LECTURE
Early Detection of Melanoma Progression by Quantitative
Real-time RT-PCR Analysis for Multiple Melanoma Markers
Peter Arenberger,1 Monika Arenbergerova,1 Olga Vohradnikova,1and Jaromir Kremen2

Department of Dermatovenereology, Charles University 3rd Medical Faculty,
Srobarova 50, CZ 100 34 Prague 10, Czech Republic2
Department of Chemistry and Biochemistry, Charles University 3rd Medical Faculty,
Srobarova 50, CZ 100 34 Prague 10, Czech Republic
1(Received for publication on June 18, 2007)(Revised for publication on September 28, 2007)(Accepted for publication on November 15, 2007)

Abstract: Standard screening of melanoma patients is a useful tool for predicting outcomeof patients, however, an instant methodology for exact detection of subclinical disease ormonitoring treatment response is under investigation.
Detection of circulating melanoma cells is, therefore, a possible novel promising staging
method. However, inconsistent data on method sensitivity and on the predicted patient outcome has been shown repeatedly.
Recently, a multimarker real-time RT-PCR methodology for quantification of five melanoma markers Melan-A, gp 100, MAGE-3, MIA and tyrosinase was described by our group. In
the current prospective trial, blood specimens of 65 patients with AJCC stage IIB-III cutaneous melanoma after surgery were periodically examined. In the above group, 27 % of subjects relapsed during the study. Prior to the disease progression we could observe a statistically significant tumor marker elevation in previous 0 to 9 months in all patients with clinical relapse. MAGE-3 became the most sensitive progression marker. During progression,
three concordant positive markers were seen in 39 % of patients, followed by two concordant positive markers in 28 % and 1 marker in 33 %.
This study supports the use of a multimarker real-time RT-PCR as a disease progression
predictor. The dynamic assessment of serially obtained blood specimens represents a useful
method for early metastasis detection and treatment response of melanoma patients. (Keio J
Med 57 (1) : 57－64, March 2008)
Key words: malignant melanoma, melanoma markers, circulating melanoma cells

Introduction

rapidly, however, nowadays imaging methods are able toinform about the presence of lymph node or visceral metastasis usually in an advanced stage only. Therefore,
new, improved non-invasive methods, which would enable to detect tumor cells in the blood stream of the patient, are urgently demanded. The detection of melanoma cells in the peripheral blood could serve as an important method for predicting clinical outcome of the patient.
Usually, only limited amount of tumor cells may be

Melanoma belongs to the most malignant tumors of theskin and mucous membranes due to its aggressive biological behavior and tendency to generate early metastases. The development of metastasis in lymphatic system
is described in 70 % of metastatic melanoma; the haematogenous metastazing affects usually liver, lung, brain
and bones.
The sensitivity of current diagnostic methods increases

Presented at the 1513rd Meeting of the Keio Medical Society in Tokyo, April 3, 2007
Corresponding Author: Prof. Peter Arenberger, MD, PhD, DSc, MBA, Chairman at the Dept. of Dermatovenereology, Charles University Third Medical Faculty, Srobarova 50, CZ 100 34 Prague 10, Czech Republic, E-mail: pa@avemedica.cz
57

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Arenberger P, et al: Real-time RT-PCR for blood melanoma marker detection

