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  Table of Contents    
CASE REPORT  
Year : 2015  |  Volume : 58  |  Issue : 2  |  Page : 241-245
Anaplastic lymphoma kinase-positive large B-cell lymphoma: A potential diagnostic pitfall


1 Department of Oncology, Taizhou People's Hospital, Taizhou 225300, Jiangsu Province, China
2 Department of Pathology, Cancer Hospital of Fudan University, Shanghai 200032, China

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Date of Web Publication17-Apr-2015
 

   Abstract 

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK + LBCL) is a rare variant of LBCL with an abnormal immunophenotype, which is easily misdiagnosed as carcinoma or plasma cell neoplasm. Here, we present two cases of ALK + LBCL in the lymph node; describe their clinical, pathological, and immunophenotype features; and compare them with those of the reported cases.

Keywords: Diagnosis, differential diagnosis, large B-cell lymphoma, anaplastic lymphoma kinase

How to cite this article:
Yu H, Huang J, Sun J, Wang C, Lin M, Li H. Anaplastic lymphoma kinase-positive large B-cell lymphoma: A potential diagnostic pitfall. Indian J Pathol Microbiol 2015;58:241-5

How to cite this URL:
Yu H, Huang J, Sun J, Wang C, Lin M, Li H. Anaplastic lymphoma kinase-positive large B-cell lymphoma: A potential diagnostic pitfall. Indian J Pathol Microbiol [serial online] 2015 [cited 2019 Dec 13];58:241-5. Available from: http://www.ijpmonline.org/text.asp?2015/58/2/241/155332



   Introduction Top


Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (ALK + LBCL) is a rare variant of LBCL. ALK + LBCL rarely expresses B-lineage antigens (e.g., CD20, CD79a) while showing an abnormal immunophenotype, such as positive for ALK, epithelial membrane antigen (EMA), CD38, CD138. Only 55 cases of ALK + LBCL have been reported so far in the published literature. [1] We present two additional cases of ALK + LBCL, which were sent to our department for consultation from other hospitals. One was initially diagnosed as metastatic poorly differentiated carcinoma, and the other was not able to be diagnosed. The two cases were finally diagnosed as "ALK + LBCL" at our department of pathology (see below). The chemotherapy with cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) was given, respectively. Case 1 was given radiation therapy (XRT) after chemotherapy, and case 2 was not. Case 1, followed-up for 16 months, was free of disease. Case 2 died 8 months after the diagnosis.

Due to the abnormal immunophenotype and rareness, ALK + LBCL is often difficult to diagnose, even being misdiagnosed as carcinoma or plasma cell neoplasm. However, the treatment for these diseases is quite different from each other. Avoiding misdiagnosis of ALK + LBCL is of great clinic importance. In this report, we describe the clinical, morphological, immunohistiochemical, and cytogenetic features of the two ALK + LBCL cases, reviewed 55 published cases, and compared the features with those of the reported cases. We came to the conclusions:

  1. ALK + LBCL can affect nonadult and adult with a bimodal age distribution, with an average age of 12.5 years in the nonadult, and 43.5 years in the adult.
  2. ALK + LBCL is a potential diagnostic pitfall for pathologists. Its diagnosis clues are that neoplastic cells are immunoblastic or/and plasmacytoid with prominent central nucleoli, positive for ALK, EMA, and some markers of late (plasma cell-like) B-cell differentiation, such as CD138, VS38 and immunoglobulins, negative for B-cell markers such as CD20 and CD79a.
  3. The investigation of involved genetic abnormality (CLTC-ALK fusion or NPM-ALK rearrangement) contributes to confirm ALK + LBCL.
  4. ALK + LBCL has highly aggressive biobehavior and poor response to standard therapies. More patient case collection and prospective studies are needed.



