Haemato-Oncology

Within the HaemOnc test directory a number of rare diagnostic tests will be delivered by specified GLHs as part of a national network.

Where these tests are not completed within our GLH, please refer samples via your local SIHMDS where they will be appropriately routed to the agreed and appropriate GLH within the national network.

Tests that will be routed outside of the GLH include:

• t(15;17) MRD testing
• Rare non-standard breakpoint cases of t(8;21) and inv(16) for MRD testing
• Rare NPM1 transcript MRD testing
• FIP1L1/PDGFRA MRD testing
• Rare BCR/ABL1 MRD testing

Those rare tests delivered within the NE&Y GLH as part of this national network are shown below:

Histiocytosis Service:

The Newcastle Histiocytosis Service is led by Prof Matt Collin, Chair of the National Histiocytosis Advisory Group.   The Newcastle team also manage the UK Histiocytosis Registry (funded by Histio UK and CRUK).

What is Histiocytosis?

Histiocytic disorders are a diverse group of rare neoplasms caused by somatic mutation and fusions of MAP kinase genes in cells of the macrophage/dendritic cell lineage.  The clinical entities include:

• Langerhans cell histiocytosis,
• Erdheim Chester disease,
• Rosai Dorfman Destombes disease
• Juvenile Xanthogranuloma
• Indeterminate cell histiocytosis

 Why is testing useful?

The clinical indications for genotyping in histiocytosis are:

• BRAF inhibitors are highly effective for BRAF V600E-mutated disease
• MEK inhibitors work for many BRAF-wild-type patients (depending upon the exact mutation)
• Other kinase mutations are also directly targetable by an increasing array of small molecules.
• Genotyping also has prognostic implications for risk of progression and neurological sequelae.

Genomic changes and testing

Clinically significant variants and fusions have been described in up to 90% of cases with histiocytic disorders.  About two-thirds of all cases have the BRAF V600E mutation that is detectable using a sensitive allele-specific PCR test.

The remainder of cases include BRAF and MAP2K1 insertion-deletion mutations (indels), mutations of CSF1R, RAS, PIK3CA, ARAF, MAP2K2, JAK3, KIT and fusions of BRAF, NTRK1, ALK and RET that require targeted NGS analysis.

Histiocytosis Service

In order to detect the spectrum of genetic abnormalities in histiocytic disorders, we offer a multi-component service:

1. Highly sensitive targeted BRAF assay (allele specific PCR)
2. 13 gene NGS panel (sub panel of paediatric NGS panel)
3. RNA fusion panel

Samples required

Frozen Fresh tissue is recommended where possible.

FFPE:  send two separate samples –
5x 10uM curls for DNA extraction (for BRAF and NGS panel testing) and
5x 10uM curls for RNA extraction (for RNA fusion panel testing) with a stated tumour cell content.

ALL MRD

MRD in paediatric ALL is completed in small number of centres (including Sheffield) in the UK. This testing is extensive and bespoke and adheres to strict quality control measures set by the Euro MRD group.

NHS England are keen that the distribution of ALL MRD testing fits the geography associated with the GLHs. This has involved a process of repatriation of cases from the Newcastle and Leeds region to Sheffield which began in March 2020 and was completed in December 2020. A small number of cases from Nottingham and Leicester we managed through the Sheffield service, but have also now been repatriated to the service associated with the East GLH.

Currently, discussion are underway as to what the process will be for the adult ALL MRD testing and whether this will follow a similar pathway to the paediatric MRD or whether a centralised process will be available.

AML MRD

The AML MRD testing includes the common NPM1 changes (types A, B & D) and t(8;21) and inv(16). These tests have been validated in Leeds in order to comply with the repatriation and consolidation requirements of these tests to the NE&Y GLH.

Myeloid and Lymphoid panels

In January 2019, all myeloid NGS testing laboratory work was transferred to Leeds making use of the existing Fluidigm myeloid NGS panel.  Distributed reporting is in place for this panel following agreement to a standardised format for analysis and reporting.

