A comparison of DNA sequencing and gene expression profiling to assist tissue of origin diagnosis in cancer of unknown primary

Abstract

Cancer of unknown primary (CUP) is a syndrome defined by clinical absence of a primary cancer after standardised investigations. Gene expression profiling (GEP) and DNA sequencing have been used to predict primary tissue of origin (TOO) in CUP and find molecularly guided treatments; however, a detailed comparison of the diagnostic yield from these two tests has not been described. Here, we compared the diagnostic utility of RNA and DNA tests in 215 CUP patients (82% received both tests) in a prospective Australian study. Based on retrospective assessment of clinicopathological data, 77% (166/215) of CUPs had insufficient evidence to support TOO diagnosis (clinicopathology unresolved). The remainder had either a latent primary diagnosis (10%) or clinicopathological evidence to support a likely TOO diagnosis (13%) (clinicopathology resolved). We applied a microarray (CUPGuide) or custom NanoString 18-class GEP test to 191 CUPs with an accuracy of 91.5% in known metastatic cancers for high-medium confidence predictions. Classification performance was similar in clinicopathology-resolved CUPs - 80% had high-medium predictions and 94% were concordant with pathology. Notably, only 56% of the clinicopathology-unresolved CUPs had high-medium confidence GEP predictions. Diagnostic DNA features were interrogated in 201 CUP tumours guided by the cancer type specificity of mutations observed across 22 cancer types from the AACR Project GENIE database (77,058 tumours) as well as mutational signatures (e.g. smoking). Among the clinicopathology-unresolved CUPs, mutations and mutational signatures provided additional diagnostic evidence in 31% of cases. GEP classification was useful in only 13% of cases and oncoviral detection in 4%. Among CUPs where genomics informed TOO, lung and biliary cancers were the most frequently identified types, while kidney tumours were another identifiable subset. In conclusion, DNA and RNA profiling supported an unconfirmed TOO diagnosis in one-third of CUPs otherwise unresolved by clinicopathology assessment alone. DNA mutation profiling was the more diagnostically informative assay. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: cancer diagnostic; cancer of unknown primary; gene expression profiling; mutation profiling; targeted therapy; tissue of origin classification.

Figure 1

Gene expression profiling (GEP) tissue of origin classification of known metastatic cancers and SUPER cancer of unknown primary (CUP) tumours. (A) The NanoString GEP classifier was tested on 188 known origin metastatic tumours with confusion matrix showing concordance of tissue of origin prediction and known cancer type. (B) The GEP classifier tested on clinicopathology‐resolved CUPs showing concordance between the likely tissue of origin and the predicted cancer type. Latent primary and clinicopathology‐resolved CUPs representing cancer types not represented in the classifier model were removed from the analysis. (C) Fraction of cases within confidence probability score grouping contrasting classification of Clinicopathology‐unresolved CUPs and clinicopathology‐resolved CUPs combined with known metastatic tumours (unclassified <0.5, low ≥0.5 and ≤0.7, medium confidence ≥0.8 and ≤0.9, high confidence = 1). (D) GEP cancer class predictions of all clinicopathology‐unresolved CUPs with high–medium confidence predictions.

Figure 2

DNA mutational profiling of the SUPER cancer of unknown primary (CUP) cohort. The Oncoplot shows somatic mutations in CUPs in descending order of frequency. Genes and mutational features coloured red are actionable and targeted in the CUPISCO trial. The proportion of mutations in the SUPER CUPs was compared with the AACR Project GENIE CUP cohort (right‐hand bar plot). The left‐hand plot shows the average variant allele frequency (VAF) distribution per gene. The top bar plot shows the number of coding mutations per sample. Annotations include MSKCC OncoTree class for clinicopathology‐unresolved CUPs or the assigned tumour class for latent primary and clinicopathology‐resolved CUPs; detection of COSMIC mutational signatures (V2): smoking signature, ultra‐violet (UV) signature, DNA mismatch repair signature; oncoviruses: human papillomavirus 16 (HPV16) and Epstein–Barr virus (EBV); and tumour mutation burden (TMB) status: high >10 mutations/Mb or low <10 mutations/Mb.

Figure 3

Identification of diagnostically useful DNA features using AACR Project GENIE mutation data and summary of evidence used to support tissue of origin (TOO) diagnosis among SUPER cancer of unknown primary (CUP) cases. (A) Proportion and number of cases where each genomic feature supported a putative TOO for clinicopathology‐unresolved CUPs and clinicopathology‐resolved CUPs. Genomic features included single nucleotide variants (SNV), gene expression profiling (GEP), mutational signatures, oncoviral sequences, copy number alterations (CNAs), and gene fusions. (B) MSKCC OncoTree cancer classification of clinicopathology‐unresolved CUPs before and after genomic analysis. CUPs were either resolved to a putative TOO, had reduced occult diagnosis, or remained clinicopathology‐unresolved. (C) Proportion of cases where DNA and/or GEP classification supported TOO diagnosis among clinicopathology‐unresolved CUPs. (D) Detailed summary of supportive genomic features and IHC staining used to assign a putative TOO for all genomically resolved clinicopathology‐unresolved CUPs.