Detection of TP53 Mutations in Tissue or Liquid Rebiopsies at Progression Identifies ALK+ Lung Cancer Patients with Poor Survival

Anaplastic lymphoma kinase (ALK) sequencing can identify resistance mechanisms and guide next-line therapy in ALK+ non-small-cell lung cancer (NSCLC), but the clinical significance of other rebiopsy findings remains unclear. We analysed all stage-IV ALK+ NSCLC patients with longitudinally assessable TP53 status treated in our institutions (n = 62). Patients with TP53 mutations at baseline (TP53mutbas, n = 23) had worse overall survival (OS) than patients with initially wild-type tumours (TP53wtbas, n = 39, 44 vs. 62 months in median, p = 0.018). Within the generally favourable TP53wtbas group, detection of TP53 mutations at progression defined a “converted” subgroup (TP53mutconv, n = 9) with inferior OS, similar to that of TP53mutbas and shorter than that of patients remaining TP53 wild-type (TP53wtprogr, 45 vs. 94 months, p = 0.043). Progression-free survival (PFS) under treatment with tyrosine kinase inhibitors (TKI) for TP53mutconv was comparable to that of TP53mutbas and also shorter than that of TP53wtprogr cases (5 and 8 vs. 13 months, p = 0.0039). Fewer TP53wtprogr than TP53mutbas or TP53mutconv cases presented with metastatic disease at diagnosis (67% vs. 91% or 100%, p < 0.05). Thus, acquisition of TP53 mutations at progression is associated with more aggressive disease, shorter TKI responses and inferior OS in ALK+ NSCLC, comparable to primary TP53 mutated cases.


Supplementary Materials
Method S1. Diagnosis of NSCLC and detection of EML4-ALK fusion variants and TP53 mutations by next-generation sequencing The histological diagnosis of NSCLC and the quantification of tumour cell content were performed by experienced pulmonary pathologists on formalin-fixed and paraffin-embedded (FFPE) small biopsies according to the WHO and IASLC/ATS/ERS criteria [1]. Newly diagnosed cases were screened for the presence of an ALK alteration by fluorescence in situ hybridisation (FISH) and reverse-transcription polymerase-chain reaction (RT-PCR) until 2015, or by immunohistochemistry (IHC) and next-generation sequencing (NGS) afterwards, with FISH restricted to discordant results and technical failures.
For the NGS detection of ALK fusions and TP53 mutations, areas with at least 15% tumour cellularity were marked on a hematoxylin and eosin-stained slide and macrodissected manually from consequent 8 μm thick tissue sections, followed by sample deparaffinization, digestion with Proteinase K overnight and nucleic acid extraction with the automated system Promega Maxwell 16 LEV RNA FFPE Purification Kit (Promega, Madison, WI, USA). Samples with at least 1 ng/µ L DNA and/or RNA (QuBit 2.0 DNA and RNA high sensitivity kit, ThermoFisher Scientific, Waltham, MA, USA) were split into two and used for DNA and/or RNA library preparation directly and/or after treatment with DNaseI at room temperature for 15 min, respectively. For library preparation, the multiplex PCR-based Ion Torrent AmpliSeq™ technology (ThermoFisher) was used together with the RNA Lung Cancer Fusion Panel and a proprietary Lung Cancer Panel (covering the entire exons 4, 5, 6, 7, 8, 9, and 10 of TP53 including 15 flanking base pairs for each exon), as described previously [2]. Only samples achieving library concentrations > 100 pM were considered suitable for further processing. Raw sequencing data were processed using the Torrent Suite Software (version 4.2.1) and aligned against the human genome (version hg19) using the Torrent Mapping Alignment Program algorithm. ALK fusion transcripts were detected using the fusion workflow integrated in the Ion Reporter Software (versions 4.2 and 5.2) as described previously [2].

Method S2. Detection of the V1 and V3 EML4-ALK fusion variants by RT-PCR
For the RT-PCR based detection of V1 and V3 ALK fusion variants, tumour RNA was reverse transcribed using the SuperScript III One Step RT-PCR System (Invitrogen, ThermoFisher) and amplified with primers specific for the EML4 exons 6 and 13 and the ALK exon 20 (sequences given in the Online Supplements). Direct sequencing of the PCR amplicons was performed for both strands on a 3500 Genetic Analyzer using the BigDye Terminator v1.1 Cycle Sequencing Kit (both from Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Since RT-PCR represented only a temporary solution in our laboratory and was succeeded by NGS in 2015 for the typing of ALK fusion variants, we chose not to invest additional resources in the establishment of RT-PCR assays specific for less frequent variants like V2.

Method S3. ALK IHC and FISH
For the ALK IHC a sensitive and specific antibody (D5F3 clone, Roche, Mannheim, Germany) [3] was used according to current guidelines [4]. ALK FISH analyses were performed on whole block slides of FFPE NSCLC samples using a break-apart probe (ZytoLight SPEC ALK probe, ZytoVision GmbH, Bremerhaven, Germany). At least 100 cells were analysed per case, and cases were considered to be positive when 15% or more of 50 tumour cells showed classic split signals [4].

Method S4. ctDNA analysis
For ctDNA analysis, plasma was isolated from blood samples of ALK + NSCLC patients centrifuged within 30 minutes of collection and processed with the AVENIO ctDNA Analysis Kit (covering the entire TP53 exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Briefly, DNA was isolated from 2 mL of plasma using the AVENIO cfDNA Isolation Kit (Roche) and quantified with the Qubit dsDNA High Sensitivity Kit (ThermoFisher). Targeted sequencing libraries were prepared from 39.5 ng DNA in median (range 15-50 ng) using the AVENIO ctDNA Library Preparation Kit with the AVENIO Targeted Panel (both from Roche) for hybridisation-based enrichment of a 17-gene panel. All protocols were conducted according to the manufacturer's recommendations. Equal amounts of 16 libraries were pooled and sequenced on an Illumina NextSeq 550 using the High Output Kit V2 (300 cycles) according to the manufacturer's protocol (Illumina, San Diego, CA, USA) with a median unique target sequence coverage of 7,600x (range 2,013×-12,467×). Automated raw data processing and data analysis was performed with the AVENIO ctDNA analysis software (Roche).  Figure S1. Progression-free survival of patients with metastatic ALK + NSCLC under treatment with chemotherapy according to TP53 status at baseline and under therapy. The median progression-free survival (PFS) under chemotherapy treatment was 5 months for patients with TP53 mutations at baseline (TP53mut bas ) vs. 7 months for patients with initially wild-type status and detection of TP53 mutations in a subsequent biopsy (TP53mut conv ) vs. 8 months for patients without subsequent detection of TP53 mutations (TP53wt progr , logrank p = 0.60). The treatment details are given in Table 1; ns: not statistically significant. months Figure S2. Overall survival of study patients from initial diagnosis. The median overall survival (OS) from initial diagnosis was 44 months for patients with TP53 mutations at baseline (TP53mut bas ) vs. 45 months for patients with initially wild-type status and detection of TP53 mutations in a subsequent biopsy (TP53mut conv ) vs. not reached for patients without subsequent detection of TP53 mutations (TP53wt progr , logrank p=0.0012). As shown in Table 1, 10/30 TP53wt progr and 2/23 TP53mut bas patients had secondary development of metastatic disease after relapse of initially nonmetastatic ALK + NSCLC, causing the curves in this plot to differ slightly from these of Figure 1; ns: not statistically significant.