Molecular Diagnostics Centre

Transcription

1 Directorate of Central Manchester Haematology Service Molecular Diagnostics Centre Haemato-Oncology User Guide 2014 Page 1 of 13

2 Directorate of CONTENTS About Us 3 Contact Details 3 Location 3 Postal Address 3 Working Hours 4 Quality Statement 4 Accreditation 4 Services Available 4 Specimen Acceptance policy 5 Specimen Requirements 7 Reporting Laboratory Results 8 Clinical Scientific Diagnostic Advice and Results Interpretation 8 Haemato-Oncology Tests 8 Chronic Myeloid Leukaemia 8 Drug Resistance 9 Acute Lymphocytic Leukaemia 9 Acute Myeloid Leukaemia 9 CBF Leukaemias 9 Acute Promyelocytic Leukaemias 10 Normal Karyotype AML testing 10 Myeloproliferative Disorders 10 Polycythaemia Vera 10 Essential Thrombocythaemia 11 Idiopathic Myelofibrosis 11 B and T Cell Lymphoma 11 Clinical Trials samples 12 Future testing under development 12 Tests to be implemented and under evaluation 12 Tests to be offered as part of future envisaged developments 12 Service development and user engagement 13 Page 2 of 13

3 Directorate of About Us We are a nationally recognised laboratory, specialising in the field of haematological malignancies. Our remit is multi fold, contributing to the diagnosis of leukaemia and lymphoma patients using molecular genetics. Our team also monitor treatment responses, detect early signals of relapse, and prognostically profile these patients to contribute to their risk stratification for therapy. This is in line with personalised medicine in haemato-oncology, with changing therapeutic options, such as the number of chemotherapy courses administered, molecular targeted therapies and various stem cell transplantation regimens. Contact Details For results enquiries or if you have a general laboratory query please contact staff on: Office telephone: Laboratory telephone: Fax: For specialist clinical scientific advice, please contact the lead for the discipline: Dr Abida Awan Office telephone: abida.awan@cmft.nhs.uk Location The laboratory is located on the top floor of the CADET & MDC Building, which resides behind Cobbett House at the end of the adult mortuary corridor. Postal Address 2nd Floor CADET and MDC Building Central Manchester University Hospitals NHS Foundation Trust Manchester Royal Infirmary Oxford Road Manchester M13 9WL Page 3 of 13

4 Directorate of Working Hours The laboratory is open from 8.00am to 5.30pm. The nature of the genetics work involves assays on both RNA and DNA isolated from EDTA stabilised collection tubes and biopsy material. Due to the labile nature of RNA, and requirements of intact cells for DNA work, samples must be received within the recommended timescale of being taken from the patient (refer to Specimen Requirements page 7). Fresh samples Peripheral blood, bone marrow aspirate and fresh / frozen biopsy material must be received as soon as possible with a cut off limit of 3.30pm to achieve processing towards stabilisation. Staff will support clinical teams to receive a sample until a final cut off of 4.30pm in an emergency and process this out of hours. However this would need to be communicated to the team with prior notice in place to be able to implement an out of hours arrangement to accommodate this type of sample. The result and its reliability are profoundly affected by the sample transit time and its storage conditions. Fixed tissue (FFPE) Biopsy material such as tissue and bone marrow trephines, that are formalin fixed paraffin embedded tissue, can be received by 5.30pm because they are stabilised and only require immediate storage prior to testing. Quality Statement The investigations are undertaken within published guidelines of agreed standards of practise. Participation in External Quality Assurance (EQA) schemes, such as NEQAS, demonstrates that acceptable standards are achieved. Additionally, the team is a participant in the UK MRD standardisation group, the European Leukaemia Network and has been involved in European directives based upon quality improvement programmes in the field of RNA and DNA. Accreditation The laboratory is accredited with CPA (UK) Ltd as part of the Department of Haematology at Central Manchester University Hospitals. The CPA accredited laboratory reference number is Our staff are HPC registered clinical scientists and genetic technologist staff, registered on the voluntary genetics council, or are staff in training towards these. Services Available Molecular genetics services are provided for the haematological malignancies. There are genetic targets found in tumours in areas other than haematology, where the work Page 4 of 13