The myeloid NGS panel includes 26 genes listed here:

ASXL1, BCOR, CALR, CBL, CSF3R, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, MPL, KRAS, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2

SRSF2 is poorly covered on the NGS panel and is gap-filled using Sanger sequencing.

In June 2019, the lymphoid panel (liquid samples) was made available across the GLH. This panel covers genes specific to CLL, MZL and HCL, but as the Jan 2019 test directory only requests panel testing for TP53 currently, the uptake and use of this panel has remained low.

PanHaemOnc NGS panel

The NE&Y GLH HaemOnc team are working towards a bespoke panHaemOnc NGS panel designed to conform to NHS Englands plan to provide large panel NGS analysis. It will cover all genes on the Test Directory (TD), plus a number of additional genes of interest from a research or future planning perspective.

The panel was designed using Twist probes and library preparation to run on a NextSeq and has 258 genes known to be implicated in both myeloid and lymphoid disorders.

This is an ambitious aim, and we plan to manage this in a step-wise manner with the SNV analysis available on fresh samples first, and then working up the utility for FFPE samples and the use of the copy number analysis feature.

As of Dec 2020, the wet-work has been completed and is awaiting full analysis and further bioinformatics developments. We are planning to run the panel in parallel to the current myeloid and lymphoid panels, with a view to going live in April 2021.

The panHaemOnc NGS panel will cover a wide range of indications on the TD and we have been working on both testing algorithms, to ensure the most appropriate samples are tested, and , to ensure the analysis is directed, but not limited.

Virtual panels have been created to ensure that appropriate gene panels are assessed for each referral, but with aim of not limiting the analysis where differential diagnoses may be considered.

The first two panels to be implemented are the acute and myeloid panel and the lymphoid panels. All genes will have been sequenced, so there remains the option of going back to specific genes on the full panel if required.

Acute & Myeloid Virtual Gene Panel
ANKRD26	GNB1	PTPN11
ASXL1	HRAS	RAD21
BCOR	IDH1	RUNX1
BCR-ABL (TKD)	IDH2	SETBP1
CALR	IKZF1	SF3B1
CBL (NM_005188.2)	JAK2	SH2B3
CEBPA	KIT	SRSF2
CHEK2	KMT2C	STAG2
CSF3R	KRAS	STAT3
CUX1	MLL-PTD	STAT5B
DDX41	MPL	TET2
DNMT3A	NF1	TP53
ETNK1	NFE2	U2AF1
ETV6	NOTCH1	UBA1
EZH2	NPM1	WT1
FBXW7	NRAS	ZRSR2
FLT3	PPM1D
GATA1	PTEN

Lymphoid Virtual Panel
ARID1A	KLF2 )
ATM	MAP2K1
BCL2	MEF2B
BIRC3	MYD88
BRAF	NOTCH1
BTK	NOTCH2
CARD11	PLCG2
CCND3	POT1
CD79B	RHOA
CDKN1B	RPS15
CREBBP	SF3B1
CXCR4	STAT3
DNMT3A	STAT5B
EP300	TCF3
EZH2	TET2
FOXO1	TNFAIP3
ID3	TP53
IDH2

NGS RNA fusion panel

The HaemOnc Test Directory includes a number of fusion genes. It is recognised that the majority of these can be detected cytogenetically, but the NHSE direction of travel is to have the provision to detect these via NGS panel.

Fusion NGS panels are indicated by the test directory for four disease groups, AML, ALL, Myeloma and Histiocytosis.

Newcastle have experience of running the Illumina Trusight RNA Fusion Panel. This panel covers 507 genes and includes at least one gene partner for the majority of HaemOnc fusions required by the Test Directory. IGH and TCR genes are not included. Although cytogenetics will continue to be offered for the majority of indications in the first instance, referral for this panel will be available for our users.

This panel is also used in to detect NTRK fusions in solid cancer – more information on the panel and how to order it can be found below.