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Role of Elevated PHIP Copy Number as a Prognostic and Progression Marker for
Cutaneous Melanoma
Vladimir Bezrookove 1, Mehdi Nosrati 1, James R. Miller III 1, David De Semir 1, Altaf
A. Dar 1, Elham Vosoughi 1, Edith Vaquero 1, Antje Sucker 3, Alexander J. Lazar 2,
Jeffrey E. Gershenwald 2, Michael A. Davies 2, Dirk Schadendorf 3, Mohammed
Kashani-Sabet 1*
1 Center for Melanoma Research and Treatment, California Pacific Medical Center
(CPMC) Research Institute, San Francisco, CA; 2 Departments of Pathology, Genomic
Medicine, Dermatology & Translational Molecular Pathology, The University of Texas
MD Anderson Cancer Center, Houston, TX; Department of Dermatology, 3 University of
Duisburg-Essen, Essen, Germany, and German Cancer Consortium, Heidelberg,
Germany.
Running title: PHIP copy number in melanoma progression
* Corresponding Author: Mohammed Kashani-Sabet, M.D., California Pacific Medical Center Research Institute, 475 Brannan Street, Suite 130, San Francisco, CA, 94107. Phone: (415) 600-3166, Fax: (415) 600-1719; Email: [email protected] Conflicts of interest: J.R.M. has ownership interests (including patents) at MDMS LLC. J.E.G. has served on advisory boards for Merck, Syndax, and Castle Biosciences. M.A.D. has served on advisory boards for Bristol-Myers Squibb, Roche/Genentech, Novartis, Sanofi-Aventis, Syndax, and Vaccinex, as a consultant for Nanostring (without compensation), and has received grant support from Bristol-Myers Squibb, Roche/Genentech, GSK, Sanofi-Aventis, Oncothyreon, Merck, and Astra Zeneca. D.S. has served on advisory boards for and received honoraria from Bristol-Myers Squibb, Roche, Merck, Incyte, Amgen, Novartis, and EMD. M.K.S. has ownership interest in Melanoma Diagnostics, has received honoraria from Cepheid, and has received grant support from Merck. All other authors declare no conflicts.
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Translational Relevance This manuscript presents a comprehensive analysis of the role of PHIP copy number in
sequential stages of melanoma progression. We confirm that elevated PHIP copy number
is an independent prognostic marker for primary cutaneous melanoma. In addition, we
identify distinct molecular subtypes of melanoma in which PHIP is enriched. Finally, we
show that the percentage of melanoma cells with elevated PHIP copy number is
increased in the progression from primary to metastatic melanoma. Taken together, these
studies describe a key role for PHIP in melanoma progression, confirming it as a
promising target for therapy.
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Abstract
Purpose: Previous studies have indicated an important role for pleckstrin homology
domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis.
Here we aimed to confirm the role of PHIP copy number in successive stages of
melanoma progression.
Experimental Design: PHIP copy number was examined using fluorescence in situ
hybridization (FISH) in three independent cohorts by recording the percentage of cells
harboring > 3 copies of PHIP. The impact of PHIP copy number on survival was
assessed using Cox regression analysis. The enrichment of PHIP was assessed in various
molecular melanoma subtypes. PHIP expression was analyzed in The Cancer Genome
Atlas (TCGA) melanoma cohort.
Results: Elevated PHIP copy number was significantly predictive of reduced distant
metastasis-free survival (DMFS) and disease-specific survival (DSS), and increased
prevalence of ulceration in primary melanoma (cohort #1). By multivariate analysis,
PHIP FISH scores were independently predictive of DMFS and DSS. PHIP copy number
was enriched in metastatic melanomas harboring mutant NRAS or expressing PTEN
protein (cohort #2). PHIP copy number was significantly elevated in metastatic
melanomas when compared with matched primary tumors from the same patient (cohort
#3). Several of these associations were replicated using TCGA cohort analysis.
