PMC8486277_01
Pathological examination of the patient tumor tissue. Hematoxylin and eosin (HE) staining of the biopsy specimen showed a poorly differentiated tumor tissue. IHC experiments showed positive expressions of Napsin A, TTF-1, Ki-67 and negative expression of P40 (x200), revealing an adenocarcinoma origin of the lung cancer.
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The immunohistochemical analysis revealed positive expression for napsin A, Ki-67, thyroid transcription factor 1 (TTF-1), and negative expression for P40 (Figure 1), indicating an adenocarcinoma origin of the lung tumor.
Diagrams showed breaking point of SLC8A1-ALK fusion and BCL11A-ALK fusion. (A) Positions of genes ALK, SLC8A1 and BCL11A in the chromosome 2 (Chr2). (B) The breaking point of partial SLC8A1 exon2 (blue color) fused to the last 54 bp of ALK exon 19 (orange color) detected on DNA levels. (C) The breaking point of partial BCL11A exon3 (purple color) fused to the first 1 bp of ALK exon 20 detected. (D and E) Diagrams showed the sequence around the breaking point of SLC8A1-ALK fusion and BCL11A-ALK fusion, respectively.
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The sequencing results showed that the SLC8A1 exon 2 rearranged with ALK exon 19 (Figure 2A, B and D). The breakpoints and corresponding fusion sequences were further examined using reverse transcriptase-polymerase chain reaction (RT-PCR), followed by Sanger sequencing to confirm its expression pattern on RNA levels. Of note, the results of targeted RT-PCR sequencing revealed a different ALK rearrangement partner gene: a B-cell CLL/lymphoma 11A (BCL11A) exon 3 rearranged with ALK exon 20 (Figure 2A, C and E).
FISH images. (A) FISH analysis showed fused red-green signals (negative signal), split red-green signals (positive signal) and single red signals (positive signal) in the patient's biopsy specimen. (B) FISH analysis of typical EML4-ALK fusion gene expression, as the reference. The red arrows indicate cells with positive signals of ALK-fusion events and the green arrows indicate negative signals of non-ALK fusion. Both images were magnified 100 time (x100) under microscope.
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ALK rearrangement events on a chromosomal level in the biopsy specimen obtained from the patient (Figure 3A), with typical positive ALK-rearrangement signals in comparison with the positive reference EML4-ALK fusion image (Figure 3B). Apart from the aforementioned ALK fusion, there was no epidermal growth factor receptor (EGFR), TP53 and KRAS mutations or ROS1 rearrangement detected in this patient.
Thoracic computed tomography (CT) images showed tumor responses to ALK-TKIs treatment before 1 month of therapy (baseline) and 1~3 months after therapy. The CT scans demonstrated that a mass in the right lower lobe, plump mediastinal lymph nodes and pleural effusion of the right chest was obviously resolved 1 month after the crizotinib treatment. In 9 months, CT showed solid composition of mass increased (red arrow) and the tumor responded to alectinib treatment again in 13 and 17 months.
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Chest CT scan images demonstrated that the mass, plump mediastinal lymph nodes and pleural effusion had obviously resolved at 1 month and reached stable disease status at 3 months after the treatment (Figure 4). However, a chest CT scan (July 4, 2020) revealed an increased solid composition of the mass, multiple thickened nodules with right horizontal fissures, and plum per mediastinal lymph-nodes appeared in comparison with previous CT scan images (January 11, 2020) (Figure 4).