Long-distance polymerase chain reaction targeting genomic DNA to detect ALK fusion genes with various partners in ALK+ hematological tumors

Abstract

We developed genomic DNA-targeting long-distance polymerase chain reaction (LD-PCR) to detect ALK fusion genes with various partner genes in ALK+ hematological tumors. DNA was obtained from 3 anaplastic large cell lymphoma (ALCL) cases with NPM1::ALK, 2 ALCL cases with ATIC::ALK, 1 ALCL case with TRAF1::ALK, 1 ALK+ large B-cell lymphoma case with CLTC::ALK, 1 acute myeloid leukemia case with RANBP2::ALK, and 5 additional ALK+ ALCL cases, in which the ALK fusion gene had not been characterized. Forward primers were designed for exons of NPM1, TPM3, ATIC, TRAF1, TFG, CLTC, and RANBP2 and a reverse primer for ALK exon 20. We obtained PCR products representing the fusion sequences in 8 cases with known partners using the corresponding primer combination. For 5 ALCL cases with uncharacterized fusion genes, we performed LD-PCR using combinations of the forward primers for the 7 genes and ALK reverse primer. The results showed that ALK fused to ATIC in 3, TRAF1 in 1, and TFG in 1. A total of 13 ALK breakpoints were randomly distributed within intron 19 and those of the NPM1, ATIC, and TRAF1 genes were located within introns 4, 7, and 6, respectively, without creating subclusters or recurrent breakpoints. This study indicates that LD-PCR can effectively detect the ALK fusion gene in the majority of ALK+ ALCL cases and is useful for rapid diagnosis of the disease.

Translated Abstract

我々は，ALK陽性造血器腫瘍における様々なパートナー遺伝子とのALK融合遺伝子を検出するために，ゲノムDNAを標的としたlong-distance polymerase chain reaction（LD-PCR）を開発した．NPM1::ALKを有する未分化大細胞型リンパ腫（ALCL）3症例，ATIC::ALKを有するALCL 2症例，TRAF1::ALKを有するALCL 1症例，CLTC::ALKを有するALK陽性大細胞型B細胞リンパ腫1症例，RANBP2::ALKを有する急性骨髄性白血病1症例，およびALK融合遺伝子の特徴が明らかにされていないALK+ALCL 5症例からDNAを得た．NPM1，TPM3，ATIC，TRAF1，TFG，CLTC，RANBP2のエクソンに対してフォワードプライマーを，ALKエクソン20に対してリバースプライマーを設計した．既知のパートナーを持つ8症例において，対応するプライマーの組み合わせを用いてLD-PCRを実施し，融合配列を示すPCR産物を得た．融合遺伝子が未同定のALCL 5症例については，7遺伝子のフォワードプライマーとALKリバースプライマーの組み合わせでLD-PCRを行った．その結果，ALKは3例でATICと，1例でTRAF1と，1例でTFGと融合していた．合計13例のALKのブレークポイントはイントロン19内にランダムに分布し，NPM1，ATIC，TRAF1遺伝子のブレークポイントはそれぞれイントロン4，7，6内に位置し，サブクラスターやリカレントなブレークポイントは形成されなかった．本研究により，LD-PCRはALK陽性ALCL症例の大部分においてALK融合遺伝子を効果的に検出し，本疾患の迅速診断に有用であることが示された．

INTRODUCTION

The ALK (anaplastic lymphoma kinase) gene is located at the chromosomal band 2p23 and encodes the receptor-type tyrosine kinase ALK protein.^1,2 In ALK-positive (ALK+) tumors, the ALK gene fuses with various partner genes by chromosomal translocation or inversion, producing chimeric proteins composed of the protein structure of the partner gene at the N-terminus and the ALK tyrosine kinase structure at the C-terminus.^1-3 It has been reported that the chimeric proteins enhance kinase activity by forming dimers through the partner protein structure, activating downstream signaling pathways, thereby leading to tumorigenesis.⁴ In hematological tumors, the NPM1::ALK fusion gene created by t(2;5)(p23;q35) translocation was first described in ALK+ anaplastic large cell lymphoma (ALCL).³ Subsequently, not only NPM1::ALK but also ALK fusion genes with partner genes other than NPM1 have been found in various non-ALCL hematological tumors, including ALK+ large B-cell lymphoma (LBCL), acute myeloid leukemia (AML), extramedullary plasmacytoma/multiple myeloma, and ALK+ histiocytosis.^5-8

