Identification of a novel fusion, SQSTM1-ALK, in ALK-positive large B-cell lymphoma

Abstract

ALK-positive large B-cell lymphoma is a rare subtype of lymphoma, and most cases follow an aggressive clinical course with a poor prognosis. We examined an ALK-positive large B-cell lymphoma case showing an anti-ALK immunohistochemistry pattern distinct from those of 2 known ALK fusions, CLTC-ALK and NPM-ALK, for the presence of a novel ALK fusion; this led to the identification of SQSTM1-ALK. SQSTM1 is an ubiquitin binding protein that is associated with oxidative stress, cell signaling, and autophagy. We showed transforming activities of SQSTM1-ALK with a focus formation assay and an in vivo tumorigenicity assay using 3T3 fibroblasts infected with a recombinant retrovirus encoding SQSTM1-ALK. ALK-inhibitor therapies are promising for treating ALK-positive large B-cell lymphoma, especially for refractory cases. SQSTM1-ALK may be a rare fusion, but our data provide novel biological insights and serve as a key for the accurate diagnosis of this rare lymphoma.

Introduction

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+LBCL) is a rare subtype of lymphoma that was first described in 1997.1 Approximately 50 cases have been reported to date,2 with most cases (60%) following an aggressive clinical course.3 In well-characterized cases, 3 genes have been reported as a fusion partner of ALK: clathrin (CLTC-ALK),4–6 nucleophosmin (NPM-ALK),7–8 and SEC31A (SEC31A-ALK).9 In this paper, we report a case of ALK+LBCL that harbored a novel ALK fusion partner, sequestosome1 (SQSTM1).

Design and Methods

Materials

Biopsied specimens were fixed in 20% neutralized formalin and embedded in paraffin for conventional histopathological examination. We extracted DNA and total RNA from the snap-frozen specimens and subsequently purified the samples. Written informed consent was obtained from the patient. The study was approved by the Institutional Review Board of the Japanese Foundation for Cancer Research.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue was used. For antigen retrieval, we heated the slides for 40 min at 97°C in Target Retrieval Solution (pH 9.0; Dako), and subsequently detected the immune complexes with a dextran polymer reagent (EnVision+DAB system, Dako) and an AutoStainer instrument (Dako).

Isolation of ALK fusion cDNA

To obtain cDNA fragments corresponding to novel ALK fusion genes, we used an inverse reverse transcription-polymerase chain reaction (RT-PCR) method slightly modified from one previously reported.10 Double-stranded cDNA was synthesized from 2 μg of total RNA with 1 pM of the primer ALKREVex22–23 (5′-TGGTTGAATTTGCTGATGATC-3′) and a cDNA Synthesis System (Roche), and was self-ligated by incubation overnight with T4 DNA ligase (TaKaRa Bio). We subjected the resulting circular cDNA to PCR (35 cycles of 94°C for 15 sec, 62°C for 30 sec, and 72°C for 1 min) with primers ALKREV3T (5′-CTGATGGAGGAGGTCTTGCC-3′) and ALKFWDex20–21 (5′-ATTCGGGGTCTGGGCCAT-3′) in a final volume of 20 μL. We subjected 1 μL of the 1:100 diluted reaction products to a second PCR step (the same settings as above), with primers ALKREV4T (5′-GGTTGTAGTCGGTCATGATGGTC-3′) and ALKFWDex21–22 (5′-AGTGGCTGTGAAGACGCTGC-3′) in a final volume of 20 μL. The resulting products were purified by gel extraction and directly sequenced in both directions with primers ALKFWDex20–21 and ALKREV4T.

The fusion point of SQSTM1-ALK cDNA was amplified by RT-PCR with primers SQSTM1 565F (5′-AAACACGGA-CACTTCGGGT-3′) and ALK3078RR (5′-ATCCAGTTCGTCCT-GTTCAGAGC-3′).

Full-length SQSTM1-ALK cDNA was obtained from the specimen by RT-PCR with primers SQSTM1v1-F90 (5′-CTCGCTATG-GCGTCGCTCACCGTGAA-3′) and KA-W-cDNA-out-AS (5′-CCACGGTCTTAGGGATCCCAAGG-3′).

Fluorescence in situ hybridization (FISH)

We performed FISH analysis of the gene fusion for unstained slides (4 μm thick) with bacterial artificial chromosome (BAC) clone-derived DNA probes for ALK (RP11-984I21, RP11-62B19) and SQSTM1 (RP11-55M16).

