Specialist Integrated Haematological Malignancy Diagnostic Service (SIHMDS)

Transcription

1 Specialist Integrated Haematological Malignancy Diagnostic Service (SIHMDS) Operational Policy Version

2 Operational Policy for the Oxford NHS/BRC SIHMDS 1. Introduction Objectives Objectives of the SIHMDS process Objectives of this Operational policy General Operational Principles Detailed description of work- flows and procedures governing the SIHMDS Central Specimen Reception Contact details and shipment address Request forms Timing of requests Specimens Bone marrow samples Peripheral blood for DNA analysis Peripheral blood for RNA analysis Initial morphological assessment of samples Liquid samples Paraffin embedded material Molecular tests Clonality studies Clinical Trial Samples Algorithms to guide the requesting of additional tests At suspected diagnosis Suspected AML Suspected MDS Suspected MPN Suspected CML Suspected PRV/ET Suspected Myelofibrosis Suspected other MPNs Suspected ALL Suspected CLL/LPD Suspected Multiple Myeloma Suspected Lymphoma Follow- up samples AML follow- up MDS follow- up CML follow- up ALL follow- up CLL follow- up Lymphoma follow- up BMT samples ASCT ASCT for Multiple Myeloma Pre- BMT bone marrow

3 Post- BMT bone marrow Allogeneic transplants Pre- BMT samples Allogeneic transplant for AML/MDS/MF Allogeneic transplant for lymphoma Allogeneic transplant for CLL Allogeneic transplant for Multiple Myeloma Post BMT samples Non- CLL samples CLL samples Relapse post BMT Integrated Reporting Liquid samples Solid samples Appendix Contact details Shipment addresses Request forms Research Request Form...39 Requirements: WHO codes and scoring systems WHO Mature B cell neoplasms WHO Mature T cell and NK cell neoplasms WHO Myeloproliferative neoplasms (MPN) WHO Myelodysplastic syndrome (MDS) WHO Acute Myeloid Leukemia and related neoplasms WHO B Lymphoblastic leukemia/lymphoma WHO T Lymphoblastic leukemia/lymphoma MDS IPSS score Myelofibrosis DIPSS- plus Abbreviations

4 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 follow- up of patients with haematological malignancies and to produce a single interpretative report within a time frame that is of clinical usefulness. 1.1 Objectives 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, immuno- phenotyping, molecular genetics (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 GLP To guarantee speedy and accurate processing of research samples 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 4

5 2. General Operational Principles The laboratory is designated by the commissioning groups and by the Thames Valley Cancer Network and is managed by the Oxford University Hospitals (OUH) and the Oxford Biomedical Research Centre. It is directed by a single head of service who reports to the Chief Executive of OUH. His/her list of responsibilities includes the design of investigational algorithms, the use of resources for diagnostics and research, and the standards of reporting. The laboratory has a single central specimen reception and issues a single integrated report including morphology, histology, immunochemistry, flow cytometry, cytogenetics, FISH and molecular studies. The different sections of the laboratory are joined up by a lab IT system that covers all investigational pathways, live sample tracking, redirection of sample at any time point depending on results at that point, pathway report generation, diagnostic coding (according to WHO classification), summary of all results on a single screen to facilitate authorisation by a single haematologist/pathologists, and on- line communication of results with users. 5

6 3. Detailed description of work- flows and procedures governing the SIHMDS OVERVIEW OF SOLID SAMPLE PATHWAYS 6

7 OVERVIEW OF LIQUID SAMPLE PATHWAYS 4. Central Specimen Reception 4. 1 Contact details and shipment address See appendix Request forms See appendix. SIHMDS request forms are also available from our website: translational- molecular- diagnostics.org.uk/home.html and from the OUH website: em/molhaem.aspx 7

8 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 send 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 Bone marrow samples 10ml of EDTA and 2 unstained or stained bedside smears Peripheral blood for DNA analysis 4ml of EDTA Peripheral blood for RNA analysis 12ml of EDTA Histopathological samples Paraffin embedded blocks or micron cut sections in eppendorf tubes. Unstained paraffin- embedded mounted and marked 4 micron sections for micro- dissection. Stained paraffin- embedded mounted sections. NB All cases paraffin embedded specimens require morphological assessment prior to any molecular testing and, if possible, all stained and unstained material should be forwarded to Oxford, including a copy of any report and full clinical details. 8

