Recurrent genetic alterations and biomarker expression in primary and metastatic squamous cell carcinomas of the vulva

Abstract

Using a comprehensive next-generation sequencing pipeline (143 genes), Oncomine Comprehensive v.2, we analyzed genetic alterations on a set of vulvar squamous cell carcinomas (SCCs) with emphasis on the primary and metastatic samples from the same patient, to identify amenable therapeutic targets. Clinicopathologic features were reported and genomic DNA was extracted from 42 paraffin-embedded tumor tissues of 32 cases. PD-L1 expression was evaluated in 20 tumor tissues (10 cases with paired primary and metastatic tumors). Fifteen (88%) of 17 successfully analyzed HPV-unrelated SCCs harbored TP53 mutations. 2 different TP53 mutations had been detected in the same tumor in 4 of 15 cases. Other recurrent genetic alterations in this group of tumors included CDKN2a mutations (41%), HRAS mutations (12%), NOTCH1 mutations (12%) and BIRC3 (11q22.1-22.2) amplification (12%). Six HPV-related tumors harbored PIK3CA, BAP1, PTEN, KDR, CTNNB1, and BRCA2 mutations, of which, one case also contained TP53 mutation. Six cases showed identical mutations in paired primary site and distant metastatic location and four cases displayed different mutational profiles. PD-L1 expression was seen in 6 of 10 primary tumors and all 6 paired cases showed discordant PD-L1 expression in the primary and metastatic sites. Our results further confirmed the genetic alterations that are amenable to targeted therapy, offering the potential for individualized management strategies for the treatment of these aggressive tumors with different etiology. Discordant PD-L1 expression in the primary and metastatic vulvar SCCs highlights the importance of evaluation of PD-L1 expression in different locations to avoid false negative information provided for immunotherapy.

Keywords: Mutations; PD-L1; Squamous cell carcinoma; TP53; Vulva.

Figure 1.

Somatic mutations detected by next-generation sequencing and a representative case with TP53 and CDKN2a mutations. The mutated activating oncogenic and caretaker tumor suppressor genes in successfully sequenced vulvar SCCs are plotted (arranged in descending order of number of mutations, and if the same number then in an alphabetic order, A). A representative case (case no. 7) with mutations of TP53 (p.Arg175His missense mutation, B) and CDKN2a (p.Arg80Ter nonsense, C) are shown in the middle and right. Consistent with genotype, the tumor (D-a, H&E) demonstrates aberrant/mutation-type p53 over-expression (D-b) consistent with a missense mutation staining pattern. The tumor also shows a negative p16 staining with positive internal control, consistent with CDKN2a truncation mutation pattern (D-c).

Figure 2.

A representative HPV-unrelated SCC case (case no. 13) with mutations of TP53 and PIK3CA. The tumor (A-a, H&E) demonstrates aberrant/mutation-type complete loss of p53 expression (“null” pattern, A-b) because truncated p53 protein (p.Gln165fs, B) cannot be recognized by the p53 antibody. The tumor demonstrates a focal/patchy p16 staining (A-c). PIK3CA (p.Glu542Lys) mutation is also detected (C).

Figure 3.

A representative HPV-related SCC case (case no. 20) with mutations of BAP1 and PTEN. The tumor (A-a, H&E) demonstrates strong and diffuse p16 (A-b) with detected high-risk HPVs (A-c) by RNA in situ hybridization. The tumor harbors BAP1 p.Ser460Ter nonsense mutation (B) and PTEN p.Gly129Arg missense mutation (C).

Figure 4.

Semi-quantitative assessment of PD-L1 expression in paired primary and metastatic SCCs of the vulva. A representative primary vulvar SCC (case 7, A-a, H&E) shows PD-L1 expression with CPS approximately 23 (A-b); metastatic tumor (A-c, H&E) demonstrates decreased PD-L1 expression with CPS approximately 4 (A-d). A schematic graph of CPS of PD-L1 expression in 10 paired primary and metastatic SCCs of the vulva (B). * Case 19, HPV-related.

Figure 5.

Factors related to cause specific survival (CSS) in patients with vulvar SCCs by a univariate analysis. HPV-related etiology demonstrates a superior CSS (A). Negative lymph node involvement shows a more favorable CSS in all SCCs of the vulva (B) and HPV-unrelated tumors (C). CDKN2a mutations was not associated with prognosis in the patients with HPV-unrelated SCCs of the vulva (D). LN: lymph node; m: month.