PMC7400592_01
Clinical history and histopathologic findings of small cell transformation.. a Timeline demonstrating the treatment course of the patient and time points of tumor (T) collection. Numbers represent months since initial diagnosis. SBRT stereotactic body radiation therapy, FISH fluorescence in situ hybridization, IHC immunohistochemistry, NGS next-generation sequencing. b Representative axial computed tomography images demonstrating the progression of hepatic metastases during the disease course. c Schematic of the tumor samples collected. d Immunostains of the treatment-naive lung primary demonstrating thyroid transcription factor-1 (TTF-1)-positive adenocarcinoma, and of the representative autopsy tumor specimen demonstrating small cell morphology with positive stains for synaptophysin and chromogranin. H&E hematoxylin and eosin. ROS1 FISH demonstrates that the ROS1 fusion is retained in both treatment-naive and autopsy samples; representative split signals indicative of ROS1 fusion are highlighted by white arrows. ROS1 IHC illustrates detectable ROS1 protein expression in the treatment-naive tumor, but undetectable ROS1 expression in the small cell tumor. Immunostains for TP53 and RB1 reveal wild-type RB1 (red arrows indicating positive nuclear staining) and loss of TP53 expression in the treatment-naive tumor, and lack of RB1 and TP53 expression in the small cell-transformed tumor. The scale bar represents 100 microm in all panels except for the ROS1 FISH panel, in which it represents 30 microm. Magnification: x200 for H&E and TTF-1, synaptophysin, chromogranin, and ROS1, and x400 for TP53 and RB, of the initial biopsy sample; x100 for H&E and TTF-1, synaptophysin, chromogranin, ROS1, TP53, and RB immunostains of the autopsy sample.
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Fig1
A 32-year-old woman of South Asian descent presented with persistent dry cough and decreased exercise tolerance (Fig. 1a). Core biopsy of the right middle lobe lung tumor (T1, Fig. 1a) demonstrated adenocarcinoma with mixed lepidic and micropapillary patterns. Repeat biopsy of this lung nodule (T2, Fig. 1a) confirmed adenocarcinoma. South Asian population and is likely a polymorphism. The patient was treated with stereotactic body radiation therapy (SBRT) to the growing lung nodule and continued on crizotinib. Subsequent scans showed growth of an adjacent left lower lobe lung nodule, which was not biopsied, but treated with microwave ablation (Fig. 1a). Repeat imaging after 57 months on crizotinib demonstrated two new hepatic metastases (Fig. 1b). She subsequently received short courses of chemotherapy (carboplatin, pemetrexed, bevacizumab), lorlatinib (a ROS1/ALK TKI), and cabozantinib (multitargeted TKI with ROS1 activity), none of which induced a tumor response (Fig. 1b). Metastatic tumor samples were collected from the lung, liver, and portal lymph node (T3.2-3.12, Fig. 1c). Of note, six regions were sampled from a dominant right hepatic lobe metastasis (T3.3-3.8, Fig. 1c); one representative region was sampled from each of the remaining metastatic sites. Histopathology review of all of the autopsy samples revealed tumor cells with scant cytoplasm, finely dispersed chromatin and inconspicuous nucleoli, morphologically distinct from the patient's prior treatment-naive and crizotinib-resistant lung biopsy specimens (Fig. 1d). ROS1 FISH confirmed the presence of a ROS1 rearrangement in all autopsy tumor samples (Fig. 1d). However, by immunohistochemistry (IHC), all autopsy samples lacked ROS1 protein expression (Fig. 1d). By IHC, tumor cells in the original, treatment-naive biopsy demonstrated wild-type RB1 expression, but loss of TP53 expression, whereas a representative autopsy sample lacked expression of both TP53 and RB1 (Fig. 1d). Copy number landscape was also homogeneous overall (Supplementary Fig. 1; focal amplifications and deletions shown in Supplementary Data Files 2 and 3, respectively).
Genetic and phylogeny analysis of metastatic tumors.. a Circos plots for a representative autopsy tumor specimen, providing a high-level overview of genomic gains (red) and losses (blue) across all evaluable probes in all chromosomes. There are diffuse losses across chromosome 13. b A higher magnitude view of four genes on chromosome 13 demonstrating loss of RB1. c Next-generation sequencing pile-up illustrating the presence of a splice region variant in RB1 in the majority of the reads. d Branching diagram of the metastatic tumors collected at autopsy and analyzed by whole-exome sequencing. The numbers on the branches represent the number of distinct mutations (synonymous and non-synonymous). "N" refers to normal tissue. The treatment-naive tumor (T1) and the crizotinib-resistant tumor (T2) were not analyzed by whole-exome sequencing, and therefore, could not be located precisely in this diagram. e Decreased expression of lung epithelial genes and increased expression of neuroendocrine genes in the MGH968-A PDX, MGH968-B, and MGH968-C cell line models, as determined by quantitative RT-PCR. MGH9018-1 is a cell line derived from a crizotinib-resistant CD74-ROS1 fusion-positive adenocarcinoma and is shown for comparison. f Resistance of MGH968-A and MGH968-B cells to clinically available ROS1 inhibitors. The proliferation assay was performed in triplicate, and the error bars represent the standard error of the mean.
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Fig2
FLT3 (S497W; c.1490C > G); these genes had not been evaluated in the NGS analysis of the prior crizotinib-resistant lung tumor. Further analyses supported biallelic inactivation of RB1, with loss of one allele of RB1 and the presence of the splice region/intronic variant in the remaining allele (Fig. 2a-c). Clonal analysis and phylogenetic reconstruction revealed that the post-mortem metastatic sites were homogeneous, with a small number of mutations distinguishing the metastatic tumors (Fig. 2d). SCLC morphology with loss of RB and p53 expression (Supplementary Fig. 2A), and the ROS1 rearrangement was confirmed by FISH (data not shown). Similar to the original tumor, solid fusion assay did not detect expression of the SLC34A2-ROS1 transcript. C). Consistent with the clinical tumor sample and PDX model, RB and p53 expression were lost (Supplementary Fig. 2D). We also observed decreased lung epithelial gene expression (napsin A, KRT7) and increased neuroendocrine gene expression (chromogranin, synaptophysin, DLL3, ASCL1, NEUROD1) consistent with transformation from adenocarcinoma to SCLC (Fig. 2e). We were unable to detect expression of the SLC34A2-ROS1 fusion transcript in the established cell lines, despite the presence of the rearrangement in the genomic DNA (Supplementary Fig. 2C). Fig. 2B). Finally, we examined whether the SCLC-transformed MGH968 cell lines were resistant to ROS1 TKIs. Consistent with a loss of dependency on ROS1, the cell lines were insensitive to two different clinical ROS1 inhibitors (Fig. 2f).