9 5. Initial morphological assessment of samples 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. haematopathologists to discuss the case as soon as it arrives at SIHMDS. Please contact one of the 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 1-2x10 7 cells will be dispatched for each test. All samples for research will be processed immediately to guarantee cell viability. Generally, 1x10 7 cells for flow cytometry, 2x10 7 cells for cytogenetics, 1x10 6 cells for DNA preparation and 1x10 7 cells for RNA preparation and ensure that the remainder of sample is banked. Tissue bankers are expected to collect research sample as soon as the sample is 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 from 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. The registrar reviewing the marrow and arranging further tests 9

10 should print the latest clinic letter and append it to the blue folder in order to provide maximum clinical context for the interpretation of test results. 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 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 Clonality studies B and T- cell receptor clonality analysis by PCR on paraffin embedded material and cytological specimens are carried out. 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 allow speedy sample preservation. 10

11 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 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. 6. Algorithms to guide the requesting of additional tests In addition to review of morphology and histology, the SIHMDS will perform additional tests when indicated with a view of providing and integrated and interpretative report. The SIHMDS will follow national and international consensus guidelines and NICE recommendations whenever possible. Disease 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

12 6.1 At suspected diagnosis 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. d) In cases where APL is suspected, FISH for PML- RARA (Churchill CGN lab) and PML body staining (JR Molecular Diagnostics) should be performed urgently. RQ- PCR for PML- RARA is done in JR Molecular Diagnostics. e) All patients should be tested for FLT3 TKI and internal tandem repeat (ITD) mutations as well as NPM1 mutations and c- kit if inv16 positive (Molecular). f) If M2 morphology is seen, RQ- PCR for t(8;21) breakpoint, and inv(16) in M4. g) All diagnostic samples will be stored as DNA and RNA to allow quantitative molecular monitoring should this be indicated. h) All samples will be stored under MDS Bio i) High throughput sequencing of a panel of genes known to be mutated in AML (eg RUNX1, TET1/2, IDH1/2, DNMT3A etc) is in development. This may be requested on a research basis from now, with the clear understanding that the results cannot at this time be used for clinical decision making. 12

13 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. b) Where the blast count exceeds 20% AML is diagnosed and the AML flowchart to be followed. c) Cytogenetics. The person requesting the marrow will indicate whether the sample is urgent in which case it will be done in 2 weeks, or non- urgent in which case it will be done in 4 weeks. The SpR requesting the tests should then indicate this on the integrated report worksheet. d) Iron stains. These should be reported on LIMS. e) MDS Bio f) High throughput sequencing of a panel of genes known to be mutated in MDS/AML (eg RUNX1, TET1/2, IDH1/2, CBL, ASXL1 etc) is in development. This may be requested on a research basis from now, with the clear understanding that the results cannot at this time be used for clinical decision making. 13

14 6.1.3 Suspected MPN Suspected CML Samples should be evaluated as follows: a) Cytogenetics & FISH for BCR- ABL (all done at Churchill CGN) b) DNA analysis for JAK2 V617F in BCR- ABL negative cases (Molecular) c) BCR- ABL multiplex & RQ- PCR for BCR- ABL (Molecular) d) DNA and RNA samples will be stored on all cases of suspected CML e) MDS Bio 14

15 Suspected PRV/ET All samples will be tested for: a) JAK2 V617F mutations. Allelic burden will be reported as WT:mutant ratio. b) On request, JAK2 exon 12 mutation (PRV only) analysis or MPL mutation (ET only) analysis will be performed. JAK2 exon 12 should be done on marrow rather than peripheral blood if possible. c) RQ- PCR for BCR- ABL will be performed in ET JAK2 mutation negative patients. 15

16 Suspected Myelofibrosis All patients should have a) Karyotyping if MF suspected b) DNA analysis for JAK2 V617F (Molecular) c) DNA analysis for MPL if JAK2 ve (Molecular) d) MDS Bio 16

