Specialist Integrated

Haematological Malignancy Diagnostic

Service (SIHMDS)

Operational Policy

Version 7- 08.07.16

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Operational Policy for the Oxford NHS/BRC SIHMDS

1. Introduction ............................................................................................................................... 4 1.1 Objectives ................................................................................................................................ 4

1.1.1 Objectives of the SIHMDS process .......................................................................... 4 1.1.2 Objectives of this Operational policy ...................................................................... 4

2. General Operational Principles ........................................................................................... 5 2. 1 Organisation and Leadership of the SIHMDS (13-1D-101h) ................................ 5 2.2 Laboratory IT system (13-1D-104h) ............................................................................. 5 2.3 Sample Handling (13-1D-101h) ...................................................................................... 6 2.4 Turn-around times (13-1D-101h) .................................................................................. 6 2.5 Quality Assurance system for the SIHMDS (13-1D-103h) ...................................... 6

3. Detailed description of work-flows and procedures ................................................... 8 3.1 Overview of solid sample pathways ............................................................................ 8 3.2 Overview of liquid sample pathways ........................................................................... 8

4. Central Specimen Reception (13-1D-105h) .................................................................... 9 4. 1 Contact details and shipment address ......................................................................... 9 4. 2 Request forms ....................................................................................................................... 9 4.3 Timing of requests ............................................................................................................... 9 4.4 Specimens ................................................................................................................................ 9

4.4.1 Bone marrow samples ............................................................................................. 9 4.4.2 Peripheral blood for DNA analysis .......................................................................... 9 4.4.3 Peripheral blood for RNA analysis ........................................................................... 9 4.4.4 Histopathological samples ....................................................................................10

5. Initial morphological assessment of samples (13-1D-102h; 13-1D-105h; 13-2H-105) ........................................................................................................................................... 11

5.1 Liquid samples .................................................................................................................... 11 5.2 Paraffin embedded material ......................................................................................... 12

5.2.1 Molecular tests ......................................................................................................12 5.2.2 Clonality studies ....................................................................................................12

5.3 Clinical Trial Samples ....................................................................................................... 12

6. Algorithms to guide the requesting of additional tests (13-1D-102h; 13-1D-105h; 13-2H-105) ........................................................................................................................ 14

6.1 At suspected diagnosis .................................................................................................... 15 6.1.1 Suspected AML ......................................................................................................15 6.1.2 Suspected MDS......................................................................................................17 6.1.3 Suspected MPN .....................................................................................................18

6.1.3.1 Suspected CML ...............................................................................................18 6.1.3.2 Suspected PRV/ET ..........................................................................................19 6.1.3.3 Suspected Myelofibrosis ................................................................................19 6.1.3.4 Suspected other MPNs ...................................................................................21

6.1.4 Suspected ALL ........................................................................................................22 6.1.5 Suspected CLL/LPD ................................................................................................23

6.1.5.1 Peripheral blood .............................................................................................23 6.1.5.2 Bone marrow..................................................................................................24

6.1.6 Suspected Plasma Cell Myeloma ...........................................................................26 6.1.7 Suspected Lymphoma ...........................................................................................27

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6.2 Follow-up samples ............................................................................................................ 28

6.2.1 AML follow-up .......................................................................................................28 6.2.2 MDS follow-up .......................................................................................................29 6.2.3 CML follow-up .......................................................................................................30 6.2.4 ALL follow-up .........................................................................................................31 6.2.5 CLL follow-up .........................................................................................................32 6.2.6 Lymphoma follow-up ............................................................................................33

6.3 BMT samples .......................................................................................................................... 34 6.3.1 ASCT .......................................................................................................................34

6.3.1.1 ASCT for Plasma Cell Myeloma ......................................................................34 6.3.1.1.1 Pre-BMT bone marrow ...................................................................................... 34 6.3.1.1.2 Post-BMT bone marrow .........................................................................35

6.3.1.2 ASCT for Lymphoma .......................................................................................36 6.3.2 Allogeneic transplants ...........................................................................................36 6.3.2.1 Pre-BMT samples ...............................................................................................36 6.3.2.1.1 Allogeneic transplant for AML/MDS/MF ........................................................36 6.3.2.1.2 Allogeneic transplant for lymphoma ...............................................................37 6.3.2.1.3 Allogeneic transplant for CLL ..........................................................................38 6.3.2.1.4 Allogeneic transplant for Plasma Cell Myeloma .............................................39 6.3.3.2 Post BMT samples ..............................................................................................40 6.3.3.2.1 Non-CLL samples .............................................................................................40 6.3.3.2.2 CLL samples .....................................................................................................41 6.3.3 Relapse post BMT ..................................................................................................42

7. Integrated Reporting (13-1D-105h) ................................................................................ 43 7.1 Liquid samples .................................................................................................................... 43 7.2 Solid samples....................................................................................................................... 43

8. Appendix ................................................................................................................................... 44 8.1 Current senior staff ........................................................................................................... 44 8.2 Contact details .................................................................................................................... 44 8.3 Shipment addresses .......................................................................................................... 45

8.3.1 Paraffin embedded blocks:....................................................................................45 8.3.2 Liquid samples: ......................................................................................................45

8.4 Request forms and worksheets .................................................................................... 45 8.5 WHO codes and scoring systems .................................................................................. 47

8.5.1 WHO classification of tumours of haematopoietic and lymphoid tissues ..........47 8.5.2 MDS IPSS score .........................................................................................................................61

8.5.2.1 IPSS Score (Greenberg et al, 1997) ...............................................................61 8.5.2.2 IPSS-R Score (Greenberg et al, 2012) .............................................................61

8.5.3 Myelofibrosis DIPSS-plus .....................................................................................................62 8.5.4 CML response criteria (ELN website) ...................................................................63 8.5.5 Aplastic Anaemia severity score (BCSH guidelines, 2009) ...................................64

9. Abbreviations ........................................................................................................................... 65

10. References .............................................................................................................................. 67

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1. Introduction This document defines the responsibilities and operational procedures governing the Oxford

NHS/BRC SIHMDS and its referring hospitals. The aim of the SIHMDS is to ensure accurate and

speedy diagnosis and monitoring of patients with haematological malignancies, and to produce a

single interpretative report within a time frame that is of clinical utility. All referring hospitals have

agreed that where there is a clinical suspicion of a previously undiagnosed haematological

malignancy, diagnostic tissue and blood specimens should be sent direct to the relevant named

SIHMDS rather than the local pathology services for diagnosis. This has been documented in the

network constitution (13-1C-109h Clinical Diagnostic Pathways).

1.1 Objectives

1.1.1 Objectives of the SIHMDS process To ensure compliance with NICE and NCAT guidelines

To ensure quality assured sample analysis within EQA schemes

To guarantee the appropriate and cost-effective use of complex analysis (avoiding

duplication of testing, unnecessary testing etc.)

To allow formal teaching of specialist registrars in morphology, immunophenotyping, and

molecular genetics (including indications for testing, analysis, interpretation, integration of

results)

To ensure that all patients undergoing complex analyses are issued an integrated report

that is reviewed in the MDT

To co-ordinate Oxford Radcliffe Biobank (ORB) and MDS Biobank tissue banking according

to Good Laboratory Practice (GLP)

To guarantee rapid and accurate processing of research samples

1.1.2 Objectives of this Operational policy To clearly define the responsibilities of local laboratories and the SIHMDS in delivering the

service

To outline the processes involved in establishing leukaemia and lymphoma diagnostics

including sample reception, processing and reporting

To define the governance arrangements of the SIHMDS

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2. General Operational Principles

2. 1 Organisation and Leadership of the SIHMDS (13-1D-101h) The laboratory is designated by the commissioning groups and by the Thames Valley Cancer

Network and is managed by the Oxford University Hospitals (OUH).

It is directed by a single head of service who reports to the Chief Executive of OUH, via the

division of Clinical Support Services. His/her list of responsibilities includes the design of

investigational algorithms, the use of resources for diagnostics and research, and the standards

of reporting. He/she is responsible for the financial sustainability of the laboratory and reports

to the Clinical Director of the Laboratory Medicine Directorate (see Figure 1).