Because physiological expression of ALK protein is restricted to neural tissues, a positive result of ALK immunohistochemistry (IHC) provides evidence for the presence of an ALK fusion gene in the tumor and is sufficient for the diagnosis of an ALK+ tumor.³ However, as the ALK fusion genes and chimeric proteins exhibit variable properties according to the partners,³ the fusion genes should be characterized by cytogenetic analysis and/or molecular genetic methods to determine the partner. We developed long-distance polymerase chain reaction (LD-PCR) to amplify DNA fragments of several kilobases (kb) that encompass the fusion point, using unique primers designed for exons of ALK and partner genes. In this study, we applied this LD-PCR to ALK+ hematopoietic tumors and evaluated whether the method can effectively detect ALK fusion genes.

MATERIALS AND METHODS

Cases

This study included 8 cases of ALK+ hematological tumors, in which the ALK fusion gene had already been identified by cytogenetic analysis and/or reverse transcriptase (RT-) PCR: 3 ALCL cases with NPM1::ALK fusion gene representing t(2;5)(p23;q35), 2 ALCL cases with ATIC::ALK representing inv(2)(p23q35), 1 case each of ALCL with TRAF1::ALK representing t(2;9)(p23;q33-34), ALK+ LBCL with CLTC::ALK representing t(2;17)(p23;q23.1), and AML with RANBP2::ALK representing inv(2)(p23q13).^7,9-11 In addition, we included 5 additional ALK+ ALCL cases, in which the ALK fusion gene had not been characterized (Table 1). We reviewed patients’ clinical records and obtained information on their laboratory data and treatment course.

Table 1. ALK+ hematological tumors studied

Histopathological examination

Pathological specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and then subjected to histopathological examination. Monoclonal antibodies used for IHC were: anti-CD3 (clone PS1, Novocastra), anti-CD4 (1F6, Novocastra), anti-CD30 (Ber-H2, Dako), anti-ALK (ALK1, Dako), anti-EMA (E29, Dako), anti-granzyme B (11F1, Leica Biosystems), and anti-Ki-67 (MIB-1, Dako).

Multi-parametric flow cytometry (mFCM)

Cells prepared from specimens were resuspended in phosphate-buffered saline and aliquots were subjected to mFCM. Fluorescence was captured using a NAVIOS 3L flow cytometer and analyzed by Kaluza Flow Cytometry Analysis Software (Beckman Coulter, Brea, CA, USA).

Fluorescence in situ hybridization (FISH)

Cytospin smears were prepared from cytogenetic specimens and hybridized with the ALK dual-color break-apart probe (Abbott Laboratories, Abbott Park, IL, USA). Denaturing of the chromosome/probe, hybridization, and washing were conducted as recommended by the manufacturer. FISH results were analyzed using fluorescence microscopes (Nikon Corporation, Tokyo, Japan) equipped with DAPI, fluorescein isothiocyanate (FITC), and tetramethylrhodamine B isothiocyanate (TRITC) fluorescence filters as well as a DAPI/FITC/TRITC triple band-pass filter.

LD-PCR, PCR for T-cell receptor gene rearrangement, and nucleotide sequencing

Genomic DNA was isolated from specimens using proteinase K and phenol/chloroform. PCR parameters for LD-PCR to amplify long DNA targets were as previously described.¹² We designed the reverse primer to be complementary to ALK exon 20 and the forward primers to be complementary to exons of the partner genes that come immediately 5′ of ALK exon 20 after translocation/inversion: exon 3 for NPM1,¹³ exon 8 for TPM3,¹⁴ exon 7 for ATIC,^10,11,15,16 exon 6 for TRAF1,^11,17 exon 31 for CLTC,^9,18,19 and exon 18 for RANBP2 (Table 2).^7,20 The breakpoints of TFG were reported to occur within introns 3, 4, and 5, creating the TFG::ALK_S, TFG::ALK_L, and TFG::ALK_XL fusion genes, respectively.²¹ As TFG exons 3 and 4 are 8.6-kb apart, we designed the forward primer for exon 4 to effectively amplify the latter two fusions; therefore, the TFG::ALK_S fusion gene was excluded from this study (Table 2). The sensitivity of LD-PCR to detect the NPM1::ALK fusion gene was estimated to be 0.1% (Supplementary Figure S1). PCR to detect rearrangements of TCRG and TCRB genes was performed according to the BIOMED-2 protocol.²²

