A CAJM  investigation of the Effects of Inoculated and Intralymphatic Vaccinia Virus on Primary and Secondary Deposits of Malignant Melanoma.The six patients included in this investigation were selected on the basis of histologically-proven malignant melanomas of the lower extremity, with either local intradermal metastases or regional lymph node involvement. If unvaccinated in the previous five years, vaccination was performed at least 14 days before using the virus therapeutically.
Vaccinia virus was prepared from stock vacccine obtained from the South African Institute for Medical Research. Virus was inoculated on the Chorio-allantoic membrane of 12 day embryonated hens’ eggs in which growth was confluent on the third day. Virus was extracted in buffered saline, cleared and tested for sterility. The titre of the undiluted material was 107 pock forming units per ml. This was used at a dilution of 1:50 for both the superficial and deep lesions

Cruickshank, J.G

Dent, R.I

Gordon, J.A

Swanepoel, R.

Central African Journal of Medicine

English

IDS OpenDocs

The Central African Journal of Medicine
Editor: L
MICHAEL GELFAND, C.B.E., M.D., FR.C.P.
Assistant Editor: 
JOSEPH RITCHKEN, M.D.
Volume Eighteen  
JANUARY- DECEMBER 
1972
Salisbury R hodesia
T he • Central African
J ournal of Medicine
An Investigation of the Effects 
of Inoculated and Intralymphatic 
Vaccinia Virus on Primary and 
Secondary Deposits of 
Malignant Melanoma
BY
R. I. DENT, m .b ., F.R.C.S.;
J. G. CRUICKSHANK, 
m.b., B.s.(Lond.); m .a. (Cantab.); m .r.c. Path; 
m .r.c.s .; L.R.C.P.; M.D.(Birm.).
J. A. GORDON, 
m .b ., Ch.B.fc.T.), F.R.c.s.(Edin.).
R. SWANEPOEL,
b.v.sc. (Pret.); Ph.D. (Edin.), d.t .v.m ., m .r .c.v.s .
I n trod uction
The therapeutic value of vaccinia virus in malig­
nant melanoma using direct, intraderm'al inocula­
tion, intravenous injection and injection of 
regional lymph node metastases has already been 
reported.ns Results of treatment of intradermal, 
in-transit metastases have been encouraging. 
i,3,4,5,6. This led to the further investigation of 
vaccinia virus in the treatment of regional metas- 
' tases. However, there has been little success with 
regional metastases by direct lymph node inocula­
tion.5 The use of the intra-lymphatic route of 
administration has not hitherto been reported. 
This paper reports experiences With this technique 
in, regional metastases and in the inoculation of 
intradermal lesions and the primary tumour.
M eth od
; The six patients included in this investigation 
were selected on the basis of histologically-proven 
malignant melanomas of the lower extremity, with 
either local intradermal metastases or regional 
lymph node involvement. If unvaccinated in the 
previous five years, vaccination was performed at 
least 14 days before using the virus therapeutically.
Vaccinia virus was prepared from stock vacccine 
obtained from the South African Institute for 
Medical Research. Virus was inoculated on the 
Chorio-allantoic membrane of 12 day embryonated 
hens’ eggs in which growth was confluent on the 
third day. Virus was extracted in buffered saline, 
cleared and tested for sterility. The titre of the 
undiluted material was 107 pock forming units 
per ml. This was used at a dilution of 1:50 for 
both the superficial and deep lesions.
Address for reprints: Mr. J. A. Gordon, Harari Central 
Hospital, P.O. Box ST. 14, Southerton, Salisbury, 
Rhodesia.
Vol. 18. No. 9. September, 1972.
Fig. 1.—A typical malignant melanoma of the right foot 
of an African patient.
Fig. 2.—A Lymphangiogram of a patient with a malignant 
melanoma of the right foot showing ,that the 
dye has reached the lumbar para-aortic chain 
of lymph glands. Vaccinia virus was' injected 
ihtra-lymphatically prior to the injection of the 
dye.
173
September, 1972! VACCINIA VIRUS AND MELANOMA T h e  Central AfricanJ ournal of Medicine
The superficial lesions were treated as one 
would a re-vaccination — a drop (approximately 
0,02 ml.) of virus suspension was placed on each 
nodule and inoculated by the multiple pressure 
technique using approximately 20 pressures. 
Excess was removed and the area left exposed to 
the air.
Primary lesions were treated similarly but the 
amount of virus suspension used was sufficient to 
cover the lesion and was introduced by multiple 
puncture.
In those patients treated by the intralymphatic 
route, cannulation of a large lymphatic vessel on 
the dorsum of the foot on the side of the lesion 
was performed, and 0,5 cc. of the 1/50 dilution of 
the suspension of vaccinia virus was injected. The 
primary lesion, surrounding the satellite lesions 
and intradermal in-transit lesions were directly 
inoculated as above at the same time. A lymph- 
angiogram was performed through the same 
cannula. The site of cannulation was then covered 
