TP53 mutations are considered early genetic events in Barrett’s esophagus associated with an increased risk of progression to cancer [2, 3, 8]. In general, it is difficult to assess whether a somatic mutation involves only one copy of a gene or both alleles in whole tumor tissue samples, due to stromal cell contamination. According to our data, the TP53 mutant allele was completely mutated in the primary cancer, indicating that the TP53 locus might have been involved in an early LOH event, which can explain the homozygous state of the p.Arg282Trp mutation. This mutation is frequently reported in several cancers (COSM10704, COSMIC; http://cancer.sanger.ac.uk/) and, as constitutive mutation, causes Li Fraumeni syndrome (ClinVar id12347; https://www.ncbi.nlm.nih.gov/clinvar/), an inherited cancer disease, characterized by autosomal dominant inheritance due to heterozygous mutations in TP53, with early onset and multiple tumors within an individual, including soft tissue sarcomas and osteosarcomas, breast cancer, brain tumors, leukemia and adrenocortical carcinoma (OMIM #151623). In our case, the variant was somatic, as indicated by its absence from peripheral blood-derived DNA (Fig. 1b).
The complete loss of TP53 wild-type protein in the studied tumor provides a significant impact on prognosis and therapeutic options, since the p.Arg282Trp mutation abolishes specific DNA binding, allowing evasion from apoptosis and accelerating tumor progression [8]. The pharmacological reactivation of mutant TP53 emerged as a promising strategy using molecules that restore its wild-type activity, such as APR-246/PRIMA-1Met, which is already under clinical trials for different cancers, including EAC [9, 10]. This molecule restores TP53 activity in presence of missense mutations and regulates several TP53-related pathways [9]. Therefore, precise identification of the TP53 mutational status in EAC could be instrumental for selecting more efficient therapies. In the present case, the TP53 mutation was shared by the EAC primary tumor and metastases (suggestive of an early origin) and we propose that restoring TP53 wild-type activity could be effective for metastases. From a technical perspective, the high-throughput sorting of the tumor cells led to the identification of somatic alterations without a “diluting” effect due to the presence of normal stromal cells. In addition, the capability of sorting pure stromal cells provides a convenient internal control [4]. Analyzing only the tumor but not matched normal tissue can yield many false-positive alterations that are not specific to the patient’s tumor. When matched normal tissue is unavailable, as may be the case for archival samples, this method can provide a valuable surrogate.
Standard tests measure Her-2 CNV to guide the use of the anti-Her-2 drug trastuzumab in patients with metastatic disease secondary to gastric/gastroesophageal cancer, since ~ 24% of gastroesophageal adenocarcinomas overexpress Her-2 [11]. However, trastuzumab-responsive patients may develop resistance, due to Her-2-dependent mechanisms such as the overexpression of proteins that mask the Her-2 receptor (e.g., MUC1), Her-2 alternative splicing, or Her-2-independent mechanisms [12]. In the present case, compared to the primary tumor, the Her-2 copy numbers detected by the selective cell sorting coupled to NGS were significantly lower in the metastases developed after trastuzumab therapy (Fig. 2a). This decrease was not detected by SISH, which showed clusters of the Her-2 amplified region in the primary and metastatic tissues. Notably, the areas of metastatic tissues sampled for the CNV and ISH tests were morphologically homogeneous. The lower copy number in the metastases indicates a selection of sub-clones more resistant to treatment, although the histological appearance of the cells in the tumor areas was homogeneous. CNV analysis with WES data also showed a gain in RNF146-ECHDC1 copy number, in the second metastasis. RNF146 encodes for a E3 ubiquitin ligase ring finger protein 146, a critical regulator of Wnt/β-catenin signaling, whose overexpression, for example reported in non-small cell lung cancer, enhanced cell growth, invasion, and survival [13, 14]. ECHDC1 encodes for a proofreading enzyme involved in lipid metabolism, with an increased expression observed in resistant bladder cancer cells [15]. We hypothesize that the acquired RNF146-ECHDC1 copy gain in the cells giving raise to the second metastasis, coupled to the loss of cells with HER2 amplification, might contributes to resistance and progression in metastatic EAC cancer. Sub-clone molecular heterogeneity is also revealed by the detection of somatic events in other cancer genes at different degrees.
