Oncocytoma 0 0
F 79 Parotid Oncocytoma 0 0
F 47 Parotid Oncocytoma + 75 + 45
− indicates negative immunohistochemistry; +, positive immunohistochemistry; Ad., adenoma; ADK, adenocarcinoma without other specification; Carc., carcinoma; Ex AP carc., carcinoma ex-pleomorphic adenoma; IHC, immunohistochemistry.

IHC Results and Intermethod Agreement for A7H6R-based and EPR17341-based Analyses

Pan-TRK IHC using the EPR17341 clone was positive (ie, H score ≥5) in 23/71 tumors (32.4%) consisting in 1 secretory carcinoma, 2 adenoid cystic carcinomas, 1 epithelial-myoepithelial carcinoma, 1 myoepithelioma, 3 basal cell adenomas, and 15 pleomorphic adenomas. Using A7H6R pan-TRK clone, IHC was positive in 20/71 tumors (28.2%) including 1 secretory carcinoma, 1 epithelial-myoepithelial carcinoma, 1 poorly differentiated adenocarcinoma, 1 low-grade mucoepidermoid carcinoma, 1 myoepithelioma, 3 basal cell adenomas, and 12 pleomorphic adenomas.

Given the “positive” versus “negative” result of EPR17341-based and A7H6R-based pan-TRK IHC, the intermethod comparison result in a κ value of 0.7 (95% confidence interval: 0.520; 0.881), reflecting a “good agreement.” A number of 9 tumors had discrepant conclusions between the 2 tests: 6 EPR17341-positive but A7H6R-negative tumors (4 pleomorphic adenomas and 2 adenoid cystic carcinomas), and 3 EPR17341-negative but A7H6R-positive tumors (1 pleomorphic adenoma, 1 poorly differenciated adenocarcinoma, and 1 low-grade mucoepidermoid carcinoma). The single NTRK-rearranged case of our series (ie, a NTRK3-rearranged salivary secretory carcinoma) was positive with the 2 antibodies (Fig. 1).

FIGURE 1
FIGURE 1:
Pan-TRK immunohistochemistry results using EPR17341 (A) and A7H6R clones (B) in the NTRK3-rearranged secretory carcinoma of the parotid gland (C). A and B, ×20 magnification, DAB revelation and hematoxylin counterstaining. C, ZytoLight SPEC NTRK3 Dual Color Break Apart FISH Probe, ZytoVision GmbH, Bremerhaven, Germany, DAPI counterstaining, ×100 magnification.

Considering the H scores, the intraclass coefficient correlation was 0.5399 (95% confidence interval: 0.3521; 0.6859) reflecting a “moderate agreement” between the 2 methods related to the differences about the percentage, and staining intensity of IHC positive cells (see Fig. 2 for variations across tumors than were positive for at least 1 IHC test). We also observed very different staining patterns in terms of stained cells between A7H6R and EPR17341 clones as illustrated in Figure 3 with a more diffuse staining using EPR17341 clone, almost equal in myoepithelial and epithelial cells, whereas the staining was more intense but also more “patchy” and predominant in myoepithelial cells using A7H6R clone.

FIGURE 2
FIGURE 2:
Comparison of the H scores for EPR17341 and A7H6R pan-TRK immunohistochemistry across the 26 samples with at least 1 pan-TRK positive results. *Indicates the H scores for the NTRK3-rearranged secretory carcinoma of the parotid gland.
FIGURE 3
FIGURE 3:
Examples of pan-TRK immunohistochemistry results using EPR17341 (A, C, E, G, I, K) and A7H6R clones (B, D, F, H, J, L) in the NTRK1-rearranged Spitz tumor (control, A and B), 2 pleomorphic adenoma (C and D; and E and F), a basal cells adenoma (G and H), an adenoid cystic carcinoma (I and J), and a myoepithelioma (K and L). Examples of different staining intensity are provided with 0 (no staining: D, J), 1+ (weak staining: E, G, K), 2+ (moderate staining: C, H, I), and 3+ (strong staining: A, B, F, L) (×20 magnification, DAB revelation, and hematoxylin counterstaining, the areas illustrated are the same for each tumor for EPR17341 and A7H6R staining).

DISCUSSION

Several studies have recently report on the potential interest of pan-TRK IHC in screening for NTRK-rearranged tumors of various origins and histologic subtypes. EPR17341 is by far the most reported clone in the literature in this field but at least another clone, A7H6R, has been also proposed as a premolecular screening tool. Both antibodies target the C-terminal region of the TRK-A protein and are said to react with TRK-A, TRK-B, and TRK-C proteins and with the chimeric proteins issued of gene fusions preserving the 3′-part of any of the NTRK genes. Nevertheless, beyond our previous study dedicated to melanoma, no study has compared the staining of these 2 antibodies in a set of tumors containing a sufficient number of TRK proteins-expressing tumors.11

In our study dedicated to a set of salivary glands tumors, about one third of tumors presented a positive pan-TRK IHC with at least 1 of the 2 antibodies, mainly in pleomorphic adenomas (but not in all of them at the difference of a previous study by Hung and colleagues) and in basal cell adenomas. Among salivary gland malignancies, TRK protein expression is known to be a hallmark of secretory carcinomas in relation with a frequent ETV6-NTRK3 rearrangement in these tumors (as for 1 case in our study) but other tumors without NTRK gene rearrangement can also express these proteins as polymorphous adenocarcinomas and mucoepidermoid carcinomas.12,14 In our study, we also observed pan-TRK IHC positive results in 2/5 adenoid cystic carcinomas that was concordant with the data reported in a large set of various tumors of different organs and histologic subtypes by Solomon et al,15 reporting 8/12 adenoid cystic carcinomas positive for EPR17341 pan-TRK IHC despite the absence of NTRK rearrangement in this histologic subtype of tumor.

Beyond the analysis of NTRK-rearranged tumors (comprising notably secretory carcinomas only in the field of salivary gland tumors), it is worth for pathologists to know that pan-TRK IHC can be positive in tumors lacking any NTRK rearrangement for at least 2 reasons. First, not to conclude as a candidate for TRK-targeted therapy a patients with a cancer expressing TRK proteins without underlying NTRK rearrangement, the molecular confirmation of this rearrangement being required for the targeted therapy to be indicated. Second, to identify potential positive controls (beyond the very rare cases of NTRK-rearranged tumors) permitting the development and implementation of pan-TRK IHC testing as a first line and premolecular screening tool in pathology laboratories: in this field, we believe that the benign, frequent, and very often pan-TRK positive pleomorphic adenomas could be of interest.

CONCLUSIONS

To conclude, after a previous study dedicated to melanoma, the present study about salivary gland tumors confirms that EPR17341 and A7H6R clones can stain tumor samples in a very different manner in terms of intensity, percentage, nature, and location of stained cells.11 Given these differences, it would be interesting in future works to investigate whether these different staining patterns are also encountered in tumors series with NTRK1, NTRK2, or NTRK3 rearrangements, and if the staining vary from 1 rearranged NTRK gene to another. This comparison, requiring to collect a sufficient number of the rare NTRK-rearranged tumors, is beyond the scope of the present study but will be of crucial importance building diagnostic strategies screening for NTRK-rearranged cancers at the era of TRK-dedicated targeted therapies.

ACKNOWLEDGMENTS

The authors would like to acknowledge the technical staff of the Department of Pathology of Brest University Hospital and the Local tumor tissue biobank BB-0033-00037 (“CRB Santé/Tumorothèque de Brest”) for their collaboration in this study, and the Omnium group for support.

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Keywords:

NTRK; salivary gland tumor; immunohistochemistry; reproducibility

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