present in blood. Therefore, molecular biology methods
using reverse transcription (RT) followed by polymerase
chain reaction, characterized by high sensitivity andspecificity for detection of mRNAs encoding protein
have been used. These are specific for certain tumor cell
subtypes. Real-time RT-PCR methodology used in thepresent work enables even more accurate quantificationof mRNA genes.
The detection of circulating melanoma cell has been
proposed as a sensitive method for selecting patients whoare in a high risk to relapse.1, 2 However, after the original description of molecular monitoring of circulating
melanoma cells by reverse transcriptase-PCR (RT-PCR)
had been published,3 inconsistent data on sensitivity andclinical relevance of this method have been reported4-15.
Two possible reasons for the conflicting results may exist: (a) a single marker detection, (b) conventional or
semiquantitative RT-PCR assay. Explaining the firstpoint (a), heterogeneity of the expression of melanoma
antigens poses one of the major problems in detection of
circulating melanoma cells.1 Single marker detection has
a limited value as a prognostic marker and the multiple
marker assay including other tumor associated antigens
is suggested to increase the sensitivity and specificity ofthis method.1 6 Moreover, the majority of these studies
investigated tyrosinase as a unique marker, however its
usefulness as a melanoma marker is highly debated.17, 18
Objections to the second argument (b) are as follows.
There are limitations of conventional or semiquantitative
RT-PCR as a prognostic marker. In contrast, a quantitative real time PCR offers a quick and accurate approach.
It measures housekeeping gene expression and expression of melanoma associated RNA and thus allows rapid
and reproducible quantitative analysis of melanoma
markers.19, 20
Only a limited number of tumor associated markers expressed by melanoma cells specifically have been identified.
In this study, we investigated five presumablepredictive markers for primary and metastatic melanoma:
melanoma antigen recognized by T cells (Melan-A/
MART-1), melanosomal matrix protein (gp 100), melanoma antigen-A3 family (MAGE-3), melanoma inhibitory protein (MIA) and key enzyme in biosynthesis of melanin (tyrosinase). Additional serum tumor markers havebeen shown to be relevant for prognosis of patients withdifferent types of cancer.21-24
In the prospective study 65 melanoma patients stage
IIB -III were enrolled. Peripheral blood samples werecollected every three months for following 18 months
and circulating melanoma cells were examined and compared with clinical staging results. Eighteen patients relapsed during the trial and showed different types ofmelanoma progression. All these patients experienced
concomitant statistically significant tumor marker elevation in the period up-to 9 months before the disease pro-

gression. Our experience showed that MAGE-3 was themost sensitive progression marker. In patients with progression we found three concordant positive markers in
39 % of cases, two concordant positive markers in 28 %and finally one marker in 33 %.
We established a quantitative multimarker real time
RT-PCR assay, giving exact information about the levelof different markers in the blood. This method provides
information on the frequency of melanoma-associatedgene transcripts and helps to monitor their dynamics inthe blood during a certain time period. The final aim of
the present study is the routine usage of this method for
rapid screening of early metastasis and treatment response in high risk melanoma patients.
Methods
Patient selection
In total 65 patients participated in the study. The groupconsisted of 39 men and 26 women at the age of 20-75
years. The average age of the patients was 55 years.
Only patients with histologically verified resected melanoma in stage IIB-IIIC were enrolled. The stage of thedisease was determined according to the American Joint
Committee on Cancer guidelines.2 5-2 7 We selected patients after surgical resection of melanoma who weretreated during the whole study period with immunotherapy with interferon alpha 2a (3x 6 MIU subcutaneously
every week). Patients were enrolled into the study after
4 weeks of interferon alpha run-in treatment and wereclinically examined at the enrollment day and then every
three months during a period of 18 months. At the sametime the trial blood collection and complete staging were
performed.
RNA isolation from blood samples
Seven ml of peripheral blood was collected into EDTA
coated tube and the blood sample was processed within
following 2 hours. Total cellular RNA was extracted
from non-erythrocytic cellular population obtained bycentrifugation (30 min, 1200x g) using Histopaque Sigma 1119 and 1077 (2:1) gradient. For RNA isolation
High Pure RNA Isolation Kit (Roche Diagnostics GmbH,Germany) or MagNA Pure Compact (Roche Diagnostics
GmbH, Germany) device assisted methodology together
with MagNA Pure Compact RNA Isolation Kit (Roche
Diagnostics GmbH, Germany) were used. RNA quality
was determined electrophoretically in 1.5% agars gelwith ethidium bromide and quantity was checked spectrophotometrically.
Melanoma cell lines
RNA from the cell line SK-MEL 28 was kindly provided by M. Vejražka, MD., PhD., Institute of Medical Biochemistry, Prague. This cell line is expressing the panel