Conclusions: These results underscore the important role of PHIP copy number
elevation in melanoma progression, and identify molecular subtypes of melanoma in
which PHIP is enriched. Finally, as elevated PHIP copy number appears to be selected
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for during the progression of primary to metastatic melanoma, these results confirm PHIP
as a promising therapeutic target for melanoma.
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Introduction
Melanoma represents the fifth most common malignancy in the United States (1).
As the clinical behavior of melanoma is unpredictable, the development of prognostic
markers has represented an important area of research. Histological prognostic factors are
the mainstay of prognostic assessment for primary cutaneous melanoma (2). While many
factors may play a role in refining melanoma prognosis, tumor thickness and ulceration
are the only factors currently incorporated within the melanoma staging classification for
primary melanoma (3).
Molecular factors represent the next frontier in prognostic factor research. The
development of molecular prognostic factors for melanoma has been hampered largely by
the lack of sufficient validation of promising markers in independent tissue sets. In
addition, the role of a putative marker in melanoma progression has rarely been explored
in the context of the same patient. Finally, given the identification of dominant
mutational drivers of melanoma, information regarding the role of a given marker in the
context of the known mutational landscape of melanoma would be important (4). As a
result, no molecular markers are routinely utilized in the prognostic assessment of
melanoma patients, and none are incorporated into its staging classification.
Our previous studies identified both a functional and a biomarker role for PHIP in
melanoma progression, and as a molecular prognostic factor for melanoma. PHIP was
identified as the top gene overexpressed in melanoma metastases when compared with
primary tumors using cDNA microarray analysis (5). Subsequent studies showed an
independent prognostic role for PHIP in primary melanoma (6,7). Functional studies
demonstrated that increasing PHIP expression enhanced the distant metastatic potential
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of melanoma (6,7). Here, we aimed to confirm the prognostic role of PHIP in primary
melanoma, to identify the molecular subtypes of melanoma in which PHIP is enriched,
and to determine whether PHIP is selected for during the progression from primary to
metastatic melanoma.
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Materials and Methods
Study population
We developed three non-overlapping patient cohorts to assess PHIP’s biomarker role: (i)
cohort #1- unstained primary melanoma tissue sections obtained from 204 stage I/II
patients in the German Cancer Consortium (see Table 1 for characteristics); (ii) cohort
#2- tissue microarrays (TMA) from a previously described cohort of 130 stage III
melanoma patients in the MD Anderson Cancer Center (8); and (iii) cohort #3- 37
matched primary and metastatic melanomas obtained from the same patient (N=15) who
developed both regional lymph node and distant metastasis, identified from the CPMC
Center for Melanoma Research and Treatment archives (see Supplemental Table 1 for
characteristics). In cohort #3, 21 samples were from 7 patients with triple-matched
primary melanoma, regional lymph node and distant metastasis, and 16 samples were
from 8 patients where the primary melanoma was matched either with a regional lymph
node or distant metastasis. The mean follow up of cohort #1 was 4.58 years. Information
regarding mitotic rate was not available in cohort #1. These analyses were complemented
by analysis of PHIP expression levels obtained from TCGA (4), available from 66
primary tumors and 264 metastases. The investigations described here were conducted in
accordance with recognized ethical guidelines (i.e., Declaration of Helsinki), were
approved by the Sutter Health Institutional Review Board, and informed consent was
obtained from each subject.
FISH analysis
FISH for PHIP copy number was performed as previously described (6,7) using bacterial
artificial chromosome (BAC) clones RP11-767O1and CTD-2297E14 to detect the PHIP
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locus and clones RP11-26M18 and RP11-136K2 to detect 6q11.1 and 6p11.1,
respectively (interpreted as chromosome 6 centromere). FISH for PTEN copy number
was performed using BAC clones RP11-79A15 and RP11-813O3. For a chromosome 10-
specific probe, BAC clone RP11-96F8 was used to detect 10p11.1 (all BAC clones were
obtained from the Children's Hospital Oakland Research Institute). The quality and
mapping of all probes were verified by hybridization to normal metaphase spreads in
combination with a commercially available centromeric probe for chromosomes 6 and 10
(Open Biosystems, Lafayette, CO) before tissue analysis. Z-stacked images were
acquired using a Zeiss Axio Image Z2 microscope controlled by AxioVision software
(Zeiss, Jena, Germany). At least 30 nuclei from each case were evaluated, and the signals
were interpreted according to guidelines described previously (9). Signals from BAC
clones detecting 6q11.1, 6p11.1 and 10p11.1 were interpreted as the centromeric signal
for chromosome 6 and 10, respectively.
