Deletion at chromosome arm 9p in relation to BRAF/NRAS mutations and prognostic significance for primary melanoma

Abstract

We report an investigation of gene dosage at 9p21.3 and mutations in BRAF and NRAS, as predictors of relapse and histological markers of poor melanoma prognosis. Formalin-fixed primary melanomas from 74 relapsed and 42 nonrelapsed patients were sequenced for common BRAF and NRAS mutations (N = 71 results) and gene dosage at 9p21.3 including the genes CDKN2A (which encodes CDKN2A and P14ARF), CDKN2B (CDKN2B), and MTAP was measured using multiplexed ligation-dependant probe amplification (MLPA), (N = 75 results). BRAF/NRAS mutations were detected in 77% of relapsers and 58% of nonrelapsers (Fisher's exact P = 0.17), and did not predict ulceration or mitotic rate. There was no relationship between BRAF/NRAS mutations and gene dosage at 9p21.3. Reduced gene dosage at MTAP showed a borderline association with BRAF mutation (P = 0.04) and reduced gene dosage at the interferon gene cluster was borderline associated with wild type NRAS (P = 0.05). Reduced gene dosage in the CDKN2A regions coding for CDKN2A was associated with an increased risk of relapse (P = 0.03). Reduced gene dosage across 9p21.3 was associated with increased tumor thickness, mitotic rate, and ulceration (P = 0.02, 0.02, and 0.002, respectively), specifically in coding regions impacting on CDKN2B and P14ARF and CDKN2A. Loss at MTAP (P = 0.05) and the interferon gene cluster (P = 0.03) on 9p21 was also associated with tumor ulceration. There was no association between reduced gene dosage at 9p21.3 and subtype or site of tumor. This study presents supportive evidence that CDKN2B, P14ARF, and CDKN2A may all play a tumor suppressor role in melanoma progression.

Figure 1

MLPA for analysis of gene dosage at chromosome 9p21. A, a diagrammatical representation of the 9p21 region covered by the P024A MLPA kit. Locations of probed genes are shown in green and MLPA probes in orange with probe names outlined in bold. Probe groupings used in data analysis are represented by orange bars with black dots at top of the picture. B, gene dosage ratios for a representative primary melanoma sample showing an almost total loss of gene dosage (representing ≥80% loss) from MTAP exon 6 to CDKN2B intron.

Figure 2

Heatmap of gene dosage at chromosome region 9p21 by relapse status. Gene dosage ratios are shown for tumors from 45 relapsed patients (above) compared with tumors from 27 patients who had not relapsed. Blue coloration indicates reduced gene dosage, red coloration indicates increased gene dosage.

Figure 3

Heatmap of gene dosage at chromosome region 9p21 by Breslow thickness. Gene dosage ratios are shown for 18 tumors with Breslow thickness <2 mm (above), 30 tumors with Breslow thickness 2–4 mm (middle) and 27 tumors of thickness >4 mm. Blue coloration indicates reduced gene dosage, red coloration indicates increased gene dosage.

Figure 4

Immunohistochemistry of CDKN2A in primary tumors. (A) Bladder tumor control minus primary antibody. Some nonspecific stromal staining is visible in the stroma. (B) Strong specific cytoplasmic and nuclear staining in a bladder positive control in presence of primary antibody. (C) Results from representative tumors show very low cytoplasmic staining of CDKN2A from a tumor with a large homozygous deletion of CDKN2A. (D) Strong nuclear and cytoplasmic staining of CDKN2A and unstained normal stromal cells from a tumor which retained 2 copies of CDKN2A. Scale bar = 25 μm.