A Novel Immunohistochemical Classifier to Distinguish Hodgkin Lymphoma From ALK Anaplastic Large Cell Lymphoma

Claudia Döring; Martin-Leo Hansmann; Claudio Agostinelli; Pier P Piccaluga; Fabio Facchetti; Stefano Pileri; Ralf Küppers; Sebastian Newrzela; Sylvia Hartmann

Disclosures

Mod Pathol. 2014;27(10):1345-1354. 

In This Article

Results

Gene Expression Analysis

In a reevaluation of gene expression analyses performed by Brune et al[19] and Eckerle et al[20] a supervised comparison of the gene expression profiles of microdissected tumor cells of classical Hodgkin lymphoma and ALK anaplastic large cell lymphoma was performed. Eighteen transcripts were found to be overexpressed in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma compared with the tumor cells of ALK anaplastic large cell lymphoma and 15 transcripts were overexpressed in the tumor cells of ALK anaplastic large cell lymphoma (fold change<−1.8 or >1.8, P-value<0.05, false discovery rate<0.1, Table 3, Figure 1). Based on significant differential expression, macrophage-derived chemokine/chemokine ligand 22 (MDC/CCL22) (39.8-fold upregulated in Hodgkin and Reed-Sternberg cells), tubulin beta 2B (TUBB2B) (9.9-fold upregulated in Hodgkin and Reed-Sternberg cells), CD83 (8.4-fold upregulated in Hodgkin and Reed-Sternberg cells), signal transducer and activator of transcription 3 (STAT3) (4.2-fold upregulated in Hodgkin and Reed-Sternberg cells), and calpain 2 (CAPN2) (5.0-fold upregulated in the tumor cells of ALK anaplastic large cell lymphoma) were selected for immunohistochemical studies (Figure 1).

Figure 1.

Heat map of the genes differentially expressed in the microdissected tumor cells of classical Hodgkin lymphoma and ALK anaplastic large cell lymphoma from the studies by Brune et al 19 and Eckerle et al. 20 NA, not annotated; red color: high expression; black color: intermediate expression; green color: low expression. Genes selected for immunohistochemical studies are highlighted by arrows.

Immunohistochemical Investigation of Differentially Expressed Genes on Protein Level

In majority of the 30 classical Hodgkin lymphoma cases of the first series, stained on whole tissue sections, Hodgkin and Reed-Sternberg cells were positive for TUBB2B (80%) as well as STAT3, CD83, and MDC/CCL22 (83% each, Table 1 and Figure 2). MDC/CCL22 and CD83 were strongly expressed in the cytoplasm and Golgi field of Hodgkin and Reed-Sternberg cells, whereas TUBB2B showed a homogeneous expression in the cytoplasm. An enhanced STAT3 expression was observed in the nucleus and cytoplasm of Hodgkin and Reed-Sternberg cells. A weak expression of STAT3 was also observed in histiocytes and the tumor cells of some anaplastic large cell lymphoma cases (Figures 2e and f). Only 10% of the 31 anaplastic large cell lymphoma in the first series presented with a strong nuclear and cytoplasmic STAT3 expression and cytoplasmic TUBB2B expression. Like the Hodgkin and Reed-Sternberg cells, a subgroup of macrophages/dendritic cells stained positive for CD83 and MDC/CCL22 in some cases, whereas anaplastic large cell lymphoma tumor cells were consistently negative in this first series. Interestingly, among 10 classical Hodgkin lymphoma cases with expression of granzyme B, Hodgkin and Reed-Sternberg cells were less frequently positive for the Hodgkin classifier markers, when compared with the typical classical Hodgkin lymphoma cases being negative for cytotoxic molecules. In these cases, MDC/CCL22 was positive in only 5/10 cases (50%), CD83 was positive in 7/10 cases (70%), TUBB2B and STAT3 were each positive in 8/10 cases (80%).

Figure 2.

Immunhistochemical stainings differentiating classical Hodgkin lymphoma and anaplastic large cell lymphoma. (a and b) CD83; (c and d) MDC/CCL22; (e and f) STAT3; (g and h) TUBB2B; (i and j) CAPN2. (a, c, e and g) Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma show expression of the respective immunohistochemical markers. (b, d, f and h) Tumor cells of ALK-negative anaplastic large cell lymphoma are negative for the respective markers. (i) Hodgkin and Reed-Sternberg cells are negative for CAPN2, whereas a subset of lymphocytes is positive. Inset: classical Hodgkin lymphoma case with CAPN2-positive Hodgkin and Reed-Sternberg cells. (j) Tumor cells of ALK anaplastic large cell lymphoma show expression of CAPN2.

CAPN2 was expressed in the tumor cells of both classical Hodgkin lymphoma and ALK anaplastic large cell lymphoma cases (23 and 45%, respectively) and was therefore not further considered.

In the training set, stained on tissue microarray, Hodgkin and Reed-Sternberg cells were also positive in majority of the 30 classical Hodgkin lymphoma cases for TUBB2B and MDC/CCL22 (77% each), STAT3 (93%), and CD83 (90%). In contrast, tumor cells of the 13 ALK anaplastic large cell lymphoma cases were only infrequently positive for TUBB2B (8%), STAT3 (15%), and CD83 (31%). They were always negative for MDC/CCL22.

Establishment of a Classifier to Efficiently Distinguish Classical Hodgkin Lymphoma and ALK Anaplastic Large Cell Lymphoma

A scoring system (0–4) was created reflecting the number of positive stainings (MDC/CCL22, CD83, STAT3, and TUBB2B) per case (0, no positive stainings; 4, all four stainings positive). The first case series, stained on whole tissue sections, was evaluated applying this scoring system. A threshold of ≥2-positive stainings proved to be optimal for the classification as Hodgkin lymphoma, whereas cases with a score 0–1 were regarded as anaplastic large cell lymphoma. Applying this scoring system to the second series of cases stained on tissue microarray, all 30 classical Hodgkin lymphoma cases were correctly classified and only one out of 13 ALK anaplastic large cell lymphoma cases was misleadingly classified as Hodgkin lymphoma (42/43, 97%, Table 4). Applying the classifier to all cases, two out of 60 Hodgkin lymphoma cases and two out of 34 nodal ALK anaplastic large cell lymphoma were misclassified, yielding an accuracy of 96% (90/94). The misclassified Hodgkin lymphoma cases were two cases with granzyme B expression. In an unsupervised hierarchical clustering by the classifier, two large branches were observed (Figure 3).

Figure 3.

Unsupervised hierarchical clustering by the classifier of four immunohistochemical stainings for MDC/CCL22, CD83, STAT3, and TUBB2B. Red color: tumor cells positive, green color: tumor cells negative. Color code: yellow: classical Hodgkin lymphoma, green classical Hodgkin lymphoma with granzyme B expression, red: ALK anaplastic large cell lymphoma, orange: cutaneous anaplastic large cell lymphoma, blue: ALK+ anaplastic large cell lymphoma.

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