Primary Sclerosing Cholangitis–Associated Cholangiocarcinoma Demonstrates High Intertumor and Intratumor Heterogeneity

Abstract

INTRODUCTION: Intertumor and intratumor heterogeneity may explain the diagnostic challenge and limited efficacy of chemotherapy for primary sclerosing cholangitis–associated cholangiocarcinoma (PSC-CCA). In this study, tumor heterogeneity was assessed through p53 and p16 protein expression analysis and next-generation sequencing (NGS) of TP53 and CDKN2A genetic alterations in PSC-associated CCA. METHODS: Formalin-fixed paraffin-embedded tissue samples from resection material of patients with PSC-CCA or patients with PSC diagnosed with biliary dysplasia were selected. Sections with CCA and foci with dysplastic epithelium were identified by 2 independent gastrointestinal pathologists. Immunohistochemical evaluation of p53 and p16 protein expression and NGS of TP53 and CDKN2A genetic alterations were performed. RESULTS: A total of 49 CCA and 21 dysplasia samples were identified in the resection specimens of 26 patients. P53 protein expression showed loss of expression, wild type, and overexpression in 14%, 63%, and 23% CCA and in 19%, 62%, and 19% dysplasia samples, respectively. P16 protein expression showed negative, heterogeneous, and positive results in 31%, 57%, and 12% CCA and in 33%, 53%, and 14% dysplasia samples, respectively. NGS showed high intertumor and intratumor heterogeneity of TP53 mutations and CDKN2A loss. Nearly 70% of the samples with a TP53 missense mutation demonstrated p53 overexpression, whereas all samples with a TP53 nonsense mutation demonstrated loss of p53 protein expression. DISCUSSION: PSC-associated CCA is characterized by high intertumor and intratumor heterogeneity of both p53/p16 protein expression and genetic alterations in TP53 / CDKN2A , indicating that these tumors consist of multiple subclones with substantially different genetic makeup. The high intertumor and intratumor heterogeneity in PSC-CCA should be acknowledged during the development of diagnostic and therapeutic strategies.

Genetic alterations during the neoplastic cascade towards cholangiocarcinoma in primary sclerosing cholangitis

“…The majority of these mutations in PSC‐CCA resection specimens and biopsies are well‐known pathogenic mutations [ 39 ]. In our study, several regions per specimen were investigated to avoid the risk of missing alterations due to intratumor molecular heterogeneity [ 40 ]. In addition to these known mutations in PSC‐CCA, the assessment of a considerable number of LGD and HGD samples in this study provides insight into the genetic alterations occurring at specific stages in the progression towards PSC‐CCA.…”

Mutational Landscape of Cholangiocarcinoma According to Different Etiologies: A Review

“…TP53 mutations were detected in eleven PSC-associated CCAs and three PSC-associated dysplasia samples; in particular, one PSC-associated CCA sample carried two different TP53 missense mutations and one PSC-associated dysplasia sample showed the combination of a splice site and a TP53 missense mutation. Loss of the CDKN2A gene was detected in 8 PSC-associated CCAs and 3 PSC-associated dysplasia samples [30]. However, because of the small sample size of patients analyzed in this study, no definitive conclusions can be drawn about the genomic landscape in PSC-associated CCA.…”

“…Currently, less is known about the mutational spectrum of PSC-associated CCA. A recent study on 26 patients with PSC, including 24 cases with CCA and 2 cases with biliary dysplasia, showed a high intertumor and intratumor heterogeneity in TP53 mutations and CDKN2A gene loss [30]. TP53 mutations were detected in eleven PSC-associated CCAs and three PSC-associated dysplasia samples; in particular, one PSC-associated CCA sample carried two different TP53 missense mutations and one PSC-associated dysplasia sample showed the combination of a splice site and a TP53 missense mutation.…”

Diagnosis Biomarkers of Cholangiocarcinoma in Human Bile: An Evidence-Based Study

“…p16INK4a is one of the four isoforms of p16 transcription, which is a tumor suppressor protein that acts as a negative regulator of the proliferation of normal cells that induce g1 cell cycle arrest by interacting strongly with CDK4 and CDK6 [ 23 ]. p16 is one of the possible biomarkers for assessing cervical lymph node metastasis in oral squamous cell carcinoma, and an NGS study of 26 patients with cholangitis-associated cholangiocarcinoma (PSC-CCA) or primary biliary cholangitis (PSC) showed the aberrant expression of p16 consistent with heterogeneity in PSC-CCA [ 24 , 25 ]. Moreover, this NGS result also showed the potential of p16 to distinguish between PSC-CC and PSC.…”