Table 2. Designation and sequences of primers used for LD-PCR to amplify ALK fusion genes

All PCR procedures were conducted using Veriti 96 Well Thermal Cycler (Applied Biosystems, Inc., Forester City, CA, USA). PCR products were visualized by ethidium bromide (EtBr)-stained agarose gel electrophoresis, purified using MinElute^® PCR Purification Kit (QIAGEN, Hilden, Germany), subjected to the cycle sequencing reaction (BigDye^TM Terminator v3.1 Cycle Sequencing Kit; Thermo Fisher Scientific), and then sequenced using SeqStudio^TM Genetic Analyzer (Thermo Fisher Scientific). The data obtained were applied to BLASTn programs to identify closely related sequences.

RESULTS

Application of LD-PCR to ALK+ hematological tumors with known ALK fusion genes

We first performed LD-PCR for 8 cases of ALK+ hematological tumors with known ALK fusion genes (Table 1), using forward primers designed for each partner gene in combination with the ALK/02 reverse primer. As shown in Figure 1, we successfully obtained LD-PCR products in all 8 cases, ranging from 1.3 to 5.0 kb. Among 3 NPM1-ALK cases and 2 ATIC-ALK cases, the amplified products varied in size, indicating that the products were specific for each case. It remained to be determined whether the intensity of the LD-PCR products on EtBr-stained gel electrophoresis was related to the percentage of tumor cells in each material.

Figure 1.

EtBr-stained gel electrophoresis of LD-PCR for 8 cases of ALK+ hematological tumors with known partner genes. LD-PCR products were run through 1% agarose gel. Lane M, molecular size marker.

Application of LD-PCR to ALK+ ALCL with uncharacterized ALK fusion genes

We next applied LD-PCR to 5 additional ALK+ ALCL cases to investigate whether our LD-PCR strategy could efficiently detect ALK fusion genes with unspecified partners. Seven Eppendorf microtubes were prepared for each case, and LD-PCR was performed using combinations of the forward primer for one of the 7 test genes (NPM1, ATIC, TRAF1, CLTC, RANBP2, TFG, and TPM3) and the ALK/02 reverse primer; for the last two test genes, positive control DNA was not available. As a result, we obtained LD-PCR products representing the ATIC-ALK fusion gene in 3 cases, TRAF1-ALK fusion gene in 1 case, and TFG-ALK_L fusion gene in 1 case (Figure 2). The products varied in size and intensity on the EtBr-stained gels. No LD-PCR products were obtained with NPM1, CLTC, RANBP2, or TPM3 primers. We then extracted RNA from O.C.T. compound-embedded frozen sections (partner-unknown case 1), the formalin-fixed paraffin-embedded biopsy specimen (case 5), or the same materials from which DNA was extracted (cases 2 and 3), and confirmed the ATIC::ALK and TFG::ALK_L fusion genes by RT-PCR in each case (data not shown).^11,21

Figure 2.

EtBr-stained gel electrophoresis of LD-PCR for 5 cases of ALK+ ALCL with unknown partner genes (partner-unknown [uk] cases 1 to 5). Positive amplification by the test primer in each case is indicated by an arrow. Positive control (PC) materials for each amplification were selected from cases presented in Figure 1, except for TFG::ALK and TPM3::ALK fusion genes. Lane M, molecular size marker.

Nucleotide sequencing of the partner gene::ALK junctions

We finally sequenced LD-PCR products obtained from all 13 cases and determined sequences that encompassed the partner gene::ALK junction in each case. Comparing the sequences with each of the corresponding germline sequences, 2 cases had a single nucleotide insertion at the junction, 7 cases had an overlap (microhomology) of 1 to 5 nucleotides, and in the remaining 4 cases, two genes were precisely joined without nucleotide insertion or overlap (Figure 3). Next, we found that all ALK breakpoints were located within intron 19, and they were distributed without creating subclusters or regions specific to any partner gene (Figure 4). On the other hand, the breakpoints for the NPM1, ATIC, and TRAF1 genes were within introns 4, 7, and 6, respectively, and again, they appeared to be randomly distributed within each intron without creating subclusters or recurrent breakpoints (Figure 3).

Figure 3.

Diagram showing the positions of breakpoints and primers on partner genes: NPM1 (A), ATIC (B), TRAF1 (C), TFG (D), CLTC (E), and RANBP2 (F), and nucleotide sequences encompassing the fusion points in a total of 13 ALK+ hematological tumors. Vertical lines indicate the nucleotide identity and the nucleotides are numbered according to GRCh38.