   Case Reports Top


Case 1

A 25-year-old man presented a mass on the right neck that had been noticed for about 1-month. Physical examination showed the mass on the right neck was 1.5 cm × 1.2 cm × 1.0 cm in size with a tender. No fever was reported. Hematologic studies, lactate dehydrogenase, and serum protein electrophoresis were all within normal limits. An excisional biopsy of the mass was done, and the diagnosis of ALK + LBCL was finally made (see below). Bone marrow biopsy, computed tomography (CT), and magnetic resonance imaging of the thorax and abdomen were performed. The results showed bone marrow was negative for involvement by lymphoma, and no additional mass lesion or lymphadenopathy was detected anywhere else in the body. The disease was in stage I. The patient underwent six cycles of CHOP chemotherapy followed by right neck irradiation. Follow-up imaging studies showed no evidence of recurrence. Sixteen months later, he was free of disease.

Case 2

A previously healthy 32-year-old man complained of a rapidly enlarging tender lump on his left neck with a low fever for about 2 months. Physical examination showed he had superficial lymphadenopathies in his axillary and inguinal areas besides the mass of the left neck. CT imaging showed no other mass or lymphadenopathy in anywhere else in the body. The range of the lymphadenopathies in size is from 1.5 cm to 3.0 cm in the maximum diameter. An excisional biopsy of the neck mass was done, and the diagnosis of ALK + LBCL was finally made (see below). A bone marrow biopsy was performed, and no involvement by lymphoma was found. Other laboratory tests showed no distinct abnormalities. The disease was in stage III. The patient underwent six cycles of CHOP chemotherapy therapy and died 8 months after diagnosis.

The clinical features of the two patients are summarized in [Table 1].
Table 1: Clinical features of the two patients with ALK + LBCL


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   Materials and Methods Top


Preparation of specimen

The two cases were sent to our department for consultation from other hospitals with the samples of hematoxylin and eosin (H and E) slides and corresponding paraffin blocks. The submitted paraffin-embedded tissue blocks were, respectively, cut into 3 μm, 4 μm, and 6 μm sections for H and E staining, immunohistochemical staining, and DNA extraction, respectively. In addition, the tumor tissue of case 1 from the submitted paraffin-embedded tissue blocks was cut 6 sections in 10 μm thickness for RNA extraction. Clinical and laboratory data for each of the two patients were obtained through physician interview and medical chart review.

Immunohistiochemical analysis

Immunohistochemical analysis was performed for the two cases on 4 μm formalin-fixed, paraffin-embedded tissue sections using EnVision (Dako, Denmark) two-step method. Briefly, the sections were deparaffinized with xylene and rehydrated using graded ethanol concentrations. After heat-induced antigen retrieval in 0.01 mol/L citrate buffer (pH 6.0), the slides were incubated with primary antibodies (Dako) leukocyte common antigen (LCA), ALK1, EMA, CD57, IgA, CD138, VS38C, CD3, CD45RO, CD20, CD79a, CD68, and CD30 [Table 2] at 4°C overnight. The next day, the sections were washed 3 times with phosphate-buffered saline, incubated with the EnVision reagent (Dako) at room temperature for 30 min, visualized with 3,3Ͳ-diaminobenzidine-chromagen solution and finally counterstained with hematoxylin (Sigma). Appropriate positive and negative tissue control samples were used with each run.
Table 2: Antibodies used for immunohistochemical analysis


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Clone rearrangement of immunoglobulin heavy chain Gene by DNA polymerase chain reaction

Five 6 μm paraffin-embedded sections of tumor tissues of the two cases were deparaffinized. The 6 μm sections were lightly stained with hematoxylin for microdissection. The microdissections were performed under a dissection microscope with a scalpel. Tumor cells should account for at least 80% of the total cells isolated. The microdissected tissues were transferred directly into an Eppendorf tube with 200 μl cell lysis buffer (0.5 mol/L Tris-HCl, 20 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L NaCl, 10 g/L sodium dodecyl sulfate (SDS), 0.5 g/L Proteinase K). The subsequent DNA extraction was performed according to the protocol of the DNA extraction kit (Qiagen). Polymerase chain reaction (PCR) amplification was performed with the condition described by Tan et al. [2] employing commercially available PCR-based kits (InVivoScribe Technologies, San Diego, CA). No template DNA was used as a negative control. PCR products were analyzed by electrophoresis using 1.0% agarose gels, stained with ethidium bromide.