Statistical Analysis
The impact of elevated PHIP copy number on melanoma progression was analyzed in
several ways. In cohort #1, consistent with our previous analysis (6), elevated PHIP copy
number was measured by recording the percentage of cells harboring 3 or more copies of
the PHIP locus. The cut-point utilized to define high PHIP copy number was that which
maximized the average of sensitivity and specificity for predicting DSS (determined to be
greater than or equal to 19%). The same cut-point was uniformly applied to analyses of
DMFS and ulceration status. Subsequently, a previously identified cut-point for PHIP
copy number (greater than or equal to 16%) (6) was applied to the analysis of DMFS,
DSS, and ulceration in this cohort. The association between elevated PHIP copy number
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and ulceration status was determined using logistic regression analysis. The association
between elevated PHIP copy number and survival outcomes (i.e., DMFS and DSS) was
determined using Kaplan-Meier analysis, and univariate and multivariate Cox regression
analyses. In cohort #2, the enrichment of PHIP in various molecular subtypes of
melanoma was assessed using the T test or the one-way analysis of variance (ANOVA).
In cohort #3, for the analysis of 7 triple-matched tissue specimens, the significance of
monotonically increasing PHIP copy number in melanoma progression was assessed
using the Friedman two-way analysis of variance by ranks test. The significance of
increasing PHIP copy number between primary and metastatic melanoma in the 8
double-matched specimens was assessed using the binomial sign test. As a control,
identical analyses were performed for PTEN copy number in the same 15-patient
matched cohort. Except for the directional analyses just described, all P values reported
are two-sided.
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Results
In this study, we aimed to perform a comprehensive analysis of the role of PHIP
levels in distinct, sequential stages of melanoma progression, and to confirm its role as a
prognostic marker for primary melanoma. Initially, we aimed to replicate the prognostic
role of elevated PHIP copy number (assessed as percentage of cells harboring 3 or more
copies of PHIP) in primary melanoma in cohort #1. We assessed the potential association
between PHIP copy number and survival using univariate Cox regression. Increasing
PHIP FISH scores (as a continuous variable, using the entire scale) were significantly
predictive of distant metastasis-free survival (DMFS, P<0.03) and disease-specific
survival (DSS, P=0.025). By Kaplan-Meier analysis, high PHIP scores (assessed as a
dichotomous variable) were also significantly predictive of DMFS (P=0.0017, Fig. 2A)
and DSS (P=0.0002, Fig. 2B). In addition, we assessed the association between PHIP
FISH scores and both tumor thickness and ulceration status (6). Increasing PHIP copy
number (using the entire scale) was associated separately with increased tumor thickness
(P=0.0001) and with a higher prevalence of ulceration (P<0.0001) by univariate logistic
regression analysis. High PHIP FISH scores (assessed as a dichotomous variable) were
significantly associated with increased prevalence of ulceration (P<0.00005).
We then assessed the independent impact of elevated PHIP copy number on
survival associated with melanoma using multivariate stepwise Cox regression analysis
with backward elimination. We included in the model the following covariates included
in the legacy prognostic analyses performed by the AJCC melanoma committee (10):
tumor thickness (assessed by T category), ulceration status, Clark level, patient age,
patient gender, and tumor site. In the analysis of DMFS, elevated PHIP copy number (as
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a dichotomous variable) emerged as significantly predictive of survival (P=0.025), along
with tumor thickness and gender (Table 2). In the analysis of DSS, PHIP FISH scores
were also significantly predictive of survival (P=0.01), along with tumor thickness (Table
2). Thus, elevated PHIP copy number was independently predictive of both DMFS and
DSS, replicating our original analysis (6).