17 Suspected other MPNs These need to discussed by the clinical consultant with Dr Schuh 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 testing 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 17

18 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) DNA and RNA will be stored should BCR- ABL monitoring be indicated. e) ORB 18

19 6.1.5 Suspected CLL/LPD Peripheral blood a) All cases with suspected LPD, ie with persistent lymphocytosis of >5x10 9 /l for longer then 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. This is done in Molecular Diagnostics. 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 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. 19

20 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. 20

21 6.1.6 Suspected Multiple Myeloma All cases should have: a) Morphology to confirm >5% plasma cells b) Flow cytometry and CD138 sort if >5% plasma cells c) Cytospin CD138 cases for FISH analysis for del17p and t(4;14) d) Storage of CD138 cells under ORB 21

22 6.1.7 Suspected Lymphoma The diagnosis of NHL and Hodgkin s lymphoma relies on histological assessment of tissue biopsies and immunostaining. Pathologists might 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 for rearrangements of: c- MYC t(8;14) cases with features diagnostic or suspicious of Burkitt s lymphoma; diffuse large B- cell lymphomas with a proliferation index >80% to identify double hit lymphomas. 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; diffuse large B- cell lymphomas with a proliferation index >80% to identify double hit lymphomas (request at the same time as requesting FISH for a MYC rearrangement). 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, provided these are treated by Oxford or regional haematologists. If the patient is being treated for a gastric marginal zone lymphoma of MALT type, the Oxford gastroenterologists do not want us to do this test. ALK- 1 t(2;5) suspected cases of ALK- 1+ anaplastic large cell lymphoma, in which immunohistochemistry is inconclusive. P53 cases of chronic lymphocytic leukaemia/ small lymphocytic lymphoma at the request of the MDT meeting only. 22

23 6.2 Follow- up samples 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 (JR Molecular). The person 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. NPM1 molecular monitoring will be available shortly, and assay validation is in progress. 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) Patients who had been in previous CR and now have relapsed should have all the tests as indicated in diagnostic samples e) MDS Bio 23

24 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, flow cytometry with the full panel of ELN markers to be used. c) Cytogenetics d) Iron stains for patients who had ring sideroblasts e) MDS Bio 24

25 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 (JR Molecular) 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 at least be considered as per ELN guidelines d) Suboptimal responders and treatment failures: 3 monthly monitoring by the appropriate test. Kinase mutations should be excluded. T315I will be routinely done. If negative, sequencing of the kinase domain will be carried out. e) MDS Bio 25

26 6.2.4 ALL follow- up BCR- ABL positive cases should be monitored for BCR- ABL transcript levels by RQ- PCR (paired PB and BM- JR Molecular). Patients with abnormal cytogenetics should be monitored by cytogenetics/fish until they become negative. 26

27 6.2.5 CLL follow- up Follow- up samples prior to treatment should be tested for del17p and TP53 mutation analysis (JR Molecular). MRD monitoring by flow cytometry is not performed routinely (except post transplant, see relevant section). However, on request of the treating physician s samples can be forwarded to Leeds HMDS. 27

28 6.2.6 Lymphoma follow- up NHL that tested positive at diagnosis for a particular marker might be followed- up by PCR or FISH: T- cell and B- cell receptor rearrangement studies (by PCR). If possible, aspirates should be sent for this analysis, as bone marrow trephines often do not yield sufficient DNA quality. In JR Molecular Diagnostic lab, FISH studies for rearrangements of: c- MYC(8;14) bcl- 2(18;14) cyclin D1(11;14) MALT1(11;18) ALK- 1(2;5). The necessity for this will be considered on an individual basis, often following MDT discussion. 28

29 6.3 BMT samples ASCT ASCT for Multiple Myeloma Pre- BMT bone marrow The aim of the marrow sample is to assess disease burden. This is done by morphological assessment only (% plasma cells on aspirate). The sample should not be sent for cytogenetics, FISH or flow cytometry Post- BMT bone marrow The aim of the marrow sample is to assess disease burden. This is done by morphological assessment only (% plasma cells on aspirate). If the patient was poor risk (p53 del, t(4,14)) at diagnosis, there is no need to repeat the FISH. If, however, the patient was good risk, do need to do the FISH. 29

