PMC9356229_01
Case 1. (A) Pathology results. (a) H&E staining shows lung adenocarcinoma (40x). (b, c) IHC shows positive expression of ALK and BRAF-V600E after alectinib resistance (40x). (B) Identification of the fusion sequence by NGS. (a) The identification of GCKR-ALK and unknown-ALK fusion by DNA sequencing, visualized by the Integrative Genomics Viewer (IGV). (b) The identification of EML4-ALK by RNA sequencing. (c) Schematic diagram illustrating molecular rearrangements that explain the emergence of the EML4 and ALK mRNA based on the DNA and RNA NGS results. (C) Radiological evaluations. (a) Before treatment; yellow circle represents mediastinal lesion, yellow arrow represents primary lung tumor. (b) Efficacy evaluation was SD after 3 months of first-line aletinib treatment. (c) Disease progression after 4.5 months of aletinib treatment, the mediastinal lesion invaded bilateral main bronchus, and the left main bronchus was nearly occluded; red arrow represents intratracheal invasion. (d) First efficacy evaluation was SD after 1 month of second-line dual-target combination therapy. (e) Disease progression after 2 months of dual-targeted combination therapy with partial enlarged mediastinal lesion and a suspicious new lymph node metastasis in the right neck; red circle represents new lymph node metastasis. (f) The timeline of treatment. (D) Changes of tumor infiltration under bronchoscopy. (a) The mediastinal lesion invaded the carina and bilateral main bronchus after 4.5 months of first-line alectinib treatment. (b) The tumor infiltration was slightly relieved after 1 month of second-line dual-target combination therapy. (E) Dynamic monitoring of tumor markers during treatment. NGS, next-generation sequencing; SD, stable disease.
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The pathologic examination showed poorly differentiated adenocarcinoma (cT1bN3M1a, stage IVA) ( Figure 1A ). Progression of the disease was observed 4.5 months later due to mediastinal lesion invading bilateral main bronchus ( Figures 1C, D  ), thus bronchus stents were placed to alleviate tracheal stenosis. We then re-evaluated the alterations of the coughed-up tissues by IHC, of note, IHC revealed positive expression of ALK-D5F3 and BRAF-V600E ( Figure 1A ). D  ). Considering the resistance of this patient to alectinib, and the fact that lorlatinib was not accessible, alectinib was then replaced with ceritinib, 450 mg daily, combined with dabrafenib and trametinib. Despite switching to second-line treatment, tumor markers increased sharply ( Figure 1E ) and progression of disease was observed again 2 months later ( Figure 1C ), the patient died shortly owing to rapid disease progression.
PMC9356229_02
Case 2. (A) Pathology results. (a) H&E staining shows lung adenocarcinoma (40x). (b) IHC shows positive expression of ALK (40x). (B) Identification of the fusion sequence by NGS. (a) The identification of IGR-ALK by DNA sequencing, visualized by the Integrative Genomics Viewer (IGV). (b) The identification of EML4-ALK by RNA sequencing. (c) Schematic diagram illustrating molecular rearrangements that explain the emergence of the EML4 and ALK mRNA based on the DNA and RNA NGS results. (C) Radiological evaluations. (a) Before treatment; the blue arrow represents primary lung tumor, the blue circle represents intracranial metastasis, which maintained stable throughout the course of treatment. (b) First efficacy evaluation was PR after 1 month of first-line alectinib treatment. (c) Lung lesion progression after 5 months of first-line alectinib treatment (d, e) Continuing response to second-line chemotherapy in combination with bevacizumab. (f) The timeline of treatment. (D) Dynamic monitoring of tumor markers during treatment. NGS, next-generation sequencing; PR, partial response.
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IVB) ( Figure 2A ). IHC revealed positive expression of ALK-D5F3 ( Figure 2A ). A rare IGR (downstream ZIC4)-ALK exon20 (abundance: 22.6%) fusion was found by a NGS assay based on 425-gene panel (GENESEEQ TP53, were also been detected in the same tumor tissue (see  Supplementary Material  for more detailed genetic informations). The patient consented to take alectinib at 600 mg orally twice daily, in combination with intracranial gamma knife therapy for brain metastases. Five months later, the efficacy evaluation was progress disease (PD) due to progression of lung lesions ( Figure 2C ). To clarify the genetic alterations after alectinib resistance, a NGS assay based on a 139-gene panel (GENESEEQ Technology The patient's best response was partial response (PR) and maintained SD during the treatment, with intracranial metastasis maintained stable throughout the course of treatment ( Figures 2C, D  ), achieving a PFS of over 1 year up to the last follow-up assessment.