In addition, given that the cut-point identified in this analysis (19%) was very
close to that identified in our original analysis (16%) (6), we examined whether use of the
previously-identified cut-point would be predictive of outcome in cohort #1. High PHIP
scores using the original cut-point were also significantly predictive of DMFS (P=0.03),
DSS (P=0.0069), and ulceration status (P<0.00005) by univariate analysis.
Next, we assessed the role of PHIP in melanoma nodal metastases in cohort #2.
We initially assessed the potential prognostic role of elevated PHIP copy number in this
cohort. There was no significant association between PHIP copy number (assessed as a
continuous variable) in metastatic tumors and survival in stage III melanoma. The dataset
from this cohort contained information regarding three important molecular markers in
melanoma: BRAF and NRAS mutation status, and PTEN expression level (8). Our
analysis identified a significant association between elevated PHIP copy number and
NRAS mutation status in melanoma. The mean percentage of melanoma cells harboring >
3 copies of PHIP was 45.7% in NRAS-mutant melanomas, compared with 19.3% in
BRAF-mutant melanomas, and 15.4% in BRAF/NRAS-wild-type melanomas to
(P<0.00005, ANOVA, Fig. 3A). Separately, our analysis indicated that PHIP copy
number was significantly higher in melanomas with intact PTEN protein expression
versus those with PTEN loss. Specifically, the mean percentage of melanoma cells
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harboring > 3 copies of PHIP increased from 13.5% in melanomas with PTEN loss to
22.6% in PTEN-expressing melanomas (P=0.004, T test, Fig. 3B). In addition, in BRAF-
mutant melanomas, there was a trend toward increased PHIP copy number in PTEN-
expressing melanomas vs. those with PTEN loss (P=0.086, T test, Fig. 3C). Finally, cases
with a mean PHIP copy number of > 3 were present in each molecular subtype of
melanoma investigated, including in wild-type BRAF/wild-type NRAS/PTEN-expressing
melanomas. Taken together, these results indicate that PHIP levels are enriched in NRAS-
mutant and PTEN-expressing melanoma, and that PHIP copy number elevations are
observed in both BRAF-mutant and BRAF wild-type/ NRAS wild-type/PTEN-expressing
melanomas.
Subsequently, we assessed the role of PHIP as a progression marker in
melanoma. We performed FISH analysis of PHIP copy number on a matched cohort of
15 patients from whom the primary tumor and at least one metastatic tumor were
available (cohort #3, Supplemental Table 1). Among the 7 triple-matched patients (in
whom the primary tumor, a lymph node metastasis, and a distant metastasis were
available for analysis), PHIP copy number increased monotonically in the transition from
primary to lymph node to distant metastasis in 4 patients (Fig. 4). In two patients, the
lymph node metastasis had a lower copy number than the primary tumor, but the distant
metastasis had a higher copy number than both the primary tumor and the lymph node
metastasis. In one patient, the lymph node metastasis had a slightly higher copy number
than the distant metastasis, but both the lymph node and distant metastasis had higher
copy numbers than the primary tumor. There was a significant increase in PHIP copy
number in the transition from primary tumor to lymph node to distant metastasis
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(P=0.008, Friedman test). The mean percentage of cells with elevated PHIP copy number
increased monotonically from 13.5% in the primary tumor to 21.6% in the lymph node
metastases, and to 55.5% in the distant metastases. In the matched-pair analysis, for each
of the 8 cases, PHIP copy number was higher in the metastatic lesion when compared to
the corresponding primary tumor (P=0.0039, binomial sign test, Fig. 4). To determine
whether this systematic change was unique to PHIP, we performed an identical analysis
of PTEN copy number on the same matched-specimen cohort. There was no significant
change in PTEN copy number in the transition from primary tumors to either lymph node
or distant metastases or both (Supplemental Fig. 1). By comparison, the mean percentage
of cells with elevated PTEN copy number was 9.1% in the primary tumor, 12.7% in the
lymph node metastases, and 11.8% in the distant metastases (P=0.19, Friedman test
applied to the same 7 matched triplets).
