Comprehensive analysis of cancers of unknown primary for the biomarkers of response to immune checkpoint blockade therapy


Joanne Xiu, Ph.D., Semir Vranic, M.D., Ph.D., Zoran. Gatalica, M.D., D.Sc.


Carcinoma of unknown primary (CUP) accounts for approximately 3% of all malignancies. Identification of common cancer pathway alterations (hallmarks of cancer) in diverse cancer lineages offers a rationale for search for biomarkers of targeted therapies in patients with CUP. Avoiding immune destruction is a more recently recognized common cancer characteristic and biomarkers associated with immune check-point blockade were explored in this study.


389 cases of CUP were tested with NextSeq platform with a 592-gene panel. Tumor mutational load (TML) was calculated using only somatic nonsynonymous missense mutations; microsatellite instability (MSI) was evaluated with NGS by direct analysis of known MSI loci in the target regions of the sequenced genes. ArcherDx FusionPlex Assay was used to detect gene fusions and 52 gene targets were analyzed for 156 tumors. IHC was used to detect tumor expression of PD-L1 (SP142 antibody). All tests were done in a CLIA/CAP/ISO-certified lab.


Average patient’s age was 62.7 years; 52% were female. TML high was seen in 11.8% (46/389) tumors using a cutoff of 17 mutations/Mb. MSI-high was detected in 7/384 (1.8%) of tumors. A total of 69 different genes were found mutated with the incidence ranging from 0.5% to 54%; the most frequent were TP53 (54%), KRAS (22%) and ARID1A (13%). Additional notable targetable mutations include PIK3CA (9%), CDKN2A (8%), SMARCA4 (7%), APC and RB1 (5%), PTEN and BRAF (4%), and ERBB2 (1.5%). Targetable gene fusions identified included FGFR2 fusions (N=2), RET (N=2) , RAF1 (N=1), NTRK3 (N=1).Tumors with detected fusions carried a significantly lower TML (average 5.9 mutations/megabase) than the complete cohort (11.0, p<0.001) with no MSI-high seen in this subgroup. Tumor expression of PD-L1 was seen in 22.1% (80/362) of the cases while PD-1 tumor infiltrating lymphocytes were detected in 58.7% (37/63). Commonly amplified genes included CCND1 (5%), FGF3, FGF4, FGF19, HER2, MYC (3%, respectively) and AKT2, KRAS and MDM2 (2%, respectively). Of note, CD274 (PD-1) gene was rarely amplified (1.4%).


Using a multiplex testing approach, 28% of CUPs had biomarkers (TML-H, MSI-H and/or PD-L1) of response to the immune check-point blockade were identified, making CUP one of the most likely candidate to benefit from immune checkpoint inhibitor.

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