Keio J Med 2008; 57 (1): 57－ 64

59

Table 1 Primer and Light Cycler probes sequences
HTYR-FHTYR-RHTYR-FLHTYR-LC

Melan-se
Melan-as
Melan-FL
Melan-LC

hu TYR, mRNA
TCTTCTCCTCTTggCAgATTgTCAgTAAgTggACTAgCAAATCCTTC
FRET probe hu TYR, mRNA
CCAAggCTCCCCTCTTCAgCTg X
TgTAgAATTTTgCCTgAgTTTgACCCAAT p
Primer hu Melan A, mRNA
TggATAAAAgTCTTCATgTTggC
gTggAgCATTgggAACCAC
FRET probe hu Melan A
gCTgTCCCgATgATCAAACCCTTC X
TgTgggCATCTTCTTgTTAAggCACA p

hu Primer MAGE3, mRNA
gAgAAgATCTgCCAgTgggTCT
gCCTCCTgCTCCTCAgTAgC
FRET probe hu MAGE3
MAGE3-FL gAggCATgATgACTCTggTCAgggC X
MAGE3-LC ACAggCgggAgTgTgggCA p
MAGE3-S
MAGE3-A

gp100 Fgp100 R
gp100 FL
gp100 LC

Primer hu gp100 mRNA
AAggTCCAgATgCCAgCTCAATCAAggATCTCggCACTTTCAATACCC
FRET probe hu gp100 mRNA
AgTCCCCCTggATTgTgTTCTgTATCgA X
TggTTCCTTTTCCgTCACCCTggAC pof melanoma markers tested in the present work. RNA
obtained from the cell suspension diluted to concentration of 105 - 101 was used for sensitivity testing of thismethod.
Negative control
Blood samples from 23 healthy donors served as anegative control.
Melanoma markers examined
Expression of the following mRNA markers was examined using a quantitative real-time RT-PCR: MIA(melanoma inhibitory antigen), Melan-A/ MART-1 (melanoma antigen recognized by T cells), MAGE-3 (melanoma antigen gene-3), gp100 (melanosomal matrix glycoprotein) and hTYR (tyrosinase).
Quantitative real-time RT-PCR
Amplification of target sequences of examined markers
was performed by 2 step quantitative RT-PCR with primers and probes listed in Table 1. Primers and LightCyler
probes were designed and synthesized at TIB-MolBiol(Berlin, Germany). The first probe was labeled by fluorescein, as donor fluorophore, at the 3´-end whereas thesecond one by R-640, as acceptor fluorophore, at the 5´SA

NM_000372
1305-1326
1559-1535

Tm
56.2°C
56.3°CS
S

1430-1451
1453-1481

65.2°C
64.2°CS
A

NM_005511
226-248357-339Tm
55.9°C
57.6°CA
A

305-282
280-255

64.3°C
65.2°CSA

NM_175742
135-156322-303Tm
58.3°C58.3°CA
A

216-192
190-172

66.3°C
66.6°CS
A

NM_006928
1341-1364
1527-1504

Tm
63.6°C
61.7°CS
S

1441-1468
1471-1495

66.5°C
67.9°C-end and the 3´-end was blocked by phosphate.
In the first step, cDNA was synthesized from 2.0 µg of
total cellular RNA via First Strand cDNA Transcriptor
Synthesis Kit (Roche Diagnostics GmbH, Germany) according to producer recommended conditions, using random hexanucleotides primers. The use of random hexamer primers provides priming throughout the length ofRNA for uniform representation of all RNA sequences
and, in addition, allows reverse transcription of RNAs that
do not carry a poly(A) tail.
In the second step, 2.5 µl of marker cDNA was amplified in 10 µl of total volume of LightCycler FastStart
DNA MasterPLUS Hybridization Probes Kit (Roche Diagnostics GmbH, Germany) supplemented by above listed
primers, LightCycler probes, and LC-Uracil-N-glycosylase (Roche Diagnostics GmbH, Germany) for the selective destruction of carryover products.
For kinetic real-time PCR LightCycler Instrument(Roche Diagnostics GmbH, Germany) under following
conditions was used: 37°C/10 min incubation with LC
uracil-N-glycosylase; 95°C/10 min inactivation of uracilN-glycosylase and concomitant activation of LC Fast
TaqPolymerase; amplification 55 cycles: 95°C/10 s, 55°
C/10 s (fluorescence measurement), 72°C/13 s. The
product check was performed by Tm determination. Ex-