2.2 Laboratory IT system (13-1D-104h) The different sections of the laboratory are working according to the present operational policy.

This policy covers all investigational pathways and defines situations where redirection of samples

at any time point depending on results at that point may be required.

The laboratory generates an integrated report including diagnostic coding (according to WHO

classification and ICD003) that is up-loaded into the network-wide e-prescribing system ARIA, into

EPR (in progress) and into a secure web-based portal for access to all customers.

The purchase or design of a purpose-built LIMS system has been considered and funds are

allocated. The SIHMDS has been in negotiations with Leeds to lease their HILIS system. A review of

the current and future demands (including extensive mapping of laboratory processes) was

Clinical Director of Laboratory Medicine

Haematology clinical

Lead/SIHMDS Lead

Lymphoid Lead Myeloid Lead Haematopathology

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undertaken by Charles Crichton (Davies Group, Oxford Computer Sciences). The conclusions of this

review are that contractual issues and information governance make it unlikely that the lease of

the HILIS system will go ahead. Multiple modern IT solutions are already in place that can easily be

linked together to provide and in-house solution, that will further facilitate integrated reporting

and live sample/result tracking.

2.3 Sample Handling (13-1D-101h) Sample handling and transport from regional hospitals to the SIHMDS includes booking out and

booking in of samples according to CPA standards, transport via once daily hospital transport or via

taxi for urgent samples. Details on sample types are found below. All samples arrive in a single

central specimen reception, are assessed by morphology/histopathology and then referred for

specialist testing by immunochemistry, flow cytometry, cytogenetics, FISH and molecular studies

on the basis of the initial assessment.

2.4 Turn-around times (13-1D-101h) Every effort will be made to allow a turn-around time for integrated reports of not more than 2-4

weeks (see table). Results of individual tests will be issued as soon as they become available and

are detailed in the service level agreement (SLA).

2.5 Quality Assurance system for the SIHMDS (13-1D-103h) The SIHMDS is fully CPA accredited and participates in the national external quality assurance

schemes (NEQAS). The laboratory performs regular audits and the SIHMDS lead attends clinical

governance meetings. As part of the CPA, the laboratory records data which can provide a

complete audit trail of all laboratory activity.

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Sample Type Turnaround time

Urgent A fully integrated report will be issued within 2 weeks of the sample arriving in the laboratory.

Non urgent A fully integrated report will be issued within 4 weeks of the sample arriving in the laboratory.

Individual Tests Reports on individual test within a menu request and results from

“a la carte” requests will be issued according to turnaround times of individual tests in order to avoid delays in reporting.

Molecular Genetics Urgent samples (all acute leukaemia diagnostics):- Fusion genes – 3 working days NPM1, FLT3 ITD and D835 TK – 1 week JAK2, Clonality studies, STR Chimerism and BCR-ABL:- 2 weeks

Immunophenotyping Urgent samples are processed within 1 working day and results are communicated by e-mail and telephone. All results are discussed at the MDT meetings and authorised weekly.

Histopathology 2 weeks

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3. Detailed description of work-flows and procedures

3.1 Overview of solid sample pathways

3.2 Overview of liquid sample pathways

TISSUE BANKED IF CONSENTED TO SPECIFIC STUDY

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4. Central Specimen Reception (13-1D-105h)

4. 1 Contact details and shipment address

See appendix.

4. 2 Request forms

See appendix.

SIHMDS request forms are also available from our website:

http://www.oxford-translational-molecular-diagnostics.org.uk/home.html

and from the OUH website:

http://www.oxfordradcliffe.nhs.uk/forpatients/departments/labs/haematology/molhaem/m

olhaem.aspx

Integrated analysis of samples is critically dependent on clinical information. It is therefore

vital that all clinical and already available diagnostic information is clearly stated on the

request form. In the absence of clinical information or previous results, samples will be

processed on the basis of morphology and algorithms described below.

4.3 Timing of requests

Whenever possible, samples should be sent between Monday and Thursday morning.

Urgent requests should be phoned through to the laboratory to allow urgent tracking of

samples if necessary and speedy reporting of provisional results back to the requesting

physician.

4.4 Specimens

Specification for each type of specimen is as below.

4.4.1 Bone marrow samples 10ml of EDTA and 2 unstained or stained bedside smears

4.4.2 Peripheral blood for DNA analysis 4ml of EDTA

4.4.3 Peripheral blood for RNA analysis 12ml of EDTA less than 72 hours old

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4.4.4 Histopathological samples In cases where a tissue diagnosis has been made (or can be made) the tissue block (formalin

fixed paraffin embedded) and all associated material (stained and unstained slides, including

cytology specimens) should be sent for review in Oxford prior to the requesting of additional

molecular / cytogenetic tests. Copies of the original report and any relevant clinical

information should be included.

Depending on the downstream analyses required, the following specimens may be prepared:

5-10 4-micron cut sections in eppendorf tubes.

Unstained paraffin-embedded mounted 4 micron sections with a marked H&E stained slide as

a guide for macro-dissection.

Stained paraffin-embedded mounted sections.

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5. Initial morphological assessment of samples (13-1D-102h; 13-1D-105h; 13-2H-105)

Initial assessment of paraffin embedded material and blood or bone marrow smears is

performed at the local hospital. If a diagnosis of a haematological malignancy is suspected,

diagnostic material will be sent to the SIHMDS for confirmation of initial diagnosis and

additional tests if indicated. Emergency treatment should not be delayed by the SIHMDS and

should be initiated on the basis of local results if indicated. However, rapid diagnosis at the

SIHMDS is possible. Please contact one of the haematopathologists to discuss the case as soon

as it arrives at the SIHMDS.

5.1 Liquid samples Liquid samples will be processed according to the standard operating procedures. Briefly,

booking-in of samples should be performed only once. Each sample will be given a unique

identifier that is the same for all tests.

Two bedside slides will be sent for MGG staining. The remaining bone marrow will be pooled

and mixed in a 15ml Falcon, a nucleated cell count performed and 1-2x107 cells will be

dispatched for each test. Samples for research will be processed as soon as possible to

guarantee cell viability. Generally, 1x107 cells are needed for flow cytometry, 2x107 cells for

cytogenetics, 1x106 cells for DNA preparation and 1x107 cells for RNA preparation. It is

important to ensure that the remainder of the sample is banked. Tissue bankers are expected

to collect research sample as soon as possible once the sample has been divided, as the sample

has to be processed for banking on the same day as it was received. Consent forms for tissue

banking should come in the request pouches having been completed by the person taking the

marrow sample at the bedside.

Samples for diagnostic purposes will be stored according to downstream requirements and

processed in batches on the same day or in the morning of the following day for samples

arriving after 14:00.

Morphological assessment of samples will be performed by the consultant haematologist on-

duty for the laboratory. This assessment can be delegated to the laboratory registrar after an

extended period of teaching and provided that competency has been demonstrated. On the

basis of known clinical data, previous results and the current morphological assessment,

additional tests will be requested to establish the precise diagnosis.

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In cases where it is unclear at the time of diagnostic morphology review what additional

tests will be required, DNA/RNA, cytogenetic samples and cells should be prepared for

storage.

Analysis of follow-up samples for response assessment should be guided by the initial

diagnostic findings. The diagnostic worksheets (on-line or in paper form) must therefore

contain all diagnostic information previously obtained and bone marrow reports and results of

additional tests should be entered in real-time onto the worksheet and into reporting

databases.

5.2 Paraffin embedded material

5.2.1 Molecular tests Molecular tests on paraffin embedded material should be requested only by an SIHMDS

haematopathologist following morphological and immunohistochemical (+/- in situ

hybridisation) assessment of the case. One 4 micron section per rearrangement to be

investigated should be cut on to a charged glass slide.

5.2.2 Clonality studies B and T-cell receptor clonality analysis by PCR may be performed on paraffin embedded

material and cytological specimens. For paraffin embedded material, please cut 3 - 5 x 10 μm

sections per tube into two separate microtubes, depending on specimen size. For cytological

material, please cut cell blocks onto slides (multiple sections per slide are acceptable) and send

as much material (smears, touch preps etc) as possible on slides. When preparing pathological

material, please ensure the equipment (especially the microtome blade and water bath) is

completely clean between cases to prevent cross contamination. This can be achieved by

wiping the microtome blade and any other surfaces with ethanol until they are free of any

debris from earlier cases.

