THE JOURNEY OF DISCOVERING THE ETIOLOGY OF BURKITT LYMPHOMA.
Burkitt
and O’Conor published combined data on clinical and epidemiological profile of
Burkitt lymphoma in 1961. But the etiology was still a million dollar question.
In
this post, I will take you through the journey of discovering the etiology of
Burkitt lymphoma (BL).
Based
on geographical distribution, the temperature patterns and frequent rainfall in
the lymphoma belt, the possibility of a mosquito borne virus as a potential
etiology was suggested.
During
this time, Burkitt was invited to give a lecture titled ‘The Commonest Children’s Cancer in Tropical
Africa, a hitherto unrecognised syndrome’ in Middlesex Hospital, London, 22 March
1961. After his return from London, he planned to go on a trip to explore the
lymphoma belt and define it more precisely. When he was just wondering how to
fund the trip, Sir Harold Himsworth, Director of Medical Research Council in
London, came to visit Kampala, and funded a grant of £250 from Medical Research
Council for the research and travel. From
this fund, he brought a 1954 Ford Station wagon which was used to travel and
visit even the remote hospitals. He had
two great travelling companions for the journey of 10000 miles, covering 57
hospitals in 8 countries. The first was Ted Williams, who was a British mission
doctor and an expert in car maintenance. The other companion was Cliff Nelson,
a Canadian doctor. Both Williams and Nelson were good friends of Burkitt and
they were working in hospitals in Uganda.
It
was 7 October, 1961, their trip, later came to be popularly known as “ the long
safari” started. It was a three months long trip and Burkitt
planned every step in advance and so meticulously. They filled the car with
food, water, medical supplies, spare engine parts, and two spare tyres. Though
they travelled mostly by road on an 8-year old Ford station wagon, sometimes on
goods trains and once they also used
lakesteamer, when there were torrential rains as they could not use roads. What
a dedication and interest they had, to travel 10000 miles and travel even during
torrential trains. They regarded this ‘ long safari’ as a holiday with a purpose’.
Yes and it served the purpose!!!
At
each medical centre they visited, they showed photographs of typical cases to
staff and noted it down. When they came back, he mapped down the regions
affected. The results were dramatic and unexpected. The tumour appeared to be restricted
by altitude. With the help of Alexander Haddow, Director of the East African Virus
Research Institute, at Entebbe, Uganda, he could conclude that the actual limiting
factor was not altitude per se, but temperature sensitivity with a minimum temperature of about 60 degree F
(15.5 degree C). These findings led to the hypothesis of a tumour-inducing
infectious agent being involved.
While
this epidemiological search towards etiology of BL was ongoing in Africa,
something equally interesting was going on in London…
Taking
you through the journey which happened in London…
Anthony
Epstein, a virologist from Bland Sutton Institute attended the talk titled ‘The
Commonest Children’s Cancer in Tropical Africa, a hitherto unrecognised
syndrome’ which Burkitt gave in March in London. He was more interested to find
out if there is a possible viral role in the aetiopathogenesis of BL, that he
stopped his current work and decided to start working on BL. After the lecture,
they both discussed and Burkitt finally agreed to send frozen specimens of
tumours from Kampala to Epstein.
The
British Empire Cancer Campaign (now the Cancer Research UK) funded Epstein generously
for his research. For almost three years, all Epsteins attempts to isolate
virus from tumour cells were depressing. He tried all the traditional culture
methods but with negative results. He recalled that certain chicken tumour
viruses began to reproduce only when the malignant cells themselves were grown
in culture, a process that somehow activated the latent virus inside them. But
at that time, human lymphocytes could not have been grown and Epsteins try to
culture human lymphocytes also failed.
In
1963, Epstein received research grant of
$45,000 from the US National Institute of Health. He employed two research
assistants, one was a young scientist Yvonne Barr who helped him with culture
and the other was Bert Achong, who helped with electron microscopy. They tried
all possible ways to culture but none of them grew and it was very frustrating.
Epstein has even admitted that the situation was ‘extremely alarming and
scary’…….