RNA extraction and reverse transcription-polymerase chain reaction sequencing

Total RNA of case 1 was extracted from tumor tissue using Trizol reagent (Invitrogen Life Technologies). RNAs extracted from the t(2;5)-positive SU-DHL-1 and Karpas-299 cell line were used as positive controls, while diethyl pyrocarbonate water and RNA from proper negative tissue (normal lymph node) were used as negative controls. Reverse transcription of RNA into cDNA was performed by incubating 1 μg RNA (purified by DNase digestion using TURBO DNase from Ambion), 1 μL of random primer (Promega, USA), and 200 U of reverse transcriptase (Promega, USA) in a 25 μL reaction volume at 37°C for 1 h. 1 μL cDNA was then submitted to PCR amplification. To assess the quality of cDNA, the transcript of a housekeeping gene PGK was simultaneously detected as an internal control. PCR reaction was performed using specific primers (CLTC F-GAAGGAGTACTTGACAAAGGTGGAT; ALK R-CGGAGCTTGCTCAGCTTGTA). The optimized thermal cycling condition for ALK mRNA and ALK-associated fusion gene amplification consisted of an initial denaturation step at 95°C for 10 min and then 42 cycles of 94°C for 30 s, 57°C/60°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The presence of PCR products was tested using 2% agarose gels, compared with a 100 bp DNA marker. After the bands were clearly observed, and the sized was determined, the products were purified. Sequencing was performed on an ABI Prism 3730 Sequence Detector System. ALK and CLT structures were obtained from Ensembl (http://www.ensembl.org).


   Results Top


The histologic findings in the two cases were similar. They showed a diffusely infiltrating growth pattern but focally arranged in nested and gland-like construction in case 1. Neoplasm cells exhibited distinct immunoblast-like morphologic features with regular uniform big round nuclei containing large central nucleoli and moderate lightly basophilic cytoplasm. Both of the cases presented similar immunophenotypic profiles, the tumor cells were positive for LCA, ALK1, IgA, CD138, CD38, VS38c, CD57, EMA, whereas negative for CD20, CD79a, CD3, CD45RO, CD30 and CD68. With regard to ALK protein, its expression in the two cases was restricted to the cytoplasm that showed a fine granular cytoplasmic staining pattern ALK expression [Figure 1] and [Figure 2]. The rearrangement of the clone immunoglobulin heavy chain (IgH) gene was found in the two cases, and ALK-CLTC fusion was identified by direct sequencing in case 1 [Figure 3] (Test of ALK-CLTC fusion was not performed in case 2).
Figure 1: Gland-like arrangement of tumor cells was locally seen, (H and E, ×400)


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Figure 2: Immunostains of ALK (EnVision two-step, ×400)


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Figure 3: The sequence of the translocation fusion point. Arrow indicates the corresponding fusion point


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   Discussion Top


Anaplastic lymphoma kinase-positive LBCL was originally described by Delsol et al. [3] in 1997. This lymphoma was identified due to its characteristic lack of CD30 expression in an otherwise large series of classical T-/null cell ALK-positive anaplastic large cell lymphomas (ALCL). It showed very aggressive behavior, high relapse rate, and little response to standard regimens. For the time being, this rare tumor had been recognized as "Diffuse LBCL with expression of full-length ALK" by WHO classification of lymphomas (3 rd edition). [4] The disease was defined as an entity and termed "ALK-positive LBCL" in the latest WHO classification of lymphomas (4 th edition). [5] This lymphoma often exhibited a sinusoidal growth pattern and was composed of monomorphic large immunoblast-like cells with round pale nuclei containing large central nucleoli and abundant cytoplasm, or showed the plasmablastic differentiation. Atypical multinucleated neoplastic giant cells might be seen. It was revealed to derive from B-cells based on expression of monotypic light chain, but exhibited a unique immunophenotypic profile characterized by cytoplasmic, granular ALK reactivity consistently with expression of EMA, plasmacytic markers (e.g., CD38, CD138), and variable expression of CD4 and CD57, while often lacking expression of B-lineage (e.g., CD20, CD79a) and T-lineage (e.g., CD2, CD3) markers, and CD30. Genetic studies showed that the majority of ALK + LBCL cases were characterized by the CLTC-ALK fusion, and a small minority had the NPM-ALK rearrangement. [6] Although ALK + LBCL is one of B-cell lymphomas, it expresses EMA and plasma cell markers (CD38, CD138, VS38c) instead of expressing CD20 and CD79a, which may be mistaken as metastatic poorly differentiated carcinoma or plasma cell neoplasm. It is a potential challenge for pathologists to make the diagnosis due to the abnormal immunophenotypes, as well as infrequence of the entity.