5.3 Clinical Trial Samples Samples for commercial studies will be sent away to central laboratories by the research nurse

team.

Samples from investigator-led studies should also be sent off by research nurses. However,

samples requiring cDNA preparation should be sent via the SIHMDS in order to enable sample

preservation.

If a Principle Investigator (PI) uses the laboratory for tissue banking or downstream analysis, a

research request form should be filled in and submitted prior to sending the samples.

Processing of these samples has to be agreed with the head of the SIHMDS. All non-guideline

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tests being done locally above and beyond what would constitute routine clinical care should

be costed for and a research request form should be filled in and submitted prior to sending

the samples.

For sample processing, PID numbers should be recorded on trial patients’ request forms. It is

the responsibility of the trial nurse to clearly state on the request form which tests the patient

needs if these are extra to what would constitute routine clinical care. Otherwise, the registrar

reviewing the morphology will request tests as per the standard guidelines.

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6. Algorithms to guide the requesting of additional tests (13-1D-102h; 13-1D-105h; 13-2H-105)

In addition to review of morphology and histology, the SIHMDS will perform additional tests

when indicated with a view of providing an integrated and interpretative report. The SIHMDS

will follow national and international consensus guidelines and NICE recommendations

whenever possible. Diseases will be coded as per WHO classification (see appendix). It is

important to note that further investigations such as flow cytometry or molecular

tests/FISH should only be undertaken if the abnormal cell population exceeds 5%.

The following algorithms are based on the WHO classification 2007.

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6.1 At suspected diagnosis

6.1.1 Suspected AML Samples should be evaluated as follows:

a) Evaluation of ≥ 100 cells on MGG stained slide – note dysplasia and blast count.

b) Flow cytometry: full panel of ELN markers to be used.

c) Cytogenetics – full AML karyotype

d) In cases where APL is suspected, FISH for PML-RARA should be performed urgently.

RQ-PCR for PML-RARA should also be performed to establish the diagnostic

breakpoint.

e) All patients should be tested for FLT3 TKI and internal tandem repeat (ITD) mutations

and NPM1 4bp insertion mutations

f) Inv16 and AML-ETO positive patients should also be tested for the c-KIT D816V point

mutation.

g) If M2 morphology is seen, RQ-PCR for t(8;21) breakpoint should be performed,

h) If M4 morphology is seen, RQ-PCR for inv(16) breakpoint should be performed.

i) All diagnostic samples will be stored as DNA and RNA to allow quantitative molecular

monitoring should this be indicated.

j) All samples will be stored under MDS Bio where consent has been given.

k) High throughput sequencing of a panel of genes known to be mutated in AML (see

appendix for full gene list) is now available, but requests need to be agreed with the

treating consultant.

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AML on Morphology

Record blasts and dysplasia if seen

DNA/RNA storage

MDS Bio

Karyotype

/FISH

Flow cytometry

FLT3 TKI/ITD

NPM1 mutation/Myeloid Panel

C-KIT mutation analysis

RQ-PCR for PML RARA

RQ-PCR for

Inversion 16

RQ-PCR for AML-ETO

M2

morphology

M3

morphology

M4

morphology

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6.1.2 Suspected MDS Samples should be evaluated as follows:

a) Evaluation of ≥ 200 cells on MGG stained slide – note dysplasia and blast count (This

should be recorded with sufficient accuracy to enable calculation of an IPSS-R; see

section 8.4.2).

b) Where the blast count exceeds 20%, AML is diagnosed and the AML flowchart should

be followed.

c) Cytogenetics: Full karyotype. The clinican requesting the marrow will indicate whether

the sample is urgent; in which case the turn-around time will be 2 weeks, or non-

urgent; in which case a result will be available in 4 weeks. The lab SpR should then

indicate this on the integrated report worksheet (W116).

d) Iron stains. These should be reported on LIMS by the Lab SpR.

e) MDS Bio

f) High throughput sequencing of a panel of genes known to be mutated in MDS (see

appendix for full gene list) is now available, but requests need to be agreed with the

treating consultant.

MDS on Morphology

Count >200 cells and

record blasts and

morphology

Cytogenetics MDSbio Iron stain Myeloid Panel

All Patients

Consultant request only

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6.1.3 Suspected MPN

6.1.3.1 Suspected CML Samples should be evaluated as follows:

a) Cytogenetics & FISH for BCR-ABL

b) DNA analysis for JAK2 V617F in BCR-ABL negative cases

c) BCR-ABL multiplex (Molecular). RQ-PCR should ONLY be performed on peripheral

blood; therefore if a bone marrow BCR-ABL multiplex shows a rearrangement, a

peripheral blood sample should be requested for baseline quantification.

d) DNA and RNA samples will be stored on all cases of suspected CML

e) MDS Bio

CML on Morphology

Count >100 cells and record

blasts and morphology

FISH for t(9;22)

MDSBio Multiplex RT-PCR for BCR-ABL

Store DNA/RNA

Request peripheral blood for RT-qPCR

ARMS-PCR for Jak-2 V617F

Positive Negative

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6.1.3.2 Suspected PRV/ET All samples will be tested for:

a) JAK2 V617F mutation. Allelic burden will be reported as WT:mutant ratio.

b) On request, JAK2 exon 12 mutation (PRV only) analysis, CALR and MPL mutation (ET only)

analysis will be performed. JAK2 exon 12 should be performed on marrow rather than

peripheral blood if possible.

c) Multiplex PCR for BCR-ABL will be performed in ET JAK2 mutation negative patients.

d) Cytogenetics

e) RNA/DNA storage

f) MDS Biobank

PRV or ET criteria

ARMS-PCR for JAK2 V617F

Cytogenetics

RQ-PCR for BCR-ABL

(ET only) JAK2 exon 12

(PRV only)

CALR and MPL mutations (ET only)

DNA/RNA storage

MDSBio

Negative

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6.1.3.3 Suspected Myelofibrosis All patients should have

a) Karyotyping if MF suspected

b) JAK2 V617F mutations. Allelic burden will be reported as WT:mutant ratio.

c) CALR and MPL analysis if JAK2 negative (Molecular)

d) RNA/DNA storage

e) MDS Bio

f) High throughput sequencing of a panel of genes known to be mutated in myelofibrosis

(see appendix for full gene list) is now available but requests need to be agreed with

the treating consultant

Suspected Myelofibrosis

DNA/RNA storage

Karotyping

CALR and MPL mutations

ARMS PCR for JAK2 V617F

Myeloid Panel

Negative

MDSBio

Consultant request only

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6.1.3.4 Suspected other MPNs These need to be discussed by the clinical consultant with the Section Head on a case by case

basis.

Myeloid and lymphoid neoplasms with eosinophilia +/- aberrant mast cell involvement:

Chronic Eosinophilic syndrome, AML, pre-cursor T lymphoblastic lymphoma.

Possible tests available include analysis for:

PDGFalpha, PDGFbeta and FGFR1 (cryptic) translocations: detection by FISH and nested RT-

PCR

Mast cell leukaemia: c-kit mutations on D816V by nested PCR

TCR clonality

CSF3R mutation status either single gene or via myeloid panel for atypical CML / MPN (NOS)

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6.1.4 Suspected ALL

All samples should be analysed by:

a) Flow cytometry

b) Cytogenetics

c) FISH for BCR-ABL (Done in Churchill CGN).

d) Multiplex PCR for BCR-ABL

e) DNA and RNA will be stored.

f) At consultant request, samples can be sent for clonality studies.

g) ORB

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6.1.5 Suspected CLL/LPD

6.1.5.1 Peripheral blood a) All cases with suspected LPD, i.e. with persistent lymphocytosis of >5x109/l for longer

than 3 months, should have flow cytometry performed.

b) All cases of CD19+ CD5+ lymphocytosis and CLL score of ≤3 should be tested for the

t(11;14) to exclude mantle cell lymphoma..

c) In selected CLL samples, prognostic markers (IGVH mutation analysis) can be performed

on request of the treating consultant. Remaining viable cells will be stored under ORB.

d) CD5 negative persistent lymphocytosis cases should be referred for further investigations

including bone marrow examination and lymph node biopsy. Precise diagnosis should be

established on these specimens and not on the peripheral blood.

e) Cases of persistent mature T-cell lymphocytosis should be sent for clonality studies and

referred for further investigations including bone marrow examination or lymph node

biopsy.

f) All cases with confirmed CLL requiring treatment should be tested for del17p by FISH and

TP53 mutations.

