Annual Meeting Proceedings Part 1 - American Society of Clinical ...

564s Melanoma/Skin Cancers
8596 General Poster Session (Board #38F), Sun, 8:00 AM-12:00 PM
Detection of BRAF mutations in melanoma: Rate of mutation detection at
codon 600 using Sanger sequencing as compared to the cobas 4800
method. Presenting Author: Kevin Z. Qu, Nichols Institute, San Juan
Capistrano, CA
Background: Detection of BRAF V600 mutations is currently a prerequisite
for approved use of vemurafenib in patients with metastatic melanoma. The
cobas 4800 BRAF V600 Mutation Test (Roche Molecular Diagnostics), a
PCR-based assay approved to aid in selecting patients for vemurafenib
therapy, primarily detects V600E. It is also reported to detect V600K,
which has been associated with vemurafenib response as well. We
compared the mutation detection rate of the cobas assay with that of
Sanger sequencing. Methods: 125 de-identified FFPE tissues submitted for
BRAF mutation analysis that all showed histologically-confirmed melanoma
were tested. BRAF mutations were detected using both the cobas kit
and bidirectional Sanger sequencing using BigDye kits (Applied Biosystems).
DNA was extracted from 5-um sections without macrodissection
using the cobas DNA extraction kit (for the cobas test) or from 5-10-um
sections using Agencourt extraction kits (Beckman Coulter) following
macrodissection. Results: The two methods showed agreement in 104/125
(83.2%) of cases (Table). Sanger sequencing detected V600 dinucleotide
mutations in 9 samples that were negative by the cobas assay. Sanger
sequencing produced no results in 10 cases owing to suboptimal PCR,
including 2 that were positive by the cobas assay. The cobas assay
produced 2 invalid results, including 1 that was positive for V600E by
Sanger.The cobas assay detected 7/11 V600K mutations. Conclusions:
Overall agreement between cobas and Sanger sequencing was 83.2%. The
Sanger method had higher analytic sensitivity, resulting in nine additional
V600 mutations not called by cobas compared to the two seen by cobas but
not Sanger sequencing. Thus, 16% (9/57) more patients would be
identified as candidates for vemurafenib therapy using the Sanger method.
Sanger
sequencing
Not detected V600E V600K V600R Non-600 No result Total
Not detected 56 3 a
4 b
2 c
2 d
7 74
cobas detected 40 7 2 49
Invalid 1 1 2
Total 56 44 11 2 2 10 125
a 2/3 had dinucleotide mutations (GTG�GAA) and 1 had V600_K601del. b All had
dinucleotide mutations (GTG�AAG). c Both had GTG�AGG dinucleotide mutations. d 1)
G469R; 2) E501G.
8598 General Poster Session (Board #38H), Sun, 8:00 AM-12:00 PM
Pharmacodynamic activity of selumetinib to predict radiographic response
in advanced uveal melanoma. Presenting Author: Richard D. Carvajal,
Memorial Sloan-Kettering Cancer Center, New York, NY
Background: Functionally activating mutations (mut) in Gnaq or Gna11,
genes that encode for widely expressed G-protein alpha subunits, are early
oncogenic events in uveal melanoma (UM) development and result in
activation of the MAPK pathway. We previously demonstrated effective
pathway inhibition with selumetinib (AZD6244, ARRY-142866) in UM cell
lines, with decreased viability associated with pERK and cyclinD1 suppression
(Ambrosini, AACR 2010). Methods: Using paired metastatic tumor
biopsies from patients (pts) with radiographically progressing UM treated
with selumetinib 75 mg BID on a phase II trial (NCT01143402), we
correlated MAPK pathway inhibition with radiographic tumor regression
and clinical benefit. Biopsies were performed at baseline and after 14 �/-1
days of treatment. Western blotting was performed for pERK and cyclinD1,
and quantitated by densitometry. Response (RECIST 1.1) was assessed at
baseline, week (wk) 4, wk 8, and q8wks subsequently. Radiographic
regression was defined as greatest percentage shrinkage from baseline.
Clinical benefit was defined as RECIST response or stable disease �16wks.
Results: Paired tumor biopsies were assayed from 18 pts: median age 60
(range 47-81), M:F 11:7, median 1 prior therapy (range 0-2), 17 with liver
involvement, Gnaq mut:Gna11 mut:wild-type 8:9:1. Radiographic regression
was observed in 5 pts, with 2 achieving partial responses. 4 pts were
on study �16wks (16�, 20, 25, 31 wks), with one currently on study at
11� wks. Median pERK and cyclinD1 as measured by densitometry
decreased by 48% (p�.03) and 76% (p�.03), respectively. Radiographic
regression correlated with suppression of pERK (Spearmen’s rank correlation;
p�0.04) but not cyclinD1 (p�0.38). A trend towards pERK suppression
correlating with clinical benefit was observed (p�.07) with each of the
5 pts achieving PR or SD �16wks having a decrease of �30% in pERK
from baseline. Conclusions: Selumetinib can inhibit pERK and cyclinD1 in
UM and can result in tumor shrinkage. Sustained inhibition of pERK
inhibition at day 14 may be predictive of benefit. Further evaluation of MEK
inhibition in this disease is warranted.