Clinically, the average age of the reported ALK + LBCL in the literature was 37.9 years, ranging from 9 to 72 years of age with a bimodal age distribution, with an average age of 12.5 years in the nonadult and an average age of 43.5 years in the adult. The male to female ratio was about 3:1. Data on primary sites of presentation were available in 51 cases. Twenty-seven (52.9%) were exclusively nodal in origin. The lymph node was the most common primary site of involvement, particularly cervical and mediastinal areas. The remaining cases (47.1%) had some extranodal component, including bone (n = 9), liver and spleen (n = 4), head and neck (n = 4), gastrointestinal tract (n = 3), and others (n = 8) including bone marrow, CNS, gonads, and muscle. Both of our patients were male. One was 25 years, and the other was 32 years. The sites of involvement were both located in the lymph nodes.

Microscopically, ALK + LBCL is characteristic of immunoblastic/plasmablastic microscopical appearance with round nuclei, prominent single central nucleoli, and abundant cytoplasm. Sometimes, the neoplastic cells demonstrate ample eosinophilic cytoplasm, somewhat resembling epithelioid appearance. This lymphoma often shows a sinusoidal growth pattern. In this report, both of the presented cases mainly showed a diffusely infiltrating pattern instead of the sinusoidal growth. However, the neoplastic cells in case 1 showed focally nested and gland-like arrangement. In terms of morphology, differential diagnosis of ALK + LBCL should include anaplastic variant of diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, plasmablastic myeloma, and metastatic poorly differentiated carcinomas.

Immunohistochemically, this lymphoma does rarely express the usual B-lineage (e.g., CD 19, CD20, CD79a) or T-lineage markers (e.g., CD2, CD3, CD7), demonstrate the "null" phenotype with ALK expression and EMA, which strongly suggests an ALCL. ALK + LBCL, however, expresses plasmacytic differentiation markers, such as CD138, CD38, and VS38c besides ALK and EMA.

Anaplastic lymphoma kinase-positive LBCL presents 100% positivity for plasmacytic differentiation markers such as CD138, VS38c, and MUM. EMA was expressed in 97% of the cases. B-cell related antigens such as CD20 and CD79a were expressed in ALK + LBCL in 11% and 18%, respectively. These observations support the inference that ALK + LBCL is derived from postgerminal B-cell lymphocytes that have undergone class switching and plasmacytic differentiation. In addition, expression of monotypic cytoplasmic light chain occurred in 85% of all cases. Based on these findings, ALK + LBCL falls into the category of non-GC DLBCL. [7] In our study, both cases exhibited immunophenotypic profiles similar to those in the published literature. [Table 3] shows immunohistochemical features of 55 cases of ALK + LBCL reported in the literature.
Table 3: Immunohistochemical features of 55 cases of ALK-LBCL reported in the literature


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Genetically, the most frequent ALK gene rearrangement was clathrin-ALK in 75% cases; however, 17% corresponded to NPM-ALK fusion. [8] The ALK gene is located on chromosome 2p23 and it can be translocated to either the clathrin gene locus located on chromosome 17q23 or to the NPM1 gene located on chromosome 5q35, constituting the clathrin-ALK or NPM-ALK fusion products, respectively. In the literature, the clone rearrangement of IgH gene was investigated by PCR in 20 studied cases of ALK + LBCL, and the rearrangement was found in 17 of the 20 cases (85%), which confirmed the B-cell lineage of this disorder. In this report, the fusion in case 1 was studied employing analysis of direct sequencing, and CLTC-ALK fusion was found, which was identical to those reported previously. [7],[9],[10] In addition, the rearrangement of the IgH gene was detected in both cases. [Table 4] shows genetic features of investigated cases of ALK + LBCL reported in the literature.
Table 4: Genetic feature of investigated cases of ALK + LBCL reported in the literature


