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.

Taking you through the journey which happened in Africa...

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..