Finally, we examined several of these associations by analyzing PHIP transcript
levels in the TCGA cutaneous melanoma cohort (4). Increasing PHIP expression
(assessed as a continuous variable) in the primary tumor was associated with significantly
reduced relapse-free (RFS, P=0.02) and overall survival (OS, P=0.018) by univariate Cox
regression analysis. By multivariate analysis, PHIP expression levels remained
significantly predictive of RFS (P<0.05) and OS (P=0.015), even with the inclusion of
AJCC stage. Similar to the FISH analysis, there was no significant association between
increasing PHIP expression levels in metastatic tumors and survival. In addition, mean
PHIP expression levels were significantly (4.3-fold) higher in NRAS-mutant melanomas
when compared with NRAS-wild-type cases (P=0.02, Mann-Whitney test; Supplemental
Fig. 2A). Finally, mean PHIP transcript levels were significantly (3.7-fold) higher in
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melanoma metastases when compared to primary tumors (P<0.00005; Supplemental Fig.
2B).
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Discussion
We have performed a rigorous analysis to elucidate the role of PHIP copy
number elevation in sequential stages of melanoma progression, and as a prognostic
marker for primary melanoma. We analyzed the role of PHIP copy number in three
independent patient cohorts from different centers. In cohort #1, we show that elevated
PHIP copy number is an independent prognostic marker for primary cutaneous
melanoma in a cohort of patients derived from a distinct population in Germany, and
confirm a significant association with ulceration status, an important prognostic factor
incorporated into the AJCC melanoma staging system. In cohort #2, we identify distinct
molecular subtypes of melanoma in which PHIP is enriched. In cohort #3, we determine
that the degree of PHIP copy number elevation is uniformly increased in the progression
from primary to metastatic melanoma. Several of the key findings from these three
cohorts were replicated using TCGA cohort analysis.
The independent prognostic role of PHIP levels in primary cutaneous melanoma
has now been shown using FISH analysis in two distinct cohorts, replicated at the protein
level using immunohistochemical analysis (7), and replicated at the RNA level using
TCGA cohort analysis. In addition, our prior studies demonstrated a significant
correlation between PHIP copy number and expression in primary melanoma (6). There
are few (if any) molecular markers whose prognostic impact has been demonstrated both
in distinct cohorts of melanoma patients and using several distinct platforms. These
studies advance PHIP as an important molecular marker of melanoma outcome, though
additional studies in larger cohorts will be required in order for it be routinely used as a
prognostic factor.
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Beyond its independent prognostic role, our studies of cohort #1 confirmed a
powerful association between PHIP copy number and ulceration. While the prognostic
significance of ulceration is well appreciated, the biologic basis for ulceration
development in melanoma has been poorly understood. Our previous studies showed that
PHIP mediates some of its effects on melanoma metastasis by virtue of activation of the
IGF1R-PI3K pathway. PHIP is a potent activator of AKT (7), which drives both glucose
metabolism and angiogenesis (11,12). Our studies in melanoma cell lines showed that
PHIP gene silencing results in decreased glycolysis (including reduced LDH expression)
in vitro and decreased angiogenesis in vivo (6). These results are consistent with a model
in which PHIP activation, in part through elevated copy number, promotes glycolysis and
angiogenesis, resulting in increased risk of development of ulceration and distant
metastasis. Intriguingly, this model suggests that a common signaling pathway may
underlie the development of and explain the biological basis of ulceration, a key
prognostic marker in stage I-III melanoma, and LDH levels, a prognostic marker for
stage IV disease.