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6.1.5.2 Bone marrow a) Flow cytometry will only be performed if the lymphoid infiltrate is >20%

b) All cases of CD19+ CD5+ lymphocytosis and CLL score of ≤3 should be tested for the

t(11;14) to exclude mantle cell lymphoma.

c) In selected CLL samples, prognostic markers (IGVH mutation analysis) can be performed

optionally on request of the treating physician. Remaining viable cells will be stored

under ORB.

d) CD5 negative persistent lymphocytosis cases should get a precise diagnosis from

histopathology specimens (marrow or LN).

e) Cases of persistent mature T-cell lymphocytosis should be sent for clonality studies from

the histopathological specimen.

f) All cases with confirmed CLL requiring treatment should be tested for del17p by FISH

and TP53 mutations.

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6.1.6 Suspected Plasma Cell Myeloma

All cases should have:

a) Morphology to confirm >5% plasma cells

b) Flow cytometry if >5% plasma cells

c) FISH analysis for del17p and t(4;14) on sorted CD138 cells

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6.1.7 Suspected Lymphoma The diagnosis of NHL and Hodgkin lymphoma relies on histological assessment of tissue

biopsies and immunostaining. Pathologists may request molecular tests in a minority of cases:

T-cell and B-cell receptor rearrangement studies (by PCR)

These should only be requested in cases where there is diagnostic doubt or where the MDT

requests studies to identify minimal residual disease (which may involve comparison with an

earlier biopsy). PCR should not be requested routinely on all B-cell lymphomas.

FISH studies offered by Cytogenetics for rearrangements of:

c-MYC t(8;14) cases with features diagnostic or suspicious of Burkitt’s lymphoma; and in all

cases of Germinal Centre B-cell like Diffuse Large B Cell Lymphoma in which the c-myc

expression (by IHC) is greater than 40%.

bcl-2 t(18;14) – suspected follicular lymphomas in which immunohistochemistry is

inconclusive; cases where differential diagnosis lies between Burkitt lymphoma and the

intermediate category between Burkitt lymphoma and diffuse large B-cell lymphoma; and in

all cases of Germinal Centre B-cell like Diffuse Large B Cell Lymphoma in which the c-myc

expression (by IHC) is greater than 40%.

bcl-6 rearrangement - all cases of Germinal Centre B-cell like Diffuse Large B Cell Lymphoma in

which the c-myc expression (by IHC) is greater than 40%.

cyclin D1 t(11;14) – suspected mantle cell lymphomas in which immunohistochemistry is

inconclusive.

MALT1 t(11;18) – all marginal zone lymphomas of MALT type without evidence of concurrent

high grade transformation. This test is available on request.

ALK-1 t(2;5) – suspected cases of ALK-1+ anaplastic large cell lymphoma, in which

immunohistochemistry is inconclusive.

TP53 (17p) – cases of chronic lymphocytic leukaemia/ small lymphocytic lymphoma at the

request of the MDT meeting only.

Further FISH analyses may be available on request.

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6.2 Follow-up samples

6.2.1 AML follow-up Follow-up samples should be reviewed together with all previous results. The main follow up

test pathways are outlined below:

a) PML-RARA, RUNX1-ETV6 (AML1-ETO1), CBFB-MYH (inv16) positive AML: follow-up

samples should be tested by RQ-PCR. The clinican requesting tests should check

previous results to see whether this is indicated.

b) AML is currently not routinely monitored by molecular means, and will be assessed

through the SIHMDS by morphology only. This is because sensitivity of the diagnostic

assays for FLT3 and NPM1 are of insufficient sensitivity to permit MRD monitoring.

Molecular monitoring (e.g. NPM1) of AML in certain cases may be possible by external

centres on consultant request.

c) AML with complex cytogenetics will be monitored by morphology and metaphase

analysis/FISH (Churchill CGN). Other cytogenetic markers abnormal at diagnosis can

also be followed up in the same way.

d) Relapsed samples should be processed as per the diagnostic algorithm.

e) RNA/DNA storage

f) MDS Bio

AML follow-up

Count >100 cells and record

blasts and dysplasia

Store DNA/RNA

MDSBio Karotype/ FISH until negative

RT-qPCR for fusion

transcript

Molecular monitoring by external centres (on Consultant request)

CGN marker at diagnosis

t(15;17), t(8:21) or inv(16) at diagnosis

Other molecular

marker

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6.2.2 MDS follow-up

Some MDS patients will have yearly bone marrows to monitor disease. They should have:

a) Evaluation of 100 cells on MGG stained slide – note dysplasia and blast count.

b) Where the blast count exceeds 20% and AML is suspected, the sample should be

processed as per the AML protocol.

c) Cytogenetics

d) Iron stains for patients who had ring sideroblasts

e) MDS Bio

f) High throughput sequencing of a panel of genes known to be mutated in MDS (see

appendix for full gene list) is now available but requests need to be agreed with the

treating consultant.

MDS follow-up

Iron stain

Yearly cytogenetics*

MDSBio

Count >200 cells and record blasts and

morphology

Myeloid Panel

*This includes patients on azacitidine

Previous ring sideroblasts

Consultant request only

30

6.2.3 CML follow-up

Monitoring of CML should follow the European Leukaemia Net (ELN) guidelines.

a) 3 monthly bone marrow cytogenetics/FISH until absence of Philadelphia chromosome.

Alternatively, peripheral blood FISH monitoring is acceptable.

b) Once the patient has become negative for the cytogenetic abnormality t(9;22), 3 monthly

monitoring by RQ-PCR from peripheral blood should be initiated until a major molecular

response (~3 log reduction from base-line) has been achieved. Once the patient is in MMR, 6

monthly monitoring is acceptable. RQ-PCR for BCR-ABL is never performed on bone marrow,

even as part of the clinical trials.

c) Yearly bone marrow examination for conventional metaphase analysis should be considered

as per ELN guidelines

d) Suboptimal responders and treatment failures: 3 monthly monitoring by the appropriate

test. Kinase mutations should be excluded, and assessment for the T315I mutation will be

routinely performed. If this is negative, sequencing of the kinase domain will be carried out.

e) DNA/RNA storage

f) MDS Bio

31

6.2.4 ALL follow-up

a) BCR-ABL positive cases should be monitored for BCR-ABL transcript levels by RQ-PCR

(paired PB and BM). Please note this does not apply to BCR-ABL monitoring in the

context of CML.

b) Patients with abnormal cytogenetics should be monitored by cytogenetics/FISH until

these become undetectable.

c) In select cases and at the request of the treating consultant, MRD may be available

through external centres if a clonal marker was identified at diagnosis.

32

6.2.5 CLL follow-up

All patients should have FISH for del17p and TP53 mutation status determined if treatment is

indicated.

MRD monitoring by flow cytometry is not performed routinely (except post-transplant, see

relevant section). However, on request of the treating consultant samples can be forwarded to

Leeds HMDS.

33

6.2.6 Lymphoma follow-up

NHL for which a clonal marker has been identified can be followed-up by PCR or FISH at the

request of the treating consultant. This may include:

a) T-cell and B-cell receptor rearrangement studies (by PCR).

b) FISH studies as described under section 6.1.7 (the investigation of suspected

lymphoma)

34

6.3 BMT samples

6.3.1 ASCT

6.3.1.1 ASCT for Plasma Cell Myeloma

6.3.1.1.1 Pre-BMT bone marrow

The aim of the marrow sample is to assess disease burden. This is done by morphological

assessment only (percentage plasma cells on aspirate). The sample should not be sent for

cytogenetics, FISH or flow cytometry.