with a closed dressing and .the patients transferred 
' to an isolation ward for the next ten days. 
Patients were barrier nursed by staff who had 
been recently vaccinated and precautions were 
taken in the handling of dressings, bed clothes
and other items which may carry virus. <
Excision of the primary lesion was followed by 
a block dissection of regional nodes when indi­
cated, using the lymphangiogram as a guide to 
lymph node involvement and to the completeness ; 
of their clearance. Tissue obtained from the 
primary lesion and the regional lymph nodes was 
studied for evidence of persistence of viable vims. 
The remainder of the excised material was studied 
histologically to confirm the diagnosis of the 
primary lesion, to confirm the involvement of 
regional lymph nodes, and to ascertain the effects 
of virus therapy. Directly inoculated, in-transit, | 
intradermal lesions were assessed clinically and ’ 
virologically.
Where vesicle formation had taken place, fluid 
was collected in capillary tubes and was tested 
for the presence of vaccinia vims by inoculation 
into hens’ eggs. Lymph node and primary tumour 
samples were macerated in a Griffiths grinder with 
buffered saline and tested for vaccinia vims in 
the same way.
The six cases can be sub-divided into three 
groups on the basis of the, degree of dissemina­
tion of disease and the 1 treatment employed 
(Table 1). j
Table I
Degree of Dissemination of Disease and Treatment employed.
Group Case Sex Age Site of Primary 
Lesion
Spread Viral & Related Therapy
I
1 M 57 Sole of R. Foot Nil Local inoculation and excision.
2 ' M 60 L. heel Nil Local inoculation and excision.
II
3 M 60 Sole of R. Foot Local satellite dermal deposits 
inguinal node involvement.
Inoculation of primary and satellite 
lesions, intra-lymphatic injection of 
virus, local excision of primary lesion 
and block dissection of inguinal nodes.
4 F 55 Sole of L. Foot Inguinal node involvement. Intra-lymphatic injection of virus, local 
excision of primary lesion and block 
dissection of inguinal nodes.
HI
5 M 23 Sole of R. Foot Local dermal satellite and in­
transit deposits and inguinal 
node involvement.
Inoculation of dermal deposits.
6 M 49 Sole of R. Foot Local dermal satellite deposits, 
inguinal node and pulmonary 
involvement.
Inoculation of dermal deposits.
174
September, 1972. VACCINIA VIRUS AND MELANOMA T h e  Central AfricanJ ournal of Medicine
Group 1 (Cases 1 and 2) had primary lesions 
only which were treated by direct inoculation 
and later excised.
Group 2 (Cases 3 and 4) had regional lymph 
node involvement and were treated by intra­
lymphatic injection of virus followed by excision 
of the primary tumour and block dissection of the 
involved regional nodes.
Group 3 (Cases 5 and 6) had disseminated 
disease, a feature of which was numerous intra- 
dermal metastases. These were treated by direct 
inoculation.
Re s u l t s
Vaccinia virus was never recovered from the 
primary lesions though clinically, considerable 
necrosis occurred on the surfaces following inocu­
lation of the virus. However, as all these lesions 
were ulcerated before treatment and were super­
ficially infected, the necrosis can only be regarded 
as non-specific. There was no obvious clinical 
response in the regional lymph nodes treated by 
intra-lymphatic injection. However, all the treated 
dermal metatases (and up to 20 nodules were 
treated simultaneously without adverse effect) 
developed changes of an accelerated vaccination 
reaction with vesicle formation followed by the 
development of crusted, superficial ulcers which 
healed as scars typical of those usually follow­
ing smallpox vaccination. These scars showed 
little or no evidence of pigment and in no case 
was a recurrence in a treated nodule found. In 
some instances, new tumour nodules appeared in 
previously untreated areas and these again 
responded to inoculation.
During vaccinia virus therapy, no systemic 
disturbance was noted either by the patient or 
objectively. This is at variance with previously 
reported experience,3'4 which suggests systemic 
disturbance denotes successful treatment with 
vaccinia, virus. These patients however, had 
either never previously been vaccinated or a con­
siderable period had elapsed since vaccination. 
Patients in this series were vaccinated prior to 
treatment, which may have prevented any systemic 
disturbance. It is noteworthy that in the only 
other reported series5 in which patients were 
vaccinated prior to treatment, n / severe systemic 
reactions were seen although a low grade pyrexia 
and malaise did sometimes occur. Many of 
these patients showed satisfactory regression of 
dermal metastases.
Vaccinia virus was not recovered from tissue’ 
obtained from excised primary lesions or from 