Arenberger P, et al: Real-time RT-PCR for blood melanoma marker detection

60

pression of each marker was compared with the PBGD
(porphobilinogen deaminase) housekeeping gene expression using LightCycler-h-PBGD Housekeeping Gene Set(Roche Diagnostics GmbH, Germany).
Data analysis
Numbers of amplified marker RNA copies/ml were related to number of housekeeping gene copies (number ofmarker copies/ml: number of PBGD copies/ml). The
statistical analysis was performed using SPSS software(release 10.0) and the Student´s t test.
Results
Study protocol
All patients had given informed consent before entering the study. In total 65 patients with resected cutaneous malignant melanoma were enrolled into the study.
Patients were stratified according to the recent American
Joint Committee on Cancer classification.25-27 We haveenrolled 33.9 % patients in stage IIA, 24.6 % patients instage IIB, 12.3 % patients in stage IIC, 15.4 % patients instage IIIA, 9.2 % patients in stage IIIB and 4.6 % patients in stage IIIC. Blood samples for detection of circulating cells were collected every three months during aperiod of 18 months. At the same time standard staging
procedures were performed: lymph node ultrasound, abdominal ultrasound and chest x-ray. Other imaging
methods were performed individually according to thespecific need of the patient.
Expression of Melan-A, gp 100, MAGE-3, MIA andtyrosinase mRNA in blood samples of tumor progression patients
Within a follow-up of 18 months, 18 out of 65 patientsdeveloped tumor progression. We could observe elevation of 1 to 3 tumor markers in all patients with progression. Subjects were members of IIA-IIIB initial stage
groups. In particular 5 patients were in stage IIA, 3 patients in stage IIB, 4 patients in stage IIC, following 4 instage IIIA and 2 in stage IIIB. Results of tumor marker
levels were compared in each patient with cutoff marker
levels measured in the healthy volunteer group.
Tumor marker level (marker/PBGD) for MIA was
0.832 ± 0.073, for Melan-A 0.012 ± 0.002, for MAGE-3
2.75 ± 0.302 and for gp100 0.057 ± 0.006. Values represent mean ± SD. Tumor marker mean levels in patientswith progression are shown in the same format and they
are as follows: MIA 4.272 ± 2.183 (statistically significant difference compared with cutoff value, p<0.01), for
Melan-A 0.026 ± 0.016 (statistically non-significant difference compared with cutoff value, p>0.05), for
MAGE-3 332.927 ± 196.239 (statistically significant difference compared with cutoff value, p<0.01) and for
gp100 0.953 ± 0.827 (statistically significant differencecompared with cutoff value, p<0.05).
Table 2 gives an overview on marker levels in all 18
patients in progression displayed as cutoff value multiples. In MIA the mean progression/cutoff ratio was 5.08,
in MAGE-3 153.07 and in gp100 16.72. Tyrosinase waspositive in 1 patient with progression and none of the patients developed significant increase of Melan-A marker
level compared to cutoff (p>0.05).
Elevation of different markers is significantly associatedwith the disease progression.
Expression of examined markers in tumor progression patients
Samples were most often positive for MAGE-3 (17 outof 18 samples, 94.4 %), followed by gp100 (10 out of 18
patients, 55.6 %), MIA (9 out of 18, 50 %) and tyrosinase(1 out of 18, 5.6 %). Melan-A showed no statisticallysignificant positive values (samples above cutoff) in patients suffering from melanoma progression (Fig. 1). In
patients with progression the most frequent number ofconcordant positive markers was three in 39 % of cases
followed by 1 marker (33 %) and finally by 2 markers in
28 %.
Correlation with clinical parameters
Tumor marker elevation was observed in 21 out of 65
patients. Eighteen patients (86 %) suffered from diseaseprogression during the 18 months follow-up as describedin Table 1 in detail. All these patients with progression
from our group showed elevation of single marker orconcordant elevation of 2 or 3 markers. Three more patients (14 %) displayed statistically significant elevatedmarkers above the cutoff level without clinical signs ofprogression up-to now.
Elevation of the tumor markers preceded clinical progression up-to 9 months
We could observe the elevation of the tumor markers inmost cases much earlier before the first clinical signs ofprogression. According to the protocol, blood samples
were examined every 3 months for the detection of circulating cells and at the same time imaging staging methods were carried out. We could observe an elevation of
the markers 9 months in advance in 28 % of the patients,
6 months in advance in 38 % of the patients, 3 months inadvance in 17 % of the patients and only 17 % of patients displayed elevation of the markers at the same time
as clinical progression occurred.
Discussion
The AJCC classification25-27 plays an important role inthe determination of the melanoma patient prognosis andhis life expectancy. Lower AJCC index signifies a better
prognosis for the patient, whereas higher index means aworsened prognosis. Although the sensitivity of diag-Keio J Med 2008; 57 (1): 57－ 64