30 ASCT for Lymphoma The aspirate will only be done if the marrow was involved at diagnosis or relapse. If an aspirate is taken pre- transplant and the patient s marrow was not involved at diagnosis or relapse, the slides will be stored unexamined and no further tests will be requested on the aspirate. If the marrow was previously involved, the slides will be examined and flow cytometry will be carried out if the lymphoid infiltrate exceeds 20%. 30

31 6.3.2 Allogeneic transplants Pre- BMT samples Allogeneic transplant for AML/MDS/MF a) Morphology: count >100 cells and record blasts and morphology b) If abnormal karyotype/fish at diagnosis or relapse, check again pre- transplant. Otherwise send and store for cytogenetics. 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 31

32 Allogeneic transplant for lymphoma The aspirate should only be done 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 % infiltrate and morphology b) Send all aspirates for cytogenetics to detect 2 MDS. c) For a lymphoid infiltrate >20%, flow cytometry may be considered (eg poor or no trephine taken). d) ORB 32

33 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 do FISH or molecular markers in these patients as MRD monitoring is more sensitively done by flow cytometry and these samples are all sent to Leeds HMDS for this assessment. a) Morphology: count >100 cells and record % infiltrate and morphology and dysplasia b) Send all aspirates for cytogenetics to detect 2 MDS. c) No molecular markers d) All samples to Leeds HMDS. e) ORB 33

34 Allogeneic transplant for Multiple Myeloma As for autografts, Molecular tests (FISH for p53 and t(4;14))are only carried out in previously good risk myelomas. 34

35 Post BMT samples Non- CLL samples Chimerism studies are different depending on the patient group. Falling levels of % donor levels especially in T- deplete MUDs are an indication for DLI to reduce relapse. A. AML/MDS/MF patients have chimerism studies done both on peripheral blood CD3 cells and marrow CD34 sorted cells (just write chimerism studies in the molecular tests section). B. For patients with a translocation that can be followed- up by RQ- PCR across the breakpoint (PML- RARA, BCR- ABL, inv16, 8:21), then please do NOT request chimerism studies, but request RQ- PCR for that breakpoint, as this is a far more sensitive test than chimerism. THIS INCLUDES PH+ ALL. C. For non- myeloid indications, chimerism studies are only done on CD3 cells from peripheral blood (just write chimerism studies in the molecular tests section). ****Please note: Chimerism studies are NEVER carried out by cytogenetics, even where there is a sex mismatch between donor and recipient (not very sensitive technique). **** 35

36 CLL samples MRD monitoring is sensitively monitored by flow cytometry and these samples are all sent to Leeds HMDS for this assessment. Chimerism studies are done on peripheral blood CD3 cells. Cytogenetics will be stored for later analysis if myelodysplasia becomes a question Relapse post BMT At relapse, a sample should be treated as a newly diagnosed sample would be, following those workflows for ordering further tests. 36

37 7. Integrated Reporting 7.1 Liquid samples It is the responsibility of the SIHMDS haematologist to review all results on a weekly basis and to prepare an integrated report containing a clinical summary. This will be done within 2 weeks of the samples arriving in the lab. Where some test results are unavailable in this timeframe, an interim report will be issued. The reports will be uploaded into ARIA. The WHO classification will be followed, and an assessment of prognosis (where the scoring system is dependent on test results only) will be provided. 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. Only raw data of complex cases, especially those with conflicting results will be presented in the MDT. 37

38 8. Appendix 8.1 Contact details (for FFPE samples) (for liquid samples) 8.2 Shipment addresses Paraffin embedded blocks: Professor Gatter/ Professor Pezzella/ Dr Soilleux Department of Cellular Pathology Level 1, Academic Block John Radcliffe Hospital Headley Way Headington Oxford OX3 9DU Liquid samples: Laboratory Haematology Level 4 John Radcliffe Hospital Headley Way Headington Oxford OX3 9DU 8.3 Request forms Diagnostic Request Form W129 : ORH BM Request Form (Version 1) W128 : TVHMDS Request Form (Version 2) 38

