Integration of genomics and histology revises diagnosis and enables effective therapy of refractory cancer of unknown primary with PDL1 amplification

Abstract

Identification of the tissue of origin in cancer of unknown primary (CUP) poses a diagnostic challenge and is critical for directing site-specific therapy. Currently, clinical decision-making in patients with CUP primarily relies on histopathology and clinical features. Comprehensive molecular profiling has the potential to contribute to diagnostic categorization and, most importantly, guide CUP therapy through identification of actionable lesions. We here report the case of an advanced-stage malignancy initially mimicking poorly differentiated soft-tissue sarcoma that did not respond to multiagent chemotherapy. Molecular profiling within a clinical whole-exome and transcriptome sequencing program revealed a heterozygous, highly amplified KRAS G12S mutation, compound-heterozygous TP53 mutation/deletion, high mutational load, and focal high-level amplification of Chromosomes 9p (including PDL1 [CD274] and JAK2) and 10p (including GATA3). Integrated analysis of molecular data and histopathology provided a rationale for immune checkpoint inhibitor (ICI) therapy with pembrolizumab, which resulted in rapid clinical improvement and a lasting partial remission. Histopathological analyses ruled out sarcoma and established the diagnosis of a poorly differentiated adenocarcinoma. Although neither histopathology nor molecular data were able to pinpoint the tissue of origin, our analyses established several differential diagnoses including triple-negative breast cancer (TNBC). We analyzed 157 TNBC samples from The Cancer Genome Atlas, revealing PDL1 copy number gains coinciding with excessive PDL1 mRNA expression in 24% of cases. Collectively, these results illustrate the impact of multidimensional tumor profiling in cases with nondescript histology and immunophenotype, show the predictive potential of PDL1 amplification for immune checkpoint inhibitors (ICIs), and suggest a targeted therapeutic strategy in Chromosome 9p24.1/PDL1-amplified cancers.

Keywords: multifocal breast carcinoma; neoplasm of the gastrointestinal tract.

Figure 1.

PDL1 and GATA3 amplification in a patient with cancer of unknown primary. (Upper panel) Copy number plot showing chromosomal coordinates computed as a set of regions based on whole-exome sequencing data (x-axis) and the log₂ ratio of copy number changes (y-axis). Color codes of alternating green and black regions indicate segmentation between chromosomes. (Lower panel) Amplified region on chromosome 9p.23–24.1.

Figure 2.

PDL1 protein expression, PDL1 amplification, peritumoral lymphocyte infiltration, and PDL1 mRNA expression in a patient with anaplastic cancer of unknown primary. (A) (Left) Photomicrograph showing PDL1 protein expression in a metastasis (scale bar, 100 µm); (middle) representative fluorescence in situ hybridization signal pattern showing amplification of the PDL1 locus (green signals) relative to the centromere of Chromosome 9 (red signals); (right) CD4 lymphocyte staining (scale bar, 200 µm). (B) Ranking of 266 patient samples analyzed in the NCT MASTER study according to PDL1 (CD274) mRNA levels, as determined by RNA-seq. The red bar indicates the described index patient. RPKM, reads per kilobase of exon model per million mapped reads.

Figure 3.

PDL1 DNA copy number and PDL1 mRNA expression in the molecular subtypes of breast cancer (TCGA data sets). (A) PDL1 copy number gains are more frequent in triple-negative breast cancer (TNBC) (43.3%) compared to HR⁻/HER2⁺, HR⁺/HER2⁻, and HR⁺/HER2⁺ breast cancer (26.3%, 10.9%, and 10.3%, respectively). (B) PDL1 mRNA expression is higher in TNBC compared to HR⁺/HER2⁻ and HR⁺/HER2⁺ breast cancer (fold change, 1.49; p = 0.00030 and fold change, 1.42; p = 0.0068). In the beeswarm plot, each colored dot represents a tumor. The horizontal red lines indicate the first quartile, the median, and the third quartile.

Figure 4.

Response assessment after 2 and 6 mo of immunotherapy with pembrolizumab in a patient with metastatic cancer of unknown primary. (A) Maximum intensity plots computed from positron emission tomography/computed tomography (PET/CT) imaging showing multifocal tumor manifestations that almost vanish after 6 mo of PD1 inhibitor treatment. (B) Axial PET/CT images showing target lesions in the left gluteal (upper panels) and scapular (lower panels) regions. Time line from left to right: baseline, 2 mo, 6 mo.