6.3.1.1.2 Post-BMT bone marrow The aim of the marrow sample is to assess disease burden. This is done by morphological

assessment only (percentage plasma cells on aspirate). If there is evidence of disease (>5%

plasma cells), FISH is required only for patients who did NOT previously have poor risk

cytogenetics.

35

6.3.1.2 ASCT for Lymphoma

An aspirate is usually only indicated pre-transplant if there is evidence of previous marrow

involvement.

Flow cytometry, and FISH if indicated, will be carried out if the lymphoid infiltrate exceeds

20%.

36

6.3.2 Allogeneic transplants

6.3.2.1 Pre-BMT samples

6.3.2.1.1 Allogeneic transplant for AML/MDS/MF

a) Morphology: count >100 cells and record blasts and morphology

b) If a cytogenetic abnormality has been previously identified this should be reassessed

prior to transplant. If no cytogenetic abnormality has been identified and the marrow

is morphologically normal then the sample should be sent to cytogenetics for

processing and storage.

c) Molecular markers are not monitored pre-transplant, irrespective of whether they

were positive or negative at diagnosis/relapse. However, all patients should have DNA

stored for later analysis if required.

d) MDS Bio

37

6.3.2.1.2 Allogeneic transplant for lymphoma

The aspirate should only be performed if the marrow was involved at diagnosis or relapse.

However, many of these patients are heavily pre-treated and an aspirate may be taken to

assess for secondary myelodysplasia.

a) Morphology: count >100 cells and record percentage infiltrate and morphology

b) If dysplasia is detected, then follow suspected MDS protocol (section 6.1.2)

c) For a lymphoid infiltrate >20%, flow cytometry should be performed.

d) ORB

38

6.3.2.1.3 Allogeneic transplant for CLL

These patients are all generally heavily pre-treated and an assessment for 2° MDS is often

undertaken. However, there is no need to request FISH analysis or molecular markers in these

patients as MRD monitoring is more sensitively done by flow cytometry; these samples are all

sent to Leeds HMDS for this assessment.

a) Morphology: count >100 cells and record percentage infiltrate and morphology and

dysplasia

b) If dysplasia is detected, then follow suspected MDS protocol (section 6.1.2)

c) A marrow sample is sent to Leeds HMDS for MRD assessment.

d) ORB

39

6.3.2.1.4 Allogeneic transplant for Plasma Cell Myeloma

As for autografts, FISH including p53 and t(4;14) are only carried out in previously good risk myelomas.

40

6.3.3.2 Post BMT samples

6.3.3.2.1 Non-CLL samples

Chimerism studies will be performed on different samples depending on the patient group.

A. AML/MDS/MF patients have chimerism studies performed both on peripheral blood

CD3 cells and marrow CD34 sorted cells.

B. If a patient had a translocation at diagnosis which is amenable to RQ-PCR this should

also be requested post-transplant (e.g. patient with PML-RARA, BCR-ABL, inv16, 8:21).

C. For acute non-myeloid indications, chimerism studies are only performed on the total

white cells of the marrow sample.

D. Store DNA/RNA

41

6.3.3.2.2 CLL samples

Sensitive MRD monitoring is performed by flow cytometry by Leeds HMDS.

Chimerism studies are performed on total white cells of the marrow sample.

Marrow should be sent for processing and storage by cytogenetics so that analysis can be

performed at a later date if required (e.g. for developing myelodysplasia).

42

6.3.3 Relapse post BMT

Relapse samples should be processed as per the diagnostic algorithms for the appropriate

disease.

43

7. Integrated Reporting (13-1D-105h)

7.1 Liquid samples It is the responsibility of the SIHMDS haematologists to review all results on a weekly

basis and to prepare an integrated report containing a clinical summary for all samples

referred for specialist testing. This will be done within 2 weeks of urgent samples

arriving in the lab. Where some test results are unavailable in this timeframe, an

interim report will be issued. The integrated report is compiled entirely within the

Oxford SIHMDS. It summarises the results of all investigations performed, contains

interpretative comments and a final diagnosis. An assessment of prognosis (where the

scoring system is dependent on test results only) will be provided. It uses the WHO

classification of tumours of haemopoietic and lymphoid tissues (2008) with

appropriate ICD-0-3 codes. It also uses ICD-10 codes, but solely for billing purposes.

The integrated report is authorized by a single haematologist authorised for this

purpose by the head of service. The reports are uploaded into the e-prescribing system

and into the online reporting website (http://oxfordir.oxnet.nhs.uk/).

7.2 Solid samples It is the responsibility of the SIHMDS haemato-pathologist to prepare an integrated

report containing histology, immunostaining results and molecular tests.

The integrated report will be the basis of the MDT discussion. A haemato-pathologist

of the SIHMDS is present at the MDTs. Only raw data of complex cases, especially those

with conflicting results will be presented in the MDT.

44

8. Appendix

8.1 Current named senior staff

SIHMDS Lead Clinician: Anna Schuh

Laboratory Haematology Consultants: Deborah Hay and Susan Kelly

Scientific Leads

Cytogenetics: Carolyn Campbell

Molecular Haematology: Shirley Henderson

Morphology and Flow cytometry: Andrew Platt

Quality Assurance: Andrew Platt

Tumour site specific Clinical Leads

Myeloid: Tim Littlewood

Lymphoid: Chris Hatton and Wale Atoyebi

Haemato-Pathology Lead

Daniel Royston

8.2 Contact details Contact details are available from the website or the SLA/Short user guide http://www.oxford-translational-molecular-diagnostics.org.uk/home.html

Reports website: http://oxfordir.oxnet.nhs.uk/ molpath@nhs.net (for FFPE samples) oxford.molecularhaem@nhs.net (for liquid samples) T:01865 572769

45

8.3 Shipment addresses

8.3.1 Paraffin embedded blocks: Professor Pezzella/ Dr Soilleux / Dr Royston

Department of Cellular Pathology

Level 1, Academic Block

John Radcliffe Hospital

Headley Way

Headington

Oxford OX3 9DU

8.3.1 Liquid samples: Laboratory Haematology

Level 4

John Radcliffe Hospital

Headley Way

Headington

Oxford OX3 9DU

8.4 Request forms and worksheets Diagnostic Request Form

W129 : ORH BM Request Form (Version 1)

W128 : TVHMDS Request Form (Version 2)

Research Request Form

W131 : ORB/MDS Biobank Forms (Research Samples)

Standard Operating Procedure

HC 2309 : Integrated reporting of bone marrow aspirate samples

Diagnostic Worksheet Log

W116 (Version 3) : Bone Marrow Aspirate Sample – Integrated Report Log Sheet

Integrated Bone Marrow Report

46

W130 : Molecular Integrated Report Database

(Oxford BRC Hemato-Oncology Service – Bone Marrow Integrated Report)

Research Request Form Requirements:

47

8.5 WHO codes and scoring systems

8.5.1 WHO classification of tumours of haematopoietic and lymphoid tissues

ICD-03 Codes

9590/3

Lymphoma, NOS Malignant lymphoma, NOS Microglioma

9591/3

B cell lymphoma, NOS Lymphosarcoma, diffuse Lymphosarcoma, NOS Malignant lymphoma, cleaved cell, NOS Malignant lymphoma, diffuse, NOS Malignant lymphoma, lymphocytic, intermediate differentiation, nodular Malignant lymphoma, lymphocytic, poorly differentiated, diffuse Malignant lymphoma, non-cleaved cell, NOS Malignant lymphoma, non-Hodgkin, NOS Malignant lymphoma, small cell, noncleaved, diffuse

Malignant lymphoma, small cleaved cell, diffuse Malignant lymphoma, small cleaved cell, NOS Malignant lymphoma, undifferentiated cell type, NOS Malignant lymphoma, undifferentiated cell, non-Burkitt Non-Hodgkin lymphoma, NOS Reticulosarcoma, diffuse Reticulosarcoma, NOS Reticulum cell sarcoma, diffuse Reticulum cell sarcoma, NOS