39 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 W130 : Molecular Integrated Report Database (Oxford BRC Hemato- Oncology Service Bone Marrow Integrated Report) Research Request Form Requirements: Designation of the SIHMDS head Daily sample transport to regional hospitals Specimen reception open on Saturday Internal lab IT support to allow access of early reports in each section Web based or ARIA based reporting system. 39

40 8.4 WHO codes and scoring systems WHO 2008 Diseases shown in italics are newly included in the 2008 WHO classification. *These represent provisional entities or provisional subtypes of other neoplasms WHO Mature B cell neoplasms Chronic lymphocytic leukemia/small lymphocytic lymphoma B- cell prolymphocytic leukemia Splenic marginal zone lymphoma Hairy cell leukemia Splenic lymphoma/leukemia, unclassifiable Splenic diffuse red pulp small B- cell lymphoma* Hairy cell leukemia- variant* Lymphoplasmacytic lymphoma Waldenström macroglobulinemia Heavy chain diseases Alpha heavy chain disease Gamma heavy chain disease Mu heavy chain disease Plasma cell myeloma Solitary plasmacytoma of bone Extraosseous plasmacytoma Extranodal marginal zone B- cell lymphoma of mucosa- associated lymphoid tissue (MALT lymphoma) Nodal marginal zone B- cell lymphoma (MZL) Pediatric type nodal MZL Follicular lymphoma Pediatric type follicular lymphoma Primary cutaneous follicle center lymphoma Mantle cell lymphoma 40

41 Diffuse large B- cell lymphoma (DLBCL), not otherwise specified T cell/histiocyte rich large B- cell lymphoma DLBCL associated with chronic inflammation Epstein- Barr virus (EBV) + DLBCL of the elderly Lymphomatoid granulomatosis Primary mediastinal (thymic) large B- cell lymphoma Intravascular large B- cell lymphoma Primary cutaneous DLBCL, leg type ALK + large B- cell lymphoma Plasmablastic lymphoma Primary effusion lymphoma Large B- cell lymphoma arising in HHV8- associated multicentric Castleman disease Burkitt lymphoma B- cell lymphoma, unclassifiable, with features intermediate between diffuse large B- cell lymphoma and Burkitt lymphoma B- cell lymphoma, unclassifiable, with features intermediate between diffuse large B- cell lymphoma and classical Hodgkin lymphoma Hodgkin Lymphoma Nodular lymphocyte- predominant Hodgkin lymphoma Classical Hodgkin lymphoma Nodular sclerosis classical Hodgkin lymphoma Lymphocyte- rich classical Hodgkin lymphoma Mixed cellularity classical Hodgkin lymphoma WHO Mature T cell and NK cell neoplasms T- cell prolymphocytic leukemia T- cell large granular lymphocytic leukemia Chronic lymphoproliferative disorder of NK- cells* Aggressive NK cell leukemia 41

42 Systemic EBV + T- cell lymphoproliferative disease of childhood (associated with chronic active EBV infection) Hydroa vacciniforme- like lymphoma Adult T- cell leukemia/lymphoma Extranodal NK/T cell lymphoma, nasal type Enteropathy- associated T- cell lymphoma Hepatosplenic T- cell lymphoma Subcutaneous panniculitis- like T- cell lymphoma Mycosis fungoides Sézary syndrome Primary cutaneous CD30 + T- cell lymphoproliferative disorder Lymphomatoid papulosis Primary cutaneous anaplastic large- cell lymphoma Primary cutaneous aggressive epidermotropic CD8 + cytotoxic T- cell lymphoma* Primary cutaneous gamma- delta T- cell lymphoma Primary cutaneous small/medium CD4 + T- cell lymphoma* Peripheral T- cell lymphoma, not otherwise specified Angioimmunoblastic T- cell lymphoma Anaplastic large cell lymphoma (ALCL), ALK + Anaplastic large cell lymphoma (ALCL), ALK * *These represent provisional entities or provisional subtypes of other neoplasms. Diseases shown in italics are newly included in the 2008 WHO classification WHO Myeloproliferative neoplasms (MPN) Chronic myelogenous leukemia, BCR- ABL1 positive Chronic neutrophilic leukemia Polycythemia vera Primary myelofibrosis Essential thrombocythemia Chronic eosinophilic leukemia, not otherwise specified Mastocytosis Myeloproliferative neoplasms, unclassifiable Myeloid and lymphoid neoplasms associated with eosinophilia and abnormalities of PDGFRA, 42

