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Anna Schuh - Application of nanopore sequencing in clinical haematology

London Calling 2019

Blood cancers are together the 5th most common cancer and many patients are young adults. Besides, the most common cancers of childhood worldwide are acute lymphoblastic leukaemia and endemic Burkitt’s Lymphoma. Six of the 10 most lucrative cancer drugs are prescribed for haematological malignancies ($55.6billion by 2025). Their diagnosis requires a microscope and DNA-based precision diagnostics. But precision diagnostics is not always about identifying a simple base pair change, and requires the detection of all different types of mutations from one single (often small) patient sample. Examples of where DNA-based diagnostics are critical will be given and include all leukaemias and an increasing number of lymphomas. Their detection is recommended by the WHO classification of haematological malignancies. For example, the NHS test directory includes 177 different genetic aberrations.

The problem is that current multimodality testing in diagnostics laboratories is inadequate to deal with this demand on testing. Many of the conventional single gene assays lack sensitivity, speed and precision. Illumina whole genome sequencing (WGS) of tumour and paired germline has the potential to reveal all types of different mutations and global measures across the genome, but it is limited by the small fragment size and the need for large and expensive equipment. As part of the Genomics England Chronic Lymphocytic leukaemia Pilot have used information from Illumina WGS from 400 patients to develop an improved response prediction tool for chemoimmunotherapy that predicts patients who will be cured (manuscript in preparation). An alternative way specifically for diagnostics, is to combine targeted deep sequencing and error correction with shallow whole genome sequencing using the MinION. This method and variations of it can be applied globally in haematology. For example, the most common inherited anaemias, the haemoglobinopathies, are characterised by 1700 SNVs/indels and deletions across three genes. The most common of these, sickle cell disease, occurs in sub-Saharan Africa. About 10% of patients require confirmatory diagnosis by genetics. Life-saving therapies are available for this disease, and knowledge of the presence of the condition in the fetus could significantly streamline neonatal screening programmes around the world. We have clinically validated a proprietary method for non-invasive testing for sickle cell disease from maternal plasma (pre-published in BioRxiv) and have also developed a nanopore-based test for diagnosing haemoglobinopathies from germline DNA without the need for PCR amplification.

Finally, plasma-derived DNA can also be used in other clinical indications in sub-Saharan Africa. The most common childhood cancer in the region is endemic Burkitt’s lymphoma. This is caused by EBV infection in early childhood. With simple treatment, over 90% of patients can be cured. And treatment is free of charge in all African countries affected. Currently, over 90% of kids die. This is because children present late and are not diagnosed once in hospital because there is lack of trained surgeon and pathologists to establish the diagnosis from an invasive biopsy across the region. We are now clinically validating a non-invasive method to diagnose this type of lymphoma from the blood using a combination of tumour and virus sequencing.

In conclusion: haematological diseases have always spearheaded innovations and discoveries in medicine, in particular genetics. Precision medicine is a reality for an increasing number of patients with blood diseases from targeted small molecules in leukaemias and lymphomas to gene therapy in the inherited blood diseases. The next step is to leapfrog diagnostics technologies and to introduce these advances globally as expensive cancer therapies are coming off patent and are increasingly available and on the WHO list of essential medicines. Ultimately, this approach will achieve a huge impact for a large number of patients world-wide.


"Blood cancers are the fifth most common cancer worldwide"; they are the most common cancer in young people and if left untreated they are fatal. Precision medicine has already been successfully applied in the field of haematology, meaning that we are becoming increasingly able to achieve a cure or long-term disease control. Six of the ten most lucrative drugs are for haematological malignancies, and it is thought that the size of the drug market for blood cancers will reach 55.6 billion USD by 2025.

Risk stratification of blood cancers requires a detection of multiple genomic abnormalities: single nucleotide variants (SNVs), chromosomal gains and losses, and translocations. It isn't a case of identifying single genetic change, but detecting a complex combination of changes " a much more difficult question". With 605 individual tests in the 2018 NHS England Cancer test directory, of which 177 are for haematological cancers, molecular classification of cancers is becoming increasingly relevant for diagnosis and prognosis.

In chronic myeloid leukaemia, characterised by the BCR-ABL gene fusion (the Philadelphia chromosome), precision medicine has helped to increase the long-term survival rate with the development of tyrosine kinase inhibitors against this translocation. 

In acute myeloid leukaemia, improvements in patient survival over the last 50 years have been due to better patient stratification for treatment, and supportive care. Our increasing understanding of gene fusions, acquired SNVs, and complex chromosomal abnormalities, involved in the disease course, has advanced our understanding of AML prognosis and helped to direct future therapeutic avenues - "all of these are important in developing therapy". Three drugs have been approved in the last couple of years by NICE; our increased understanding has therefore "revolutionised the therapy for AML".

Anna introduced how, in chronic lymphocytic leukaemia (CLL), current accredited technologies, such as microscopy, are inadequate for precision medicine as they cannot identify all the genetic abnormalities of interest - they have limited sensitivity, slow turnaround time, are labour intensive, and often require additional supportive tests. The advance of whole-genome sequencing (WGS) with Oxford Nanopore technology has enabled the detection of SNVs and large scale abnormalities in the same run. Using the MinION platform, amplicon and shallow WGS libraries can be combined on the same flow cell; IgHV status, TP53 mutations, del(17p) detection, and additional structural variants, can be detected simultaneously. Anna stated that "we love it" because nanopore sequencing has a rapid turnaround time, and requires no centralisation of services. Anna suggested that this approach should be applied to other leukaemias, including in low-to-middle income countries. 

Anna next described the case of sickle cell disease - the most common monogenic disease worldwide. In the UK, risk of sickle cell disease is the most common reason for prenatal diagnosis. The disease can be identified using protein-based techniques, but these fail in~10% of cases in children >6 months old, and second confirmatory tests are required. The solution proposed by Anna is the detection of mutations and/or deletions in HBA1HBA2 and HBB haemoglobin genes using the MinION platform - this would be fast, with a simple library preparation protocol, and a cloud-based analysis pipeline. CRISPR/Cas9 has been used for targeted mutation detection in the HBA1HBA2 and HBB loci, with sequencing on the MinION and Flongle. Such targeted sequencing means that read depth is greatly enriched over target regions. 

Anna suggested that sequencing of cell-free plasma DNA that circulates in the blood also has great potential in clinical research, and can be used for precise quantification of fetal allele fractions; this would reduce the need for invasive prenatal testing. It is already being used in the clinic for diagnosis of trisomy 21, and the primary aim would be to use cell-free DNA for cancer diagnosis. Anna described the case of Epstein Barr virus (EBV)-driven lymphomas in children; "95% of childhood lymphomas occur Africa". These lymphomas result from early infection with EBV, in combination with exposure to malaria. Treatment for the cancer is free, yet >90% of children currently die from it in low-to-middle income countries. In comparison, the cure rate in high-income countries is 90%. The main reason for treatment failure is due to no diagnosis or misdiagnosis, this is because diagnosis requires technical expertise, yet the training of professionals required for this is lacking. Anna suggested that nanopore sequencing technology could be applied to detect lymphoma-specific mutations in peripheral blood using liquid biopsies, and Nanopore sequencing platforms have great potential to make a significant impact in low-to-middle income countries.

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