9596/3

Composite Hodgkin and non-Hodgkin lymphoma

9650/3

Hodgkin disease, NOS Hodgkin lymphoma, NOS Malignant lymphoma, Hodgkin

9651/3

Classical Hodgkin lymphoma, lymphocyte- rich Hodgkin disease, lymphocyte predominance, diffuse Hodgkin disease, lymphocyte predominance, NOS Hodgkin disease, lymphocytic-histiocytic predominance Hodgkin lymphoma, lymphocyte-rich

9652/3

Classical Hodgkin lymphoma, mixed cellularity, NOS Hodgkin lymphoma, mixed cellularity, NOS#

48

9653/3

Classical Hodgkin lymphoma, lymphocyte depletion, NOS Hodgkin lymphoma, lymphocyte depletion, NOS

9654/3

Classical Hodgkin lymphoma, lymphocyte depletion, diffuse fibrosis Hodgkin lymphoma, lymphocyte depletion, diffuse fibrosis

9655/3

Classical Hodgkin lymphoma, lymphocyte depletion, reticular Hodgkin lymphoma, lymphocyte depletion, reticular

9659/3

Hodgkin lymphoma, lymphocyte predominance, nodular Hodgkin lymphoma, nodular lymphocyte predominance Hodgkin paragranuloma, nodular Hodgkin paragranuloma, NOS

9661/3

Hodgkin granuloma 9662/3

Hodgkin sarcoma 9663/3

Classical Hodgkin lymphoma, nodular sclerosis, NOS

Hodgkin disease, nodular sclerosis, NOS Hodgkin lymphoma, nodular sclerosis, NOS

9664/3

Classical Hodgkin lymphoma, nodular sclerosis, cellular phase Hodgkin lymphoma, nodular sclerosis, cellular phase

9665/3

Classical Hodgkin lymphoma, nodular sclerosis, grade 1 Hodgkin disease, nodular sclerosis, lymphocyte predominance Hodgkin disease, nodular sclerosis, mixed cellularity

Hodgkin lymphoma, nodular sclerosis, grade 1 9667/3

Classical Hodgkin lymphoma, nodular sclerosis, grade 2 Hodgkin disease, nodular sclerosis, lymphocyte depletion Hodgkin disease, nodular sclerosis, syncytial variant Hodgkin lymphoma, nodular sclerosis, grade 2

9670/3

Malignant lymphoma, lymphocytic, diffuse, NOS Malignant lymphoma, lymphocytic, NOS Malignant lymphoma, lymphocytic, well differentiated, diffuse Malignant lymphoma, small B lymphocytic, NOS Malignant lymphoma, small cell diffuse

Malignant lymphoma, small cell, NOS Malignant lymphoma, small lymphocytic, diffuse Malignant lymphoma, small lymphocytic, NOS

9671/3

Immunocytoma

49

Malignant lymphoma, lymphoplasmacytic Malignant lymphoma, lymphoplasmacytoid Malignant lymphoma, plasmacytoid Plasmacytic lymphoma

9673/3

Malignant lymphoma, centrocytic Malignant lymphoma, lymphocytic, intermediate differentiation, diffuse Malignant lymphomatous polyposis Mantle cell lymphoma Mantle zone lymphoma

9675/3

Malignant lymphoma, centroblastic- centrocytic, NOS Malignant lymphoma, centroblastic- centrocytic, diffuse Malignant lymphoma, mixed cell type, diffuse Malignant lymphoma, mixed lymphocytic-histiocytic, diffuse Malignant lymphoma, mixed small and large cell, diffuse

9678/3

Primary effusion lymphoma 9679/3

Mediastinal large B-cell lymphoma Thymic large B-cell lymphoma

9680/3

Anaplastic large B-cell lymphoma Angioendotheliomatosis Angiotropic lymphoma Diffuse large B-cell lymphoma, NOS Histiocyte-rich large B-cell lymphoma Intravascular B-cell lymphoma Intravascular large B-cell lymphoma Malignant lymphoma, centroblastic, diffuse Malignant lymphoma, centroblastic, NOS

Malignant lymphoma, histiocytic, diffuse Malignant lymphoma, histiocytic, NOS Malignant lymphoma, large B-cell, diffuse, centroblastic, NOS Malignant lymphoma, large B-cell, diffuse, NOS Malignant lymphoma, large B-cell, NOS Malignant lymphoma, large cell, noncleaved, diffuse Malignant lymphoma, large cell, cleaved and noncleaved Malignant lymphoma, large cell, cleaved, diffuse Malignant lymphoma, large cell, cleaved, NOS Malignant lymphoma, large cell, diffuse, NOS

Malignant lymphoma, large cell, noncleaved, NOS Malignant lymphoma, large cell, NOS Malignant lymphoma, large cleaved cell, NOS Malignant lymphoma, noncleaved, diffuse, NOS Malignant lymphoma, noncleaved, NOS

50

T-cell rich large B-cell lymphoma T-cell rich/histiocyte-rich large B-cell lymphoma

9684/3

Immunoblastic sarcoma Malignant lymphoma, immunoblastic, NOS Malignant lymphoma, large B-cell, diffuse, immunoblastic, NOS Malignant lymphoma, large cell, immunoblastic Plasmablastic lymphoma

9687/3

Burkitt lymphoma, NOS Burkitt tumor Burkitt-like lymphoma Malignant lymphoma, small noncleaved, Burkitt type Malignant lymphoma, undifferentiated, Burkitt type

9689/3

Splenic lymphoma with villous lymphocytes Splenic marginal zone B-cell lymphoma Splenic marginal zone lymphoma, NOS

9690/3

Follicular lymphoma, NOS

Malignant lymphoma, centroblastic- centrocytic, follicular Malignant lymphoma, follicle center, follicular Malignant lymphoma, follicle center, NOS Malignant lymphoma, follicular, NOS Malignant lymphoma, lymphocytic, nodular, NOS Malignant lymphoma, nodular, NOS

9691/3

Follicular lymphoma, grade 2 Malignant lymphoma, mixed cell type, follicular Malignant lymphoma, mixed cell type, nodular

Malignant lymphoma, mixed lymphocytic-histiocytic, nodular Malignant lymphoma, mixed small cleaved and large cell, follicular

9695/3

Follicular lymphoma, grade 1 Follicular lymphoma, small cleaved cell Malignant lymphoma, lymphocytic, poorly differentiated, nodular Malignant lymphoma, small cleaved cell, follicular

9698/3

Follicular lymphoma, grade 3 Malignant lymphoma, centroblastic, follicular

Malignant lymphoma, histiocytic, nodular Malignant lymphoma, large cell, follicular, NOS Malignant lymphoma, large cell, noncleaved, follicular Malignant lymphoma, large cleaved cell, follicular Malignant lymphoma, lymphocytic, well differentiated, nodular

51

Malignant lymphoma, noncleaved cell, follicular, NOS 9699/3

BALT lymphoma Bronchial-associated lymphoid tissue lymphoma MALT lymphoma Marginal zone B-cell lymphoma, NOS Marginal zone lymphoma, NOS Monocytoid B-cell lymphoma Mucosal-associated lymphoid tissue lymphoma Nodal marginal zone lymphoma

SALT lymphoma Skin-associated lymphoid tissue lymphoma

9700/3

Mycosis fungoides Pagetoid reticulosis

9701/3

Sezary disease Sezary syndrome

9702/3

Lennert lymphoma Lymphoepithelioid lymphoma

Mature T-cell lymphoma, NOS Peripheral T-cell lymphoma, large cell Peripheral T-cell lymphoma, NOS Peripheral T-cell lymphoma, pleomorphic medium and large cell Peripheral T-cell lymphoma, pleomorphic small cell T-cell lymphoma, NOS T-zone lymphoma

9705/3

Angioimmunoblastic lymphoma Angioimmunoblastic T-cell lymphoma

Peripheral T-cell lymphoma, AILD (Angioimmunoblastic 9708/3

Subcutaneous panniculitis-like T-cell lymphoma 9709/3

Cutaneous lymphoma, NOS Cutaneous T-cell lymphoma, NOS

9714/3

Anaplastic large cell lymphoma, CD30+ Anaplastic large cell lymphoma, NOS Anaplastic large cell lymphoma, T cell and Null cell type