61

Fig. 1 Expression of examined tumor markers in patients with melanoma progression. MAGE-3 was the most frequent positivemarker (17x) in this group, followed by gp100 (10x), MIA (9x) and tyrosinase (1x). Melan-A did not show any significant elevationcompared with cutoff.

nostic methods is increasing rapidly, contemporary imaging methods are able to inform us about the disease onlyin its advanced stage. Therefore, we are permanently
searching for a method, which would be able to detecttumor cells in the blood stream of the patient. Detectionof circulating tumor cells is an attractive assessment approach because tumor cells in peripheral blood are essential for the formation of metastatic lesions and because
this analysis can depict real-time tumor status.
Evaluation of studies with serum markers such as lactate dehydrogenase (LDH), S100 protein or neuron-specific enolase (NSE) could show that these serologic tumor markers are not sensitive enough for an early detection of progression or therapy efficacy control.28
In this study, we could demonstrate the uselfulness ofmultimarker real-time RT-PCR as a sensitive and specificquantitative assay to detect circulating melanoma cells inthe blood. The quantitative real-time RT-PCR enables toquickly analyze large number of samples and reduces therisk of carryover contamination. Moreover, this method
offers accurate and reproducible mRNA gene quantification.
Every three months we detected melanoma markers
from peripheral blood using this methodology. During
18 months of the study run, melanoma markers significantly increased above the cutoff line in 21 patients. In
18 out of 21 patients (86 %) the clinical progression of
the disease occured also during the study run. Three patients of the above mentioned group (16 %) showed elevated marker levels but this increase was not linked tothe disease progression. The explanation for these threepatients could be a false positive marker level result or a
prolonged period between the marker level elevation anda clinical metastasis onset. All three patients are still
within the window of less than 9 months from the marker
level increase, which means that according to our knowledge we can, unfortunately, nearly expect a clinical progression of the disease. These patients are under detailed
frequent follow-up supervision. In the meantime, prostata cancer was developed by one of these patients.
In general, the elevation of the tumor marker levels occured in the majority of cases significantly earlier compared with the clinical progression. We could demonstrate that the earliest marker elevation happened 9months before the disease progression in 28 % of patients, 6 months before in 38 %, 3 months before in 17 %and at the same time matching with the progression diagnosed by clinical and/or imaging method in another 17 %.
Thus, elevation of tumor markers can be considered as
an early method for haematogenous spread and development of metastasis.
Melanoma tumors are heterogenous in tumor-associated marker expression. Heterogeneity of melanoma
marker expression in blood makes multimarker RT-PCR
advantageous compared to single-marker detection of
circulating melanoma cells.
The real-time RT-PCR assay has high throughput capacity that can analyze large number of samples without
PCR product carryover contamination. It is a very accurate and reproducible method. This assay system can detect melanoma cells among millions of peripheral blood
leucocytes without requiring cell-separation methods

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Arenberger P, et al: Real-time RT-PCR for blood melanoma marker detection
Table 2 Elevation of melanoma markers in 18 out of 65 patients with clinical progression.
Numbers listed in the table express elevation of markers as multiplicates of cutoff value.
Patient Initial MIA
Melan-A
MAGE-3
gp 100tyrosinase
Type ofNo.
stage progression / progression / progression / progression / progression / progression
cutoffcutoffcutoffcutoffcutoff(metastases)
1
IIB
13.3
normal1019.4normalnormalsatellite
2
IIA
6.4
normal40580
normalnormalregional
lymph node
6
IIA
normalnormal257.5
normalnormalsatellite
7
IIC
3.3
normal5857
normalnormalregional
lymph node
8
IIIA 109.6
normal23241
normalnormalregional
lymph node
10
IIC
2.3
normal3.3
88.0
normalliver
14
IIIB 2.2
normal6.1
normalnormalkidney,lung
17
IIB
31.9
normal623
93.1
normalin-transit
20
IIC
normalnormal
19516
normalnormalskin, brain
21
IIC
30.5
normal438
normalnormalbrain
24
IIA
normalnormal113179398.7
normalregional
lymph node
25
IIIA 7.7
normal5664
5.2
normalregional
lymph node
28
IIB
8.0
normal105.6
normalnormal
liver
35
IIA
1.5
normal36.4
normalnormalbrain
44
IIIB 3.7
normal25833
normalnormalregional
lymph node
58
IIIA 2.5
normalnormal
16.2
positive
peritoneum
60
IIA
11.9
normal
11673
normalnormaleye, satellite
62
IIIA normal
normal318
normalnormalregional
lymph node,
satellite