excised inguinal lymph nodes involved with metas­
tatic deposits. It was however easily recoverable 
from the vesicles which followed inoculation of 
dermal metastases.
Histologically, the primary tumours, all of 
which were superficially ulcerated and infected 
before being inoculated, showed superficial chronic 
inflammatory infiltration with patchy areas of 
necrosis but the deeper layers of the tumour 
appeared normal. It was not possible to deter­
mine whether the inflammatory infiltration was 
due to the vaccinia therapy, bacterial infection or 
that often associated with even un-ulcerated 
tumours. Regional lymph nodes submitted for 
histology did not differ from the normal appear­
ances of metastatic melanoma.
Fig. 3.—Intradermal transit mestastases of a malignant 
melanoma. The primary tumour has been re­
sected and grafted and a block dissection of 
lymphatic glands carried out.
175
September, 1972. VACCINE VIRUS AND MELANOMA T h e  Central African
J ournal of Medicine
D isc u ssio n
The results presented here confirm the value of 
the use of vaccinia virus in the treatment of 
dermal metastases of malignant melanoma. They 
are susceptible to inoculated vaccinia virus and 
disappear completely over a period of from 
seven to ten days. Long term follow-up has not 
yet been possible, but no recurrences have taken 
place in the inoculated areas. The primary 
tumours seem too large for this to be a successful 
method of treatment and they did not appear to 
support the growth of virus. This may be due to 
the extensive necrosis and borderline viability of 
many of the cells of the tumour, and possibly to 
the presence of bacteria in the ulcerated tissue. 
Thus, wide surgical excision of the primary 
tumour remains the treatment of choice.
Fig. 4.—The intradermal metastases healing after inocu­
lation. The. lowest metastases is still in the stage 
of vesiculation. All intradermal metastases dis­
appeared after inoculation in this case.
It is also apparent that the virus even in high 
titre, injected intra-lymphatically is unable to 
proliferate and hence destroy lymph node metas­
tases. This may be due to the presence of antibody 
in the circulation and tissue fluids of the patients. 
It is, however, felt that the danger of treating 
unvaccinated patients systematically with vaccinia 
virus at a time when they are suffering from 
dissiminated malignant disease would be too 
great a risk.
The method of direct inoculation of even ex­
tensive dermal metastases is easily carried out and 
reliably destroys these deposits in a shorter 
time than radiotherapy and without the more 
extensive tissue involvement and side effects of 
the latter.
It will perhaps be worthwhile to look into the 
possibility of using less virulent viruses in the 
inoculation treatment of primary tumours of 
malignant melanoma, in that this may enable 
repeated applications at high titre to be performed 
in the hope that this will produce more satis­
factory results. ;
At present it is felt that^the failure to produce 
either clinical, histological .'or virological evidence 
to support the use of vacpinia virus in treating 
primary or lymph node tumours of malignant 
melanoma, precludes its use in future patients 
as this delays the definitive treatment of their 
disease along generally accepted lines. However, 
in our service, direct inoculation of dermal metas­
tases with vaccinia virus has become the treatment 
of choice.
S u m m a r y
Primary melanoma and its metastases to lymph 
nodes and the skin have been treated with vaccinia 
virus introduced by direct inoculation and intra­
lymphatic injection. Existing dermal metastases 
can be eradicated but the virus could not be re­
covered from either the primary tumour after 
inoculation or regional lymph nodes after intra­
lymphatic injection.
A c k n o w led g em en t s
The Secretary for Health, Rhodesia, is thanked 
for permission to publish this paper.
References
1. Burdick, K. H., Arch, of Derm. 82. 438-439 (1960).
2. Belissario, J. C. and M ilton, G. W., Austri. Journ.
of Derm. 6. 113 (1961).
3. Burdick, K. H. and Hawk, W. A., Cancer (Phil.).
17. 708-712 (1964). "
4. M ilton, G. W. and Brown, M. M. L., Austr. and
N.Z. Journ. of Surg. 35. 286-290 ,(1966).
5. H unter-Craig, I., N ewton, K. A., Westbury, G., and
Lacey, B. W., Brit. Med. Journ. 2. 512 (1970).
6. H u nt , J. A. (1970), Personal communication.
176
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An Investigation Of The Effects Of Inoculated And Intralymphatic Vaccinia Virus On Primary And Secondary Deposits Of Malignant Melanoma

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An Investigation Of The Effects Of Inoculated And Intralymphatic Vaccinia Virus On Primary And Secondary Deposits Of Malignant Melanoma

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