In addition, our analyses of cohort #2 identified the molecular subtypes of
melanoma in which PHIP copy number elevations are present, and in which they are
specifically enriched. Elevated PHIP copy number was present in each molecular subtype
of melanoma examined, including melanomas with BRAF mutation, NRAS mutation, as
well as in melanomas without any alterations in these markers. Moreover, we found that
PHIP copy number was significantly higher in melanomas with NRAS mutation or with
intact PTEN expression. The observation that PHIP participates in the IGF1R-PI3K
signal transduction pathway in which PTEN also operates provides a rational basis for
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these results, suggesting that either molecular alteration (PHIP copy number gain or
PTEN loss) may be sufficient to activate this pathway in melanoma. Given the
importance of PTEN loss specifically in BRAF-mutant melanomas (4), our analysis also
identified a trend toward higher PHIP copy number in PTEN-expressing BRAF-mutant
tumors, introducing PHIP as a novel potential driver of this molecular subtype of
melanoma. An intriguing and unexpected finding was the enrichment of PHIP copy
number in NRAS-mutant melanoma. Given that NRAS mutations are thought to activate
both the MAPK and PI3K pathways, our results suggest the intriguing possibility of
novel biochemical functions of PHIP beyond its participation in the IGF1R-PI3K axis.
However, our results are consistent with the observation that PTEN loss is essentially
mutually exclusive with NRAS mutation. In addition, they suggest PHIP targeting as a
potential strategy for the treatment of NRAS-mutant melanoma, which has represented an
important therapeutic challenge given the modest activity of MEK inhibitors (13). Recent
studies describing the development of a small molecular inhibitor of PHIP (14)
demonstrate its druggability, thereby highlighting its role as a potential therapeutic target.
Our studies of cohort #3 analyzed the role of PHIP in the progression cascade of
melanoma by comparing copy number changes in matched primary versus metastatic
tumors from the same patient. Remarkably, the matched-specimen analysis revealed
elevated PHIP copy number in at least one metastatic lesion in every case analyzed when
compared with its corresponding primary tumor. In many instances, the copy number in
the metastasis was extremely high, with three or more copies of PHIP observed in greater
than 50% of cells in 40% of the metastases, including 56% of distant metastases
examined. In addition, in the majority of cases where we had access to the primary tumor,
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and both lymph node and distant metastases, there was a monotonic increase in PHIP
copy number elevation in the transition from primary melanoma to lymph node
metastasis, and then to distant metastasis. These results strongly suggest that elevated
PHIP copy number is selected for in melanoma progression, supporting our original
cDNA microarray analysis in which PHIP was identified, and which was further
supported by TCGA cohort analysis, showing significantly increased PHIP expression in
metastatic versus (unmatched) primary melanomas. We are unaware of any similar
studies analyzing the role of a given molecular marker in successive stages of melanoma
progression using tissues from the same patient. Similarly, a recent analysis of colon
cancer assessed the somatic variants in hypermutable DNA regions in a cohort of 19
patients with primary colon cancer and matched lymph node and distant metastasis,
enabling a conclusion that two-thirds of distant metastases have a profile distinct from
that of the lymph node metastasis (15). Our analysis underscores the potency of these
matched-tissue comparisons to determine the role of a given gene in cancer progression
within individual patients.
In conclusion, our studies to date have shown that increased PHIP levels are
predictive of melanoma distant metastasis, promote distant metastasis in mouse models of
melanoma, and are enriched in melanoma metastases. The presence of elevated PHIP
copy number in distant melanoma metastases as shown in this study indicates that this
molecular target is present and enriched in the setting of advanced melanoma. Taken
together with functional studies demonstrating an important role for PHIP in promoting
melanoma progression, these studies describe an unprecedented role for PHIP in
melanoma progression, suggesting it as a promising target for therapy.
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Acknowledgments
This work by supported by a grant from the National Institutes of Health
(R01CA175768) to MKS. The collection of the tissue set at MD Anderson was supported
by the following: The Melanoma Specialized Program of Research Excellence of the
University of Texas MD Anderson Cancer Center (P50 CA093459); and philanthropic
contributions from the Dr. Miriam and Sheldon G. Adelson Medical Research
Foundation, the AIM at Melanoma Foundation, and the MD Anderson Melanoma Moon
Shot Program.