Large cell (Ki-1+) lymphoma 9716/3

Hepatosplenic (gamma-delta) cell lymphoma 9717/3

Enteropathy associated T-cell lymphoma

52

Enteropathy type intestinal T-cell lymphoma Intestinal T-cell lymphoma

9718/3

Lymphomatoid papulosis Primary cutaneous anaplastic large cell lymphoma Primary cutaneous CD30+ large T-cell lymphoma Primary cutaneous CD30+ T-cell lymphoproliferative disorder

9719/3

Angiocentric T-cell lymphoma

Malignant midline reticulosis Malignant reticulosis, NOS NK/T-cell lymphoma, nasal and nasal-type Polymorphic reticulosis T/NK-cell lymphoma

9727/3

Lymphoblastoma Malignant lymphoma, convoluted cell Malignant lymphoma, lymphoblastic, NOS Precursor cell lymphoblastic lymphoma, NOS

9728/3

Precursor B-cell lymphoblastic lymphoma 9729/3

Precursor T-cell lymphoblastic lymphoma 9731/3

Plasma cell tumor Plasmacytoma of bone Plasmacytoma, NOS Solitary myeloma Solitary plasmacytoma

9732/3

Multiple myeloma Myeloma, NOS Myelomatosis Plasma cell myeloma

9733/3

Plasma cell leukemia Plasmacytic leukemia

9734/3

Plasmacytoma, extramedullary (not occurring in bone) 9740/1

Mast cell tumor, NOS Mastocytoma, NOS

9740/3

Malignant mast cell tumor Malignant mastocytoma

53

Mast cell sarcoma 9741/3

Malignant mastocytosis Systemic tissue mast cell disease

9742/3

Mast cell leukemia 9750/3

Histiocytic medullary reticulosis Malignant histiocytosis

9751/1

Histiocytosis X, NOS Langerhans cell granulomatosis Langerhans cell histiocytosis, NOS

9752/1

Eosinophilic granuloma Langerhans cell granulomatosis, unifocal Langerhans cell histiocytosis, mono-ostotic

Langerhans cell histiocytosis, unifocal 9753/1

Hand-Schuller-Christian disease Langerhans cell histiocytosis, multifocal

Langerhans cell histiocytosis, poly-ostotic 9754/3

Acute progressive histiocytosis X Langerhans cell histiocytosis, disseminated Langerhans cell histiocytosis, generalized Letterer-Siwe disease Nonlipid reticuloendotheliosis

9755/3

Histiocytic sarcoma True histiocytic lymphoma

9756/3

Langerhans cell sarcoma 9757/3

Dendritic cell sarcoma, NOS Interdigitating cell sarcoma Interdigitating dendritic cell sarcoma

9758/3

Follicular dendritic cell sarcoma Follicular dendritic cell tumor

9760/3

Immunoproliferative disease, NOS 9761/3

Waldenstrom macroglobulinemia 9762/3

Alpha heavy chain disease

54

Franklin disease Gamma heavy chain disease Heavy chain disease, NOS Mu heavy chain disease

9764/3

Immunoproliferative small intestinal disease Mediterranean lymphoma

9765/1

MGUS Monoclonal gammopathy of undetermined significance

Monoclonal gammopathy, NOS 9766/1

Angiocentric immunoproliferative lesion Lymphoid granulomatosis

9767/1

Angioimmunoblastic lymphadenopathy (AIC) Immunoblastic lymphadenopathy (IBL) [obs]

9768/1

T-gamma lymphoproliferative disease 9769/1

Immunoglobulin deposition disease

Primary amyloidosis Systemic light chain disease

9800/3

Aleukemic leukemia, NOS Chronic leukemia, NOS Leukemia, NOS Subacute leukemia, NOS

9801/3

Acute leukemia, NOS Blast cell leukemia

Stem cell leukemia Undifferentiated leukemia

9805/3

Acute bilineal leukemia Acute biphenotypic leukemia Acute mixed lineage leukemia

9820/3

Aleukemic lymphatic leukemia Aleukemic lymphocytic leukemia Aleukemic lymphoid leukemia

Lymphatic leukemia, NOS Lymphocytic leukemia, NOS Lymphoid leukemia, NOS Lymphosarcoma cell leukemia Subacute lymphatic leukemia

55

Subacute lymphocytic leukemia Subacute lymphoid leukemia

9823/3

B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma Chronic lymphatic leukemia Chronic lymphocytic leukemia Chronic lymphocytic leukemia, B-cell type Chronic lymphoid leukemia

9826/3

Acute leukemia, Burkitt type

Acute lymphoblastic leukemia, mature B-cell type B-ALL Burkitt cell leukemia FAB L3

9827/3

Adult T-cell leukemia Adult T-cell leukemia/lymphoma (HTLV-1 positive) Adult T-cell lymphoma Adult T-cell lymphoma/leukemia

9831/1

Large granular lymphocytosis, NOS

NK-cell large granular lymphocytic leukemia T-cell large granular lymphocytic leukemia T-cell large granular lymphocytosis

9832/3

Prolymphocytic leukemia, NOS 9833/3

Prolymphocytic leukemia, B-cell type 9834/3

Prolymphocytic leukemia, T-cell type 9835/3

Acute lymphatic leukemia Acute lymphoblastic leukemia, L2 type, NOS Acute lymphoblastic leukemia, NOS Acute lymphoblastic leukemia, precursor-cell type Acute lymphoblastic leukemia-lymphoma, NOS Acute lymphocytic leukemia Acute lymphoid leukemia FAB L1 FAB L2 Lymphoblastic leukemia, NOS

Precursor cell lymphoblastic leukemia, NOS Precursor cell lymphoblastic leukemia, not phenotyped

9836/3

c-ALL Common ALL

56

Common precursor B ALL Pre-B ALL Precursor B-cell lymphoblastic leukemia Pre-pre-B ALL Pro-B ALL

9837/3

Cortical T ALL Mature T ALL Precursor T-cell lymphoblastic leukemia Pre-T ALL

Pro-T ALL 9840/3

Acute erythremia Acute erythremic myelosis Acute erythroid leukemia Acute myeloid leukemia, M6 type AML M6

Di Guglielmo disease Erythremic myelosis, NOS Erythroleukemia FAB M6

M6A M6B

9860/3

Aleukemic granulocytic leukemia Aleukemic monocytic leukemia Aleukemic myelogenous leukemia Aleukemic myeloid leukemia Chronic monocytic leukemia

Eosinophilic leukemia Granulocytic leukemia, NOS

Monocytic leukemia, NOS Myelocytic leukemia, NOS Myelogenous leukemia, NOS Myeloid leukemia, NOS Myelomonocytic leukemia, NOS Non-lymphocytic leukemia, NOS Subacute granulocytic leukemia Subacute monocytic leukemia Subacute myelogenous leukemia Subacute myeloid leukemia

9861/3

Acute granulocytic leukemia Acute myelocytic leukemia Acute myelogenous leukemia Acute myeloid leukemia, NOS

57

Acute non-lymphocytic leukemia 9863/3

Chronic granulocytic leukemia, NOS Chronic myelocytic leukemia, NOS Chronic myelogenous leukemia, NOS Chronic myeloid leukemia, NOS

9866/3

Acute myeloid leukemia, PML/RAR-alpha Acute myeloid leukemia, t(15:17)(q22 Acute promyelocytic leukemia

Acute promyelocytic leukemia, NOS Acute promyelocytic leukemia, PML/RAR- alpha FAB M3

9867/3

Acute myelomonocytic leukemia FAB M4

9870/3

Acute basophilic leukemia 9871/3

Acute myeloid leukemia with abnormal marrow eosinophils Acute myeloid leukemia, CBF-beta/MYH11

Acute myeloid leukemia, inv(16)(p13;q22) Acute myeloid leukemia, t(16;16)(p13;q11) Acute myelomonocytic leukemia with abnormal eosinophils FAB M4Eo

9872/3

Acute myeloblastic leukemia Acute myeloid leukemia, minimal differentiation FAB M0