such as magnetic beads, separation medium or other approaches.29
In our study we concentrated on 5 melanoma markers:
Melan-A/MART-1, gp 100, MAGE-3, MIA a tyrosinase.
MAGE-3 was identified as the most sensitive progression marker (elevation in 17 out of 18 patients), followed
by gp 100 (10 out of 18), MIA (9 out of 18) and tyrosinase with 1 elevation. In Melan-A no statistically significant increase compared with cutoff for all studied patients experiencing progression was shown. In the past,tyrosinase was used as a usual and single marker for detection of circulating cells. Its role as a melanoma marker is highly debated and explanations vary in different
papers. In published studies using tyrosinase as a solemarker, the range of patients found to be positive for circulating melanoma cells varied from 6 % to 59 %.17, 30-33
We could speculate about the technical mistake in thepreviously published papers, i.e. high percentage of false
positive results because of e.g. contamination reasons ortyrosinase down-regulation in advanced stages, which ischaracterized by reduction of tumor cell differentiationand low content of melanin in the tumor cells.34 In contrast, in our study we found most frequently three concomitantly positive markers and, therefore, we can dem-

onstrate the multimarker real-time RT-PCR as useful andsensitive one.
As an interesting fact we can note that the tumor marker elevation in the peripheral blood was connected not
only to the haematogenous spread, but also to the development of lymphogenic metastasis. This finding can explain the fact that the presence of lymph node metastasis
significantly reduces an overall survival.
In our study, tumor markers were examined every threemonths. This interval is important to get the marker elevation data as soon as possible before the onset of progression. The question is, whether 3 month interval may
be considered as optimal, or too short or too long. Important limitation is as well the financial aspect of theexamination and technical difficulties concerning the patient. The RNA isolation has to be carried out two hours
after the blood collection. This means that the patient
has to travel into the study centre. On the other hand, theoptimal examination frequency between two blood collections from the medical point of view should be very
short.
Repeated monitoring of tumor marker levels is important also for another reason. In some of the blood collections in previously positive patients, tumor cells do notKeio J Med 2008; 57 (1): 57－ 64

appear. This is what we could observe also in two patients who experienced clinical progression. In both ofthem we found 4 positive and 1 negative from 5 blood
samples (in the first patient: No.1 pos., No.2 pos., No.3
neg., No.4 pos., No.5 pos. and in the second patient
No.1 pos., No.2 neg., No.3 pos., No.4 pos., No.5 pos.).
We consider the negative value as false negative. There
are several reasons for this observation: non-homogenous
distribution of tumor cells in the blood stream, direct orindirect adjuvant immunotherapy influence on tumor
marker level, variable time dependent biological behavior of the tumor tissue, and many others.35, 36 Our experience showed that 3-month intervals are just a well-balanced option considering the financial aspect on one
hand and data quality on the other hand.
It seems that circulating melanoma cell detection
should be a useful tool to quality enhancement of the diagnostic and follow-up process in malignant melanoma
patients which could be confirmed not only by our results but also by the recent meta-analysis study with
5433 patients and 53 studies.37 However, this meta-analysis points out the heterogeneity of the studies cited and,
therefore, it is of great importance to use a standard
methodology in the future.
In conclusion, the multimarker quantitative real-time
RT-PCR used in the present study is a sensitive method
for the detection of circulating melanoma cells. It could
serve as a useful tool for early detection of metastatic
process. This laboratory methodology can possibly open
new insights into the principles of tumor microenvironment and metastasizing.
We presume that results of our study may lead to theroutine usage of this method for rapid screening of risk
patients, and a better insights in the monitoring of the effects of adjuvant therapy in the near future. Evaluationof larger patient cohorts with longer follow-up should beof particular advantages to confirm the ability of realtime RT-PCR assay to predict the prognosis of melanoma
patients.
Acknowledgements
This work was supported by the IGA 1A/8243-3 Czech
Ministry of Health grant.

63

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