`IMMUNOHISTOCHEMICAL ANALYSIS OF BRAF (V600E) MUTATION AND P16 EXPRESSION IN MALIGNANT MELANOMA IN LUTH: A 10 YEAR RETROSPECTIVE STUDY (2005-2014)

Abstract

Introduction: Malignant melanoma (MM) is the most lethal of all cutaneous malignancies,
accounting for as much as 79% of skin cancer related deaths. Despite its aggressive nature, it
remains highly curable if detected early. In blacks however, it is associated with greater
morbidity and mortality compared to Caucasians and this has been attributed to advanced stage
of disease at presentation. Studies have shown that MM with BRAF V600E mutation as well as
those with loss of p16 protein expression are associated with aggressive behaviour and worse
prognosis. BRAF kinase inhibitors have also been approved for treatment of BRAF V600E
mutant cases while p16 expression is being proposed as a prognostic marker for MM.
Aim: The aim of the study was to determine the BRAF (V600E) mutation and loss of p16
expression in MM cases in our hospital within the last 10 years and to correlate these with
prognostic markers.
Materials and Methods: Formalin-fixed paraffin-embedded (FFPE) tissue blocks and
corresponding archival routine haematoxylin and eosin (H&E) stained slides of all the cases
confirmed as MM from January 2005 to December 2014 in LUTH were retrieved. The
cutaneous MM cases were classified into histologic variants according to the 2006 WHO
criteria and the Clark’s stage and Breslow thickness were determined. Fresh sections from
the FFPE tissue blocks were taken for Immunohistochemistry (IHC) studies to determine
mutant BRAF V600E and p16 protein expression using the VE1 and anti
CDKN2A/p16INK4a monoclonal antibodies respectively following standard procedures.
Results: Fifty-two MM cases were histologically diagnosed in LUTH during the study
period which represented 1.0% of total solid malignancies. Forty three of these occurred in
the skin accounting for 19.7% of all skin malignancies. The age range was from 24 to 85
years and the peak age of incidence was the 5th decade. Male to female ratio was 1: 1.5.
Fifty eightpercent of the 52 MM cases occurred in the foot. Cutaneous MM constituted
82.7% of the cases, while mucosal, metastatic and ocular melanoma constituted 7.7%, 5.8%,
and 3.8% respectively.. Histologically, the cutaneous cases were Nodular, Acral lentiginous
and Desmoplastic MM accounting for 84%, 14% and 2% respectively. Eighty six percent of
MM cases were in Clark’s stage IV while 84% had Breslow thickness ≥4mm. Ulceration
was present in 67% of the cases. Immunohistochemistry was carried out on 45 cases with
viable tissue blocks and they consisted of cutaneous MM (37), musosal MM (3), ocular (2)
and lymph node metastatasis (3). BRAF (V600E) mutation was detected in 11% while 69%
of the cases had loss of p16 expression. There was however no significant association
between the BRAF (V600E) mutation status as well as loss of P16 expression and
prognostic factors such as patient’s age, histologic variant, Clark’s level, Breslow thickness
and ulceration.
Conclusion: MM constituted a significant proportion of skin malignancy in LUTH. BRAF
(V600E) mutation was detected in only 11% of the cases while majority (69%) showed loss
of p16 expressio9n. These markers however did not show any statistically significant
association with the prognostic factors.