Transformation assay for ALK fusion protein

We analyzed the transforming activity of SQSTM1-ALK as described previously.11–13 Briefly, cDNA for SQSTM1-ALK was inserted into the retroviral expression plasmid pMXS.14 The resulting plasmid and similar pMXS-based expression plasmids for EML4-ALK variant 1 or NPM-ALK were used to generate recombinant ecotropic retroviruses, which were then used to infect mouse 3T3 fibroblasts. We evaluated formation of transformed foci after culturing the cells for 14 days. We subcutaneously injected the same set of 3T3 cells into nu/nu mice and examined tumor formation after 20 days.

PCR for IGH gene rearrangement

Genomic PCR was used for amplification of the rearranged IGH gene using the primers FR2A 5′-TGG(A/G)TCCG(A/C)CAG (C/G)C(C/T)(C/T)CNGG-3′ and LJH 5′-ACCTGAGGAGACG-GTGACC-3′. Several clones were sequenced after subcloning the PCR product into pGEM-T-Easy Vector (Promega).

Results and Discussion

Case presentation

A 67-year old man was admitted with a tumor in the left side of his neck. A systemic workup revealed swelling of cervical, mediastinal, and hilar lymph nodes. Blood counts were within normal ranges. Lactose dehydrogenase was slightly elevated (223 IU/L) in peripheral blood with high IgG (2,425 mg/dL), normal IgA (157 mg/dL) and low IgM (32 mg/dL) levels.

Figure 1.Histopathology of SQSTM1-ALK-positive large B-cell lymphoma. (A) The pattern of tumor infiltration was diffuse. The lymphoma cells were large with abundant cytoplasm and had round, vesicular nuclei, each containing a centrally located large nucleolus. These features may be consistent with immunoblasts or plasmablasts, but the size of tumor cells was extremely large compared with these typical cell types (Magnification 40×). (B) Some lymphoma cells expressed cytokeratin (AE1/AE3) (Magnification 20×). (C) Syndecan1/CD138 was strongly expressed (Magnification 20×). (D) In anti-ALK immunohistochemistry, a diffuse cytoplasmic staining pattern with ill-demarcated spots was clearly shown (Magnification 20×).

Histopathological examination of the biopsied specimen from the cervical lymph node showed a diffuse infiltrate of tumor cells with a round, vesicular nucleus containing a centrally located large nucleolus. The cytoplasm was abundant (Figure 1A). These features may be consistent with immunoblasts or plasmablasts, but the size of tumor cells was large compared with typical immunoblasts and plasmablasts. Immunophenotypically, the tumor cells were negative for CD3, CD4, CD5, CD10, CD20, CD57, CD79a, and most cytokeratins (CK5/6, CK8, CK19, CK20); focally positive for CD30 and cytokeratins (AE1/AE3, CAM5.2, CK7, CK18) (Figure 1B); weakly positive for PAX5; and positive for CD138 (Figure 1C), EMA, and ALK (Figure 1D). The positivity of focal cytokeratin, which has been reported in a small proportion of ALK+LBCL cases,15 and the cytomorphology of this case may have led to a misdiagnosis of undifferentiated metastatic carcinoma. The presence of ALK translocation was demonstrated by an ALK split FISH assay, which was performed at a commercial laboratory (data not shown). The tumor cells were positive for PAX5, which is suggestive of ALK+LBCL. However, we carefully excluded a possibility of metastasis of ALK-positive lung cancer10 because the tumor cells were positive for some cytokeratins and immunohistochemistry for immunoglobulins was not evaluable due to background staining. Immunohistochemistry for TTF1 was negative; this is usually positive in ALK-positive lung cancers.16 In addition, PCR and sequencing analyses revealed that IGH was monoclonally rearranged and somatically hypermutated (data not shown).

The patient was diagnosed as having ALK+LBCL and achieved complete remission after 6 cycles of cyclophos-phamide, doxorubicin, vincristine, and prednisone (CHOP) treatment. Four months later, however, he relapsed.

Identification of SQSTM1-ALK

The 2 major ALK fusions in ALK+LBCL are CLTC-ALK and NPM-ALK, and they show a coarse granular cytoplasmic pattern and a nuclear and cytoplasmic pattern in anti-ALK immunohistochemistry, respectively. In the present case, anti-ALK immunohistochemistry showed a diffuse cytoplasmic staining pattern with ill-demarcated spots (Figure 1D), which was different from either of the former 2 patterns. Therefore, we carried out inverse RT-PCR to examine the presence of a novel fusion of ALK. We indeed isolated a cDNA containing the exon 5 of SQSTM1 in-frame fused to the exon 20 of ALK (Figure 2A). A separate RT-PCR assay amplified the fusion point of SQSTM1-ALK cDNA (data not shown). To confirm the chromosome rearrangement, we performed SQSTM1-ALK fusion FISH. This result was consistent with the presence of a t(2;5)(p23.1;q35.3) leading to the generation of SQSTM1-ALK (Figure 2B). The complete sequences of SQSTM1-ALK are shown in the Online Supplementary Figure S1.