Figure 4.

Distribution of a total of 13 breakpoints within intron 19 of ALK (GRCh38). Breakpoints are indicated by color-coded arrows for each partner gene. Top, diagram of exon 19, intron 19, and exon 20 of ALK. Bottom, nucleotide sequences of exon 19 (bold), intron 19, and exon 20 (bold). Sequence of the ALK/02 reverse primer is indicated by the horizontal arrow.

An illustrative ALK+ ALCL case, in which LD-PCR was effective for rapid diagnosis

We describe an illustrative ALK+ ALCL case (partner-unknown case 3 in Figure 2), in which our LD-PCR strategy facilitated rapid diagnosis of the disease. The patient was a 72-year-old man who was referred to our hospital due to multiple lymph node (LN) swelling in the supraclavicular, mediastinal, para-aortic, and mesenteric regions as well as a tumor of the right lung hilum. Eleven days after the initial visit, he was admitted to the emergency department because of pulmonary atelectasis and hypoxemia; saturation of percutaneous oxygen was 93.6% and the partial pressure of arterial oxygen was 69.0 mmHg.

Bronchoscopy performed under oxygen administration revealed that the bronchus intermedius of the right lung was obstructed. Cytological examination of the specimen obtained by bronchial lavage, brushing, and washing disclosed tumor cells with a large nuclear/cytoplasmic ratio and distinct nucleoli (Figure 5A). We extracted high-molecular-weight DNA from as few as 13 × 10³ cells and residual tissue prepared from a small bronchial biopsy specimen, and detected rearrangements of the TCRG and TCRB genes by PCR tests according to the BIOMED-2 protocol (Figure 6A and B). Then, we performed the partner gene-unknown protocol for LD-PCR and obtained PCR products corresponding to the ATIC::ALK fusion gene (Figure 2), promptly providing necessary information for the clinical and pathology departments on the 3^rd day of hospitalization.

Figure 5.

Cytological and pathological examinations of materials obtained from partner-unknown case 3. (A) Papanicolaou staining of bronchial brushing specimen (original magnification of objective lens: a, 40×, b and c, 100×). Lymphoma cells are small to medium, sometimes large, with high N/C ratio and prominent nucleoli. A hallmark cell with horseshoe-shaped nucleus can be seen (arrow). (B) Histopathology of bronchial biopsy: a, hematoxylin and eosin (H&E) staining (4×); b, H&E (40×); c, H&E (100×); d, anti-ALK immunostaining (40×) showing the cytoplasmic staining pattern; e, anti-EMA (40×); and f, anti-Ki-67 showing a labeling index of 30% (40×). Arrows indicate hallmark cells. (C) Histopathology of LN biopsy: a, H&E (40×); b, anti-ALK (40×); c, anti-CD30 (40×); d, anti-GranzymeB (40×); e, anti-CD4 (40×); and f, CD3 (40×).

Figure 6.

DNA tests for partner-unknown case 3. (A) Multiplex PCR for TCRG rearrangement using Vγ and Jγ primers. The products were run through EtBr-stained 5-20% poly-acrylamide gel. (B) Multiplex PCR for TCRB rearrangement using Dβ and Jβ primers. (C) LD-PCR for the ATIC::ALK fusion gene. The products were run through EtBr-stained 1% agarose gel. M, molecular size marker; PC, positive control for TCRG/TCRB rearrangements; LN, lymph node.

The bronchial biopsy as well as cervical LN biopsy conducted on the 3^rd hospital day revealed large tumor cells, being negative for CAM5.2, CD3, CD5, CD10, CD20, CD33, CD56, and CD79a, but positive for CD4, CD30, cytoplasmic ALK, EMA, and granzyme B by immunohistochemistry. The Ki-67 labeling index was 30% (Figure 5B and C). Tumor cells were also present in the pleural fluid, which showed the CD2⁻, CD3⁻, CD4⁺, CD5⁻, CD7⁻, CD8⁻, CD16⁻, CD25^++/−, CD30^++/+, CD38⁻, CD45RO⁺⁺, and CD56^dim/− immunophenotype by mFCM (Supplementary Figure S2). Hybridization of the cytospin smear slide with ALK break-apart probe showed a split signal of ALK in 85.8% of the cell nuclei counted (Supplementary Figure S2). We finally found that tumor cells from the bronchial and LN biopsies and pleural fluid cells shared identical TCRG and TCRB rearrangements and the ATIC::ALK fusion gene (Figure 6). These data, which were obtained after the start of treatment, confirmed that the patient had ALK+ ALCL.

CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) therapy was started on day 5. Although the patient developed tumor lysis syndrome, his condition readily recovered with intensive supportive therapy. After completing following 5 cycles of A-CHP therapy, in which vincristine was substituted with brentuximab vedotin, positron emission tomography (PET) confirmed that the disease showed a complete metabolic response (Supplementary Figure S3). One year and three months after the end of treatment, the patient remained free from relapse of the disease.

DISCUSSION

Here, we present genomic DNA-targeting LD-PCR to detect ALK fusion genes with various partners. First, we showed that LD-PCR effectively amplified the ALK fusion genes with known partner genes in ALK+ hematological tumors, using a unique forward primer for each partner gene and a single reverse primer for ALK. Next, we applied LD-PCR to ALCL cases with unknown partner genes using forward primers for 7 selected genes in combination with the ALK reverse primer. As a result, we successfully obtained amplified products that represented the fusion gene in each case, confirming the validity of our LD-PCR strategy to detect ALK fusion genes with unspecified partners. Table 3 compares the results of studies that determined the diverse partner genes of the ALK fusion gene in ALCL by different methods.^3,13,23,24 Based on the fact that the 7 genes we selected in this study cover 89 to 100% of all cases and 56 to 79% of cases with non-NPM1 partners, our LD-PCR can detect the ALK fusion gene in the majority of ALCL cases. Of course, our method is invalid if any genes other than the 7 are partners and fails to detect the TFG::ALK_S fusion gene, and it cannot be used for the purpose of searching for previously unknown partner genes. Nevertheless, these disadvantages do not diminish the value of LD-PCR for diagnosing ALCL, considering that DNA can easily be extracted even from small specimens and stored for long periods, and that the amplified products are specific not only for ALCL but also for each case.

Table 3. Studies of partner gene::ALK fusion genes in ALCL

Although ALK fusion genes have mostly been detected by reverse RT-PCR targeting cDNA generated from chimeric mRNAs, some studies have analyzed the genomic structure of the fusion gene.^13,25,26 Krumbholz M et al. amplified the NPM1-ALK fusion gene in 43 clinical materials and 4 cell lines by nested multiplex PCR and found that the NPM1 breakpoints were located in intron 4, while 93% of the ALK breakpoints were in intron 19 and 7% in exon 19.¹³ The authors showed that the breakpoints of both genes were randomly distributed within the relevant regions without creating significant subclusters, and no repeat regions or sequence motifs involved in the generation of other lymphoma/leukemia-associated translocations were found in the vicinity of the breakpoints.¹³ Our study suggests that, although the number of cases was limited, non-NPM1::ALK breakpoints are also randomly distributed within each intron, without being concentrated in a specific region. On the other hand, Krumbholz et al. observed a nucleotide filler (insertion) and microhomology (overlapping) at the NPM1::ALK fusion points in 22 and 38% of cases studied, respectively, and suggested that inaccurate non-homologous end-joining repair mechanisms are involved in formation of the fusion gene.^13,27 Our study, including 5 ATIC::ALK, 2 TRAF1::ALK, and one each of TFG::ALK, CLTC::ALK, and RANBP2::ALK fusion genes, showed that the observations of Krumbholz et al. were also notable at these non-NPM1::ALK fusion points.

Isolation of the few tumor cells contained in ALCL tissue is often challenging, even with sensitive mFCM analysis, because of the difficulty in preparing a single-cell suspension due to tight cell-cell adhesion and difficulty gating the tumor cell population due to the lack of T-cell lineage markers, such as CD3, CD5, or CD7 (Supplementary Figure S2). In partner-unknown case 3, in which the immunophenotype of tumor cells was determined by mFCM of pleural fluid cells, we were unable to obtain an adequate cell preparation from the LN biopsy specimen. Although mFCM is a rapid test, it may not be effective for the diagnosis of ALCL.

In conclusion, this study showed that genomic DNA-targeting LD-PCR is able to detect the ALK fusion gene in the majority of ALK+ ALCL cases and is of value for rapid diagnosis of the disease.

ACKNOWLEDGMENTS

This study was supported by Tenri Foundation.

ETHICAL APPROVAL

The present study was performed according to the regulations of the Institutional Review Board (Approval No. 1324).

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