And
finally something considered as bad luck, turned into good fortune for Epstein happened
and it was ‘fog’…….
I
don’t find a better example than this incident relating 'Epsteins work and fog' for
the popular quote, ‘Everything happens for a reason’…
And
now moving onto the journey and the ‘Eureka moment’…
M
A Epstein, B G Achong and Y S Barr in 1964. Picture taken from Anthony
Epstein. Annotation “Burkitt lymphoma and the discovery of EBV” British Journal of Haematology, 2012, 156,
777–779
On
Friday morning, 5 December, 1963,biopsy sample taken from a 9 year old girl
with BL was expected to arrive from Kempala. Since Heathrow airport was
fogbound, all flights were directed to Manchester airport which was 200 miles
away. The sample eventually arrived late in the lab and people were busy
leaving home for weekend. The biopsy was
received in usual transport media composed of salt solution and guinea pig
serum, but the fluid was cloudy, may be caused by bacterial contamination
because of prolonged journey was considered. But something made Epstein not to
discard it as bacterial contamination and he examined the cloudy fluid under microscope
and to his astonishment he found that the cloudiness was not due to bacteria
but due to huge numbers of viable, free-floating lymphoma cells shaken from the
edges of biopsy due to unusually long flight.
For
the first time, Epstein decided to put
the freefloating cells into suspension and
to see if it made a difference. After 16 days in culture, the cells had begun
to grow in one of the culture bottles. By 26th day of culture, New years Eve,
1963, there were enough cells to divide the culture into two. By day 43, cells
were transferred to larger bottles. The cells grew up and the first Burkitt
lymphoma derived cell line was known as EB1, featuring Epstein and Barr. This
presented the first successful long term culture of a human lymphoma of any
kind…The trick was to detach the cells from tumour and grow in suspension medium
and the technique was shown to be successful in several other laboratories.
They
tried all current tests to show a virus in the cultured lymphoma cells but
failed. They prepared samples for electron microscopy though, this technique
was not then accepted as a way of demonstrating viruses.
On
24 February, 1964, Epstein examined the first culture preparation in electron
microscopy and saw unequivocal virus particles in cultured lymphoma cells in
the very first grid square. They found that the virus was a typical member of
the herpes group but could not identify which herpes virus it could be. Their discovery
was soon published in Lancet on 28 March, 1964.
Electron micrograph
of thin sectioned EBV particles. Immature virions (above) cut in various planes
in an infected cell. Inset (below) shows a mature enveloped particle. Picture
taken from Anthony Epstein. Annotation
“Burkitt lymphoma and the discovery of EBV” British Journal
of Haematology, 2012, 156, 777–779.
The
lack of detectable biological activity was
confirmed in EB cells in the laboratory of Werner and Gertrude Henle in
Philadelphia. The immunological uniqueness of the virus was demonstrated shortly
in Epsteins laboratory and Henle laboratory.
Interesting
Firsts of EBV:
Ø The Epstein–Barr virus (EBV) was the first
infectious agent of this nature to be discovered solely by electron microscopy
as the agent was inactive in all the biological essays available at that time.
Ø It was the first description of a virus
involved in the pathogenesis of a tumour in humans.
Epstein
says in his paper published in British Journal of Hematology in 2012 as annotation
on Burkitt lymphoma and the discovery of EBV, “It was indeed fortunate that
work on the lymphoma cells and the search for virus was going forward at a
centre where a rare electron microscope was in daily use, otherwise the extreme
inertness could have left EBV undiscovered.”
References:
1.
Anthony Epstein. Annotation “Burkitt lymphoma and the discovery
of EBV” British Journal of Haematology, 2012, 156,
777–779
2.
Davis Coakley. Denis Burkitt and his contribution to
haematology/oncology. British Journal of
Haematology. 2006:135; 17–25.
3.
Textbook, The
story of Epstein Barr virus. Cancer virus by Dorothy H Crawford. Alan
Rickinson, Ingolfur Johannessen.
See you all with yet another interesting topic in the coming days..
“The man with one eye and his discoveries”
Written by Dr.Priyavadhana..
Comments
Post a Comment