43 PDGFRB, or FGFR1 Myeloid and lymphoid neoplasms associated with PDGFRA rearrangement Myeloid neoplasms associated with PDGFRB rearrangement Myeloid and lymphoid neoplasms associated with FGFR1 abnormalities Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) Chronic myelomonocytic leukemia Atypical chronic myeloid leukemia, BCR- ABL1 negative Juvenile myelomonocytic leukemia Myelodysplastic/myeloproliferative neoplasm, unclassifiable Provisional entity: refractory anemia with ring sideroblasts and thrombocytosis WHO Myelodysplastic syndrome (MDS) Refractory cytopenia with unilineage dysplasia Refractory anemia Refractory neutropenia Refractory thrombocytopenia Refractory anemia with ring sideroblasts Refractory cytopenia with multilineage dysplasia Refractory anemia with excess blasts Myelodysplastic syndrome with isolated del(5q) Myelodysplastic syndrome, unclassifiable Childhood myelodysplastic syndrome Provisional entity: refractory cytopenia of childhood WHO Acute Myeloid Leukemia and related neoplasms Acute myeloid leukemia with recurrent genetic abnormalities 43

44 AML with t(8;21)(q22;q22); RUNX1- RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB- MYH11 APL with t(15;17)(q22;q12); PML- RARA AML with t(9;11)(p22;q23); MLLT3- MLL AML with t(6;9)(p23;q34); DEK- NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1- EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15- MKL1 Provisional entity: AML with mutated NPM1 Provisional entity: AML with mutated CEBPA Acute myeloid leukemia with myelodysplasia- related changes Therapy- related myeloid neoplasms Acute myeloid leukemia, not otherwise specified AML with minimal differentiation AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Acute erythroid leukemia Pure erythroid leukemia Erythroleukemia, erythroid/myeloid Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis 44

45 Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm Acute leukemias of ambiguous lineage Acute undifferentiated leukemia Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); BCR- ABL1 Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged Mixed phenotype acute leukemia, B- myeloid, NOS Mixed phenotype acute leukemia, T- myeloid, NOS Provisional entity: natural killer (NK) cell lymphoblastic leukemia/lymphoma WHO B Lymphoblastic leukemia/lymphoma B lymphoblastic leukemia/lymphoma, NOS B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities B lymphoblastic leukemia/lymphoma with t(9;22)(q34;q11.2);bcr- ABL 1 B lymphoblastic leukemia/lymphoma with t(v;11q23);mll rearranged B lymphoblastic leukemia/lymphoma with t(12;21)(p13;q22) TEL- AML1 (ETV6- RUNX1) B lymphoblastic leukemia/lymphoma with hyperdiploidy B lymphoblastic leukemia/lymphoma with hypodiploidy B lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32) IL3- IGH B lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3);tcf3- PBX1 45

46 8.4.6 WHO T Lymphoblastic leukemia/lymphoma 46

47 8.4.7 MDS IPSS score (Greenberg 1997) Prognostic variable BM blasts (%) < 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 INT INT High risk > 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 > /L, circulating blasts 1%, presence of constitutional symptoms, presence of unfavorable karyotype, platelet count < /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) 47

48 8.4.9 CML response criteria (ELN website) Aplastic Anaemia severity score (BCSH guidelines, 2009) 48

49 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 49

50 MF: myelofibrosis 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 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): 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: Tefferi A. (2011) How I treat myelofibrosis. Blood 117(13): goals- ph- cml/eln- recommendations.jsp 50