9873/3

Acute myeloid leukemia without maturation

FAB M1 9874/3

Acute myeloid leukemia with maturation FAB M2, NOS

9875/3

Chronic granulocytic leukemia, BCR/ABL Chronic granulocytic leukemia, Philadelphia chromosome (Ph1) positive Chronic granulocytic leukemia, t(9 Chronic myelogenous leukemia, BCR/ABL positive Chronic myelogenous leukemia, Philadelphia chromosome (Ph1) positive

Chronic myelogenous leukemia, t(9 9876/3

Atypical chronic myeloid leukemia, BCR/ABL negative Atypical chronic myeloid leukemia, Philadelphia chromosome (Ph1) negative

58

9891/3

Acute monoblastic leukemia Acute monocytic leukemia FAB M5 Monoblastic leukemia, NOS

9895/3

Acute myeloid leukemia with multilineage dysplasia Acute myeloid leukemia with prior myelodysplastic syndrome Acute myeloid leukemia without prior myelodysplastic syndrome

9896/3

Acute myeloid leukemia, AML1(CBF- alpha)/ETO Acute myeloid leukemia, t(8 FAB M2, AML1(CBF-alpha)/ETO FAB M2, t(8

9897/3

Acute myeloid leukemia, 11q23 abnormalities Acute myeloid leukemia, MLL

9910/3

Acute megakaryoblastic leukemia FAB M7 Megakaryocytic leukemia

9920/3

Therapy-related acute myeloid leukemia, alkylating agent related Therapy-related acute myeloid leukemia, epipodophyllotoxin-related Therapy-related acute myeloid leukemia, NOS

9930/3

Chloroma Granulocytic sarcoma Myeloid sarcoma

9931/3

Acute myelofibrosis

Acute myelosclerosis, NOS Acute panmyelosis with myelofibrosis Acute panmyelosis, NOS Malignant myelosclerosis

9940/3

Hairy cell leukemia Hairy cell leukemia variant Leukemic reticuloendotheliosis

9945/3

Chronic myelomonocytic leukemia in transformation

Chronic myelomonocytic leukemia, NOS Chronic myelomonocytic leukemia, Type I Chronic myelomonocytic leukemia, Type II

9946/3

Juvenile chronic myelomonocytic leukemia

59

Juvenile myelomonocytic leukemia 9948/3

Aggressive NK-cell leukemia 9950/3

Chronic erythremia Polycythemia rubra vera Polycythemia vera Proliferative polycythemia

9960/3

Chronic myeloproliferative disease, NOS

Chronic myeloproliferative disorder 9961/3

Agnogenic myeloid metaplasia Chronic idiopathic myelofibrosis Megakaryocytic myelosclerosis Myelofibrosis as a result of myeloproliferative disease Myelofibrosis with myeloid metaplasia Myelosclerosis with myeloid metaplasia

9962/3

Essential hemorrhagic thrombocythemia Essential thrombocythemia

Idiopathic hemorrhagic thrombocythemia Idiopathic thrombocythemia

9963/3

Chronic neutrophilic leukemia 9964/3

Chronic eosinophilic leukemia Hypereosinophilic syndrome

9970/1

Lymphoproliferative disease, NOS Lymphoproliferative disorder, NOS

9975/1

Myeloproliferative disease, NOS 9980/3

Refractory anemia Refractory anemia without sideroblasts

9982/3

RARS Refractory anemia with ringed sideroblasts Refractory anemia with sideroblasts

9983/3

RAEB RAEB I RAEB II Refractory anemia with excess blasts

60

9984/3

RAEB-T Refractory anemia with excess blasts in transformation

9985/3

Refractory cytopenia with multilineage dysplasia 9986/3

Myelodysplastic syndrome with 5q deletion (5q-) syndrome 9987/3

Therapy-related myelodysplastic syndrome, alkylating agent related Therapy-related myelodysplastic syndrome, epipodophyllotoxin-related

Therapy-related myelodysplastic syndrome, NOS 9989/3

Myelodysplastic syndrome, NOS Preleukemia Preleukemic syndrome

61

8.5.2MDS scoring

8.5.2.1 IPSS Score (Greenberg et al, 1997)

Prognostic

variable

0 0.5 1 1.5 2

BM blasts (%) <5 5-10 - 11-20 21-30

Karyotype* Good Intermed. Poor

Cytopenias 0/1 2/3

*Good: normal,-Y,del(5q),del(20q). Poor: complex (>3 abnormalities) or chromosome 7 anomalies.

Intermediate: other abnormalities.

Risk group Score Median survival (yrs) Time to AML

transformation (for

25% in yrs) Low risk 0 5.7 9.4

INT-1 0.5-1.0 3.5 3.3

INT-2 1.5-2.0 1.2 1.1

High risk >2.5 0.4 0.2

8.5.2.2 IPSS-R Score (Greenberg et al, 2012)

62

8.5.3 Myelofibrosis DIPSS-plus

The Dynamic International Prognostic Scoring System (DIPSS) plus prognostic model for primary myelofibrosis (PMF). The DIPSS and prognostic model for PMF uses 8 risk factors for inferior survival: age > 65 years, hemoglobin level < 10 g/dL, leukocyte count > 25 × 109/L, circulating blasts ≥ 1%, presence of constitutional symptoms, presence of unfavorable karyotype, platelet count < 100 × 109/L, and the presence of red cell transfusion need.40 *Please note that a transfusion-dependent patient automatically has 2 risk factors because of transfusion need (1 risk point) and hemoglobin level < 10 g/dL (1 risk point). **Constitutional symptoms constitute weight loss > 10% of baseline value in the year preceding diagnosis, unexplained fever, or excessive sweats persisting for > 1 month.38 ***Unfavorable karyotype constitutes complex karyotype or sole or 2 abnormalities that include +8, −7/7q−, i(17q), inv(3), −5/5q− 12p−, or 11q23 rearrangement. (ref: Teferri, Blood 2011)

63

8.5.4 CML response criteria (ELN website)

64

8.5.5 Aplastic Anaemia severity score (BCSH guidelines, 2009)

65

9. Abbreviations ALL: acute lymphoblastic leukaemia

AML: acute myeloid leukaemia

ASCT: autologous stem cell transplant

BRC: Biomedical Research Centre

BM: bone marrow

BMT: bone marrow transplant

CGN: cytogenetics

CLL: chronic lymphocytic leukaemia

CML: chronic myeloid leukaemia

CMML: chronic myelomonocytic leukaemia

EDTA: Ethylenediaminetetraacetic acid

ELN: European Leukaemia Network

ET: essential thrombocythemia

EQA schemes: externalquality assessment

FISH: fluorescence in situ hybridisation

GLP: good laboratory practice

IT: information technology

LIMS: laboratory information management system

LN: lymph node

LPD: lymphoproliferative disease

MDS: myelodysplastic syndrome

MDT: multidisciplinary team meeting

MF: myelofibrosis

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MGG: May-Grunwald-Giemsa

MPN: myeloproliferative neoplasms

MRD: minimal residual disease

MUD: matched unrelated donor

NCAT: national cancer action team

NHL: Non-Hodgkin’s lymphoma

NICE: national institute for clinical excellence

ORB: Oxford Radcliffe Biobank

OUH: Oxford University Hospitals

PB: peripheral blood

PCR: polymerase chain reaction

PRV: polycythaemia rubra vera

RQ-PCR: real-time quantitative PCR

SIHMDS: specialist integrated haematological malignancy diagnostic service

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10. References 1. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T,

Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J. (1997). International scoring

system for evaluating prognosis in myelodysplastic syndromes. Blood 89(6):2079-88.

2. Marsh JCW, Ball SE, Cavenagh J, Darbyshire P, Dokal I, Gordon-Smith ECKeidan J, Laurie A,

Martin A, Mercieca J, Killick SB, Stewart R, Yin JAL (2009). Guidelines for the diagnosis and

management of aplastic anaemia. Brit J Haematol 147:43-17.

3. Tefferi A. (2011) How I treat myelofibrosis. Blood 117(13):3494-3504.

4. http://www.cmlalliance.net/treating-goals-ph-cml/eln-recommendations.jsp