SQSTM1 is an ubiquitin binding protein that is associated with oxidative stress, cell signaling, and autophagy.17–20 Autophagosomal membrane protein LC3/Atg8 binds SQSTM1 and makes SQSTM1-containing protein aggregate to the autophagosome.21 Mutations within SQSTM1 are identified in patients with Paget’s disease of bone.22

SQSTM1 is located very near NPM, which is on 5q35.1. Therefore, the cytogenetic findings of the NPM-ALK-positive and the SOSTM1-ALK-positive lymphomas may be similar, and this may mean that SQSTM1-ALK occurrence in lymphoma may be underestimated. As mentioned, however, NPM-ALK and SQSTM1-ALK differ in terms of the anti-ALK immunostaining pattern. NPM has a nuclear transport signal, while SQSTM1 does not. Therefore, NPM-ALK shows a nuclear and cytoplasmic staining pattern while SQSTM1-ALK shows only a cytoplasmic staining pattern. ALK is a representative “promiscuous” molecule because of its various fusion partners. The subcellular localization of ALK fusions depends on the fusion partners. The anti-ALK immunohistochemical staining pattern is, therefore, a simple and useful means to identify the possible partner in a tested case and, in fact, has prompted the identification of many ALK fusion partners, including the present case.

Figure 2.Discovery of SQSTM-ALK fusion gene. (A) A chromosome translocation, t(2;5)(p23.1;q35.3), generates a cDNA fusion in which exon 5 of SQSTM is joined to the ALK cDNA for the intracellular region of its encoded protein (containing the tyrosine kinase domain). Numbers indicate amino acid positions of each protein. PB1: Phox and Bem1p; Z: atypical zinc finger; U: ubiquitin-associated. (B) A section of the specimen for the present case was subjected to FISH with an SQSTM1-ALK fusion assay. Nuclei are stained blue with DAPI. (C) Murine 3T3 fibroblasts were infected with retroviruses expressing SQSTM1-ALK. The cells were photographed after culture for 14 days. (D) A nude mouse was injected subcutaneously with 3T3 cells infected as in (C), and tumor formation was examined after 20 days.

Transforming activities of SQSTM1-ALK

We generated a recombinant retrovirus encoding SQSTM1-ALK and used it to infect cultured 3T3 fibroblasts. Infection with the virus, but not with an empty virus, resulted in the formation of multiple transformed foci in vitro (Figure 2C). As control experiments for formation, EML4-ALK (variant 1) and NPM-ALK similarly produced transformed foci (data not shown). The same 3T3 cells were injected into nude mice for an in vivo tumorigenicity assay. As expected, 3T3 cells expressing SQSTM1-ALK developed subcutaneous tumors at all injection sites within an observation period of 20 days (Figure 2D), confirming the transforming potential of the novel fusion kinase, SQSTM1-ALK.

All ALK fusion partners identified so far except NPM and moesin (MSN) have a coiled-coil domain(s) in their sequences, and the domain is conserved in its fusion form. The coiled-coil domain allows the protein to homodimerize. The tyrosine kinase domain of the ALK fusions is constitutively phosphorylated and activated through homodimerization via the coiled-coil domain. It has been speculated that the binding properties of MSN to cell membrane proteins lead to the dimerization of MSN-ALK proteins, enabling the constitutive phosphorylation of the chimeric MSN-ALK protein.23 SQSTM1 does not harbor a coiled-coil domain and does not bind to membrane proteins. Instead, it has the Phox and Bem1p (PB1) domain in its N-terminus and forms heteromeric and homomeric complexes mediated by this domain.24 Therefore, SQSTM1-ALK probably homodimerizes through the PB1 domain, leading to constitutive activation of the ALK kinase domain.

In conclusion, we reported a novel ALK fusion, SQSTM1-ALK, and its oncogenicity. ALK+LBCL is an aggressive lymphoma with poor prognosis;3 ALK inhibitors are promising therapeutic agents for this condition. SQSTM1-ALK may be a rare fusion, but our data provide novel biological insights and may serve as a key to the accurate diagnosis of this rare lymphoma.

Acknowledgments

We thank Drs. Masaru Hosone, Yuichi Sugisaki, Koji Izutsu, Shuji Momose, and Jun-ichi Tamaru for their advice. The nucleotide sequences of the cDNAs for SQSTM1-ALK have been deposited in the DDBJ/EMBL/GenBank databases under the accession number, AB583922.

Footnotes

• The online version of this article has a Supplementary Appendix.
• Authorship and Disclosures The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
• Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.

• Received September 12, 2010.
• Revision received November 11, 2010.
• Accepted November 29, 2010.