8597 General Poster Session (Board #38G), Sun, 8:00 AM-12:00 PM
MicroRNAs induced in melanoma treated with combination targeted
therapy of temsirolimus and bevacizumab. Presenting Author: Aubrey G.
Wagenseller, University of Virginia, Charlottesville, VA
Background: Targeted therapies directed at commonly overexpressed pathways
in melanoma have clinical activity in numerous trials. Little is known
about how these therapies influence microRNA expression, particularly
with combination regimens. A better understanding of how microRNAs are
altered with treatment may contribute to understanding mechanisms of
therapeutic effects as well as mechanisms of tumor escape from therapy.
Methods: Using microRNA arrays, we analyzed microRNA expression levels
in melanoma samples from a Cancer Therapy Evaluation Programsponsored
phase II trial of combination temsirolimus and bevacizumab in
stage III or IV melanoma, which elicited clinical benefit in a subset of
patients. Seventeen patients were treated with temsirolimus for one week,
then combination of both temsirolimus and bevacizumab. Metastatic
melanoma biopsies were evaluated days 1, 2, and 23. Tumor samples were
evaluated from 12 patients. Results: microRNA expression remained
unchanged with temsirolimus alone; however, expression of 15 microRNAs
was significantly upregulated (1.4 to 2.5-fold) with combination treatment,
compared to pre-treatment levels. Interestingly, twelve of these fifteen
microRNAs have been reported to possess tumor suppressor capabilities in
various cancer types, including melanoma. We identified 15 putative
oncogenes and B7-H3, IGF-1, and IGF-1R as potential targets of the 12
tumor suppressor microRNAs, based on published experimental evidence.
For 18 of 25 pairings of microRNA and target-mRNA, changes in gene
expression from pretreatment to post-combination treatment samples were
inversely correlated with changes in microRNA expression, suggesting a
functional effect of the microRNA changes induced by combination
therapy. Clustering analysis based on selected microRNAs revealed signatures
characteristic of clinical response to combination treatment and of
tumor BRAF mutational status. Conclusions: We have identified microRNAs
that may be involved in the mechanism of action of combination temsirolimus
and bevacizumab in metastatic melanoma, possibly through inhibition
of oncogenic pathways.
8599 General Poster Session (Board #39A), Sun, 8:00 AM-12:00 PM
Exhibition of a stem-cell like phenotype with the expression of CD271 in
drug-resistant melanoma cells. Presenting Author: Elizabeth Ann Teresa
Lieser, Mayo Clinic, Rochester, MN
Background: Chemotherapy resistance is a common, difficult problem for
melanoma patients needing long term care and those suffering from
recurrence. Neural growth factor receptor (NGFR), also known as CD271, is
commonly expressed on mesenchymal stem cells and is important for
development, survival and differentiation of cells. This has previously
shown to be expressed on melanoma stem cells. We hypothesized that
treatment with carboplatin enriches the population of tumor cells for
cancer stem cells (CSC) as a mechanism of chemotherapy resistance.
Methods: Core tumor samples from 118 patients with metastatic melanoma
were obtained from formalin-fixed paraffin embedded specimens. Slides
were stained using an anti-CD271 antibody for IHC. A375 melanoma cells
were treated with increasing concentrations of carboplatin for approximately
1 year. WT, 6 mg/mL and 10 mg/mL resistant cells were tagged with
CD271-PE antibody and run on a flow cytometer. These cells were then
tested for gene expression by microarray using an Affymetrix human 133
plus 2.0 chip, and analyzed on Partek software. Pathway analysis of these
carboplatin resistant cells was preformed using Ingenuity. Results: Virtually
all melanoma tumor samples stained positively for CD271 by IHC, with a
median of 50% cells within tumor samples expressing the cytoplasmic
marker. Flow cytometry demonstrated an increase in the percentage of cells
expressing CD271 as resistance to carboplatin increased. Wild type A375
cells contained 30% positive CD271, A375 6mg/mL resistant contained
42% positive and A375 10mg/mL resistant showed 66% positive. Microarray
also exhibited a direct correlation between CD271 gene expression with
increasing carboplatin resistance. Functional ontology enrichment revealed
development and regulation of the EMT pathway as an important
process impacted by our resistant cell line. Conclusions: The data suggests
that as melanoma cells become resistant to certain chemotherapy drugs
they essentially loose their differentiated phenotype and become more
stem cell-like. Monitoring melanoma expression of CD271 could help
individualize therapy and anticipate chemotherapeutic resistance in this
high-risk group of patients.
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