KLC1-ALK: A Novel Fusion in Lung Cancer Identified Using a Formalin-Fixed Paraffin-Embedded Tissue Only

The promising results of anaplastic lymphoma kinase (ALK) inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE), and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5′-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer

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

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

ALK fusion partners.

<p>*Histopathological evidence is lacking. Abbreviations: ALCL, anaplastic large cell lymphoma; LBCL, large B-cell lymphoma; IMT, inflammatory myofibroblastic tumor; NSCLC, non-small cell lung carcinoma; RCC, renal cell carcinoma.</p

ALK-rearranged lung adenocarcinoma without EML4-ALK and KIF5B-ALK.

<p>Panel A shows the results of anti-ALK immunohistochemistry with the iAEP method on pulmonary adenocarcinoma in situ, nonmucinous. The staining pattern was diffusely cytoplasmic. The basal side of tumor cells was more strongly stained, indicating an uneven subcellular localization of KLC1-ALK protein. FISH analyses revealed that this case was positive in the split assay for <i>ALK</i> (Panel B: individual 5′- and 3′-signals are observed) and negative in <i>EML4-ALK</i> and <i>KIF5B-ALK</i> fusion assays (Panel C: <i>EML4</i>, red; <i>ALK</i>, green; Panel D: <i>KIF5B</i>, green; <i>ALK</i>, red).</p

Synthesis of the putative KLC1-ALK full-length cDNA.

<p>Two first-round PCRs were performed separately using cDNA synthesized from a tumor tissue expressing KIF5B-ALK with the following primer sets: KLC1-NheI-M and KLC1-bpR, and ALK-bpF and ALK-EcoRI. KLC1-bpR and ALK-bpF had sequences downstream of the ALK break point (exon 20) and upstream of the KLC1 break point (exon 9) as adopter sequences, respectively. Then, the second PCR was performed using a 1/100 dilution of the mixture of the first PCR products as a template with primers KLC1-NheI-M and ALK-EcoRI. The first PCR products were annealed, extended with each other, and then amplified with the primers.</p

Identification of KLC1-ALK.

<p>Panel A shows the schematic structure of KLC1, ALK, and KLC1-ALK proteins and the cDNA sequence around the fusion point. Dark blue, orange, and red parts represent coiled-coil, transmembrane, and kinase domains, respectively. The break point exons and the number of amino acids are indicated. KLC1-ALK-specific RT-PCR using RNA extracted from the FFPE tissue of the unknown ALK fusion-positive case amplified a fragment of the expected product size (140 bp, Panel B) with the consistent fusion sequence (Panel A). A fusion FISH assay for <i>KLC1-ALK</i> revealed a fusion signal (yellow) in multiple tumor cells (Panel C). M, marker (100-bp ladder); S, sample (the unknown ALK fusion-positive case); N, no template control.</p

Transforming potential of KLC1-ALK.

<p>Upper panels: Mouse 3T3 fibroblasts were infected with retroviruses encoding KLC1-ALK or EML4-ALK or with the corresponding empty virus (Mock). The cells were photographed after 4 days of culture. Scale bar, 1 mm. Lower panels: Nude mice were injected subcutaneously with the corresponding 3T3 cells, and tumor formation was examined after 14 days. The number of tumors formed per injections is indicated at the bottom.</p