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Prognostically, the clinical course of ALK + LBCL was aggressive with primary refractory disease and high relapse rates. Its prognosis depended largely on clinical stage. The classical CHOP regimen appeared insufficient to treat this condition, which indicates newer, more intensive therapies will be needed, even though some cases could have prolonged survival times as the authors described in the article. [6] In this report, both patients were treated with six cycles of CHOP chemotherapy. Case 1, in stage I and simultaneously treated with XRT, is alive 16 months after diagnosis. Another patient, in stage III, treated with CHOP chemotherapy, died 8 months after diagnosis.

We believe that a combination of chemotherapy and radiotherapy could prolong the survival time while stage of disease could be the most important prognosis factor. To improve understanding of ALK + LBCL and develop newer therapeutic techniques for its treatment, more collection of patient cases and prospective studies are needed.

 
   References Top

1.
Beltran B, Castillo J, Salas R, Quiñones P, Morales D, Hurtado F, et al. ALK-positive diffuse large B-cell lymphoma: Report of four cases and review of the literature. J Hematol Oncol 2009;2:11.  Back to cited text no. 1
    
2.
Tan BT, Warnke RA, Arber DA. The frequency of B- and T-cell gene rearrangements and Epstein-Barr virus in T-cell lymphomas: A comparison between angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified with and without associated B-cell proliferations. J Mol Diagn 2006;8:466-75.  Back to cited text no. 2
    
3.
Delsol G, Lamant L, Mariamé B, Pulford K, Dastugue N, Brousset P, et al. A new subtype of large B-cell lymphoma expressing the ALK kinase and lacking the 2; 5 translocation. Blood 1997;89:1483-90.  Back to cited text no. 3
    
4.
Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. World Health Organization Classification of Tumors: Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2001. p. 171-4.  Back to cited text no. 4
    
5.
Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al., editors. World Health Organization Classification of Tumors: Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008. p. 254-5.  Back to cited text no. 5
    
6.
Gascoyne RD, Lamant L, Martin-Subero JI, Lestou VS, Harris NL, Müller-Hermelink HK, et al. ALK-positive diffuse large B-cell lymphoma is associated with Clathrin-ALK rearrangements: Report of 6 cases. Blood 2003;102:2568-73.  Back to cited text no. 6
    
7.
Shi M, Miron PM, Hutchinson L, Woda BA, Nath R, Cerny J, et al. Anaplastic lymphoma kinase-positive large B-cell lymphoma with complex karyotype and novel ALK gene rearrangements. Hum Pathol 2011;42:1562-7.  Back to cited text no. 7
    
8.
Stachurski D, Miron PM, Al-Homsi S, Hutchinson L, Harris NL, Woda B, et al. Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma with a complex karyotype and cryptic 3' ALK gene insertion to chromosome 4 q22-24. Hum Pathol 2007;38:940-5.  Back to cited text no. 8
    
9.
Adam P, Katzenberger T, Seeberger H, Gattenlöhner S, Wolf J, Steinlein C, et al. A case of a diffuse large B-cell lymphoma of plasmablastic type associated with the t(2;5)(p23;q35) chromosome translocation. Am J Surg Pathol 2003;27:1473-6.  Back to cited text no. 9
    
10.
Zhang D, Denley RC, Filippa DA, Teruya-Feldstein J. ALK-positive diffuse large B-cell lymphoma with the t(2;17)(p23;q23). Appl Immunohistochem Mol Morphol 2009;17:172-7.  Back to cited text no. 10
    

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Correspondence Address:
Dr. Chaofu Wang
Department of Pathology, Cancer Hospital of Fudan University, 270 Dong An Road, Shanghai 200032
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.155332

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    Figures

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    Tables

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