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Figure Legends
Fig. 1. PHIP copy number analysis by FISH. Representative images of melanoma
samples with normal (panel A) versus elevated (panel B) PHIP copy number as detected
using probes for PHIP (red) and centromere of chromosome 6 (green). Scale bars, 20 μm.
Fig. 2. Kaplan-Meier analysis of the prognostic significance of PHIP copy number in
cohort #1. Analysis of DMFS (panel A) and DSS (panel B) in patients with low PHIP
copy number (curve 1) versus patients with high PHIP copy number (curve 2).
Fig. 3. Comparison of PHIP copy number in different molecular subgroups of melanoma
in cohort #2. (A) Box plots showing PHIP copy number in melanomas with wild-type
BRAF/NRAS vs. mutant BRAF or mutant NRAS. (B) Box plots showing PHIP copy
number in melanomas expressing PTEN protein versus melanomas with PTEN loss. (C)
Box plots showing PHIP copy number in BRAF-mutant melanomas expressing PTEN
protein versus melanomas with PTEN loss.
Fig. 4. PHIP copy number in a matched cohort of 15 patients, including the primary and
at least one metastatic tumor (cohort #3). Bar graphs present the percentage of cells with
at least 3 copies of PHIP.
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Table 1. Characteristics of cohort #1 (N=204) Male gender 111 (54.4%)
Age over 50 (years) 155 (76.0%)
Ulceration present 53 (26.0%)
T category
T1 56 (27.5%)
T2 51 (25.0%)
T3 59 (28.9%)
T4 30 (14.7%)
Unknown 8 (3.9%)
Median thickness 1.85 mm
Clark level
II/III 84 (41.2%)
IV/V 100 (49.0%)
Unknown 20 (9.8%)
Tumor site
Trunk 100 (49.0%)
Extremity 74 (36.3%)
Head and neck 24 (11.8%)
Unknown 6 (2.9%)
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Table 2. Results of stepwise multivariate Cox regression analysis of the impact of various
factors on DMFS and DSS
DMFS
Covariate Risk ratio P value
Tumor thickness 2.88 < 0.0001
Gender 3.19 0.0045
PHIP copy number 1.51 0.025
DSS
Covariate Risk ratio P value
Tumor thickness 2.61 < 0.0001
PHIP copy number 1.68 0.01
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A
B
Figure 1
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A
B
Group 1Group 2
0.5 1 2.5 510665
10162
8545
4420
Number at risk by group
0 5 10 15 20
21
50
100
Surv
ival
Fra
ctio
n (%
)
0
Group 1Group 2
0.5 1 2 410259
9851
8744
6321
Elapsed Time (Years) Number at risk by group 8
278
0 5 10 15 20
21
50
100
Surv
ival
Fra
ctio
n (%
)
0p = 0.0017
p = 0.0002
Figure 2
Elapsed Time (Years)
Years
Years
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Figure 3
A
5
10
15
0
55
20
25
30
35
40
45
50
60
65
PTEN lossPTEN present
B
% c
ells
with
≥3
copi
es
WT
5
10
15
0
55
20
25
30
35
40
45
50
65
60
% c
ells
with
≥3
copi
es
BRAF MutantNRAS Mutant
C
5
10
15
0
55
20
25
30
35
40
45
50
60
65
PTEN lossPTEN present
% c
ells
with
≥3
copi
es
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0102030405060708090
100
1 a
1 b
1 c
2 a
2 b
2 c
3 a
3 b
3 c
4 a
4 b
4 c
5 a
5 b
5 c
6 a
6 b
6 c
7 a
7 b
7 c
8 a
8 b
9 a
9 b
10 a
10 b
11 a
11 b
12 a
12 b
13 a
13 b
14 a
14 b
15 a
15 b
Figure 4
Primary tumorRegional lymph node metastasisDistant metastasis
Matched triplets Matched pairs
% c
ells
with
≥3
copi
es
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Published OnlineFirst May 18, 2018.Clin Cancer Res Vladimir Bezrookove, Mehdi Nosrati, James R. Miller, et al. Progression Marker for Cutaneous MelanomaRole of Elevated PHIP Copy Number as a Prognostic and
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