5 Directorate of can be transferable to this setting. The use of protocols to extract genetic material from biopsy material, received from fresh and FFPE tissue samples, enables us to analyse these genetic lesions in other cancers if requested or is appropriate. Specimen Acceptance Policy The laboratory operates a Specimen Acceptance Policy in accordance with the requirements of the Specimen Acceptance Policy of the Directorate of Laboratory Medicine at CMFT. This policy follows national directives from laboratory associated professional bodies. It is the responsibility of the requestor to ensure that samples are correctly labelled and request forms are completed to the required standard. All samples and request cards must include sufficient information to allow us to identify unequivocally the patient that the sample has come from, to ensure that the sample and card relate to the same person and to identify which investigations are required. If appropriate information is not provided it may not be possible to analyse a sample or to interpret test results. Appropriate contact information is required to ensure that reports are provided to the requester at the correct location. Non-compliance with the sample acceptance policy will result in requests for investigations being delayed or rejected. N.B. In order to comply with the requirements of the Human Tissue Act, the referring clinician has a responsibility to ensure that appropriate informed consent has been obtained before any genetic testing is undertaken. The laboratory must be informed of any restrictions to this consent (eg consent to store sample, use of an individual s genetic information in service development protocols). Page 5 of 13

6 Directorate of Information on a test request card: Patient sample identification Inadequately labelled or unlabelled samples will not be analysed Samples and request forms MUST be labelled with: Surname Forename Date of birth Hospital record number, NHS number or other unique identifier Please state the sample type (peripheral blood, bone marrow aspirate or tissue source for biopsy samples) Other required information Investigation required Relevant clinical information this is required for appropriate interpretation of results Signature of referring clinician or authorised requester Indication of any restrictions on consent Date of sample collection Sex of patient Address for report Referring Consultant Name of requestor and contact telephone number Page 6 of 13

7 Directorate of Specimen Requirements Investigation Acute Myeloid Leukaemia Inversion 16 (Inv 16) AML1 ETO t(8;21) NPM1 * FLT3 ITD * * See section below on NPM1 and FLT3 monitoring Acute Promyelocytic Leukaemia PML-RARA t(15;17) Chronic Myeloid Leukaemia and ALL BCR-ABL t(9;22) For ABL kinase mutation screen Discuss with the lab Myeloproliferative disorders PV, ET and IMF JAK2 V617F MPL W515L/K For JAK2 exon 12 and CALR Discuss with the lab B and T Cell Lymphoma Immunoglobulin and T cell receptor clonality analysis Blood sample requirement mls EDTAanticoagulated peripheral blood and 2-3 mls EDTAanticoagulated bone marrow (see AML/APL section page 9 for sample requirements) mls EDTAanticoagulated peripheral blood and 2-3 mls EDTAanticoagulated bone marrow (see AML/APL section page 9 for sample requirements) mls EDTAanticoagulated peripheral blood and / or 2-3 mls EDTA-anticoagulated bone marrow when required (see CML and ALL section page 8 for sample requirements) mls EDTAanticoagulated peripheral blood (see MPD section page 10 for sample requirements) Formalin fixed parafin embedded tissue. (see lymphoma section page 11 for requirements) For peripheral blood screen 10-20ml EDTA PB or a bone marrow aspirate screen 2-3ml EDTA BM Result / report available 2 Weeks For clinical urgency based upon priority clinical needs, a request can be made, and a 1 week turn around time arranged with the Lab 2 Weeks For clinical urgency based upon priority clinical needs, a request can be made, and a 1 week turn around time arranged with the Lab 3 weeks for CML 2 weeks for ALL For clinical urgency based upon priority clinical needs, a request can be made, and a 1 week turn around time arranged with the Lab 3 weeks For clinical urgency based upon priority clinical needs, a request can be made, and a 1 week turn around time arranged with the Lab 10 working days Page 7 of 13

8 Directorate of It is advisable not to post samples to the laboratory on a Friday or on the day preceding a bank holiday because the laboratory does not currently offer a weekend or bank holiday service. Please store samples at 4 0 C before sending, (especially if required to store overnight prior to dispatch) do not freeze the sample at -20 o C. The accurate monitoring of MRD from RNA requires the samples to be fresh, to reach us ideally within 24 hours of phlebotomy and no later than 48 hours. Assays performed on samples received after 48 hours of being taken has a profound effect on the sensitivity / reliability of the result. The above also applies to samples where DNA is isolated for optimum results. However samples for DNA analysis received up to 72 hours after being taken may yield data, which will be assessed on an individual basis to ensure they meet internal QC requirements to be issued. NB: For all other test requests, please contact the MDC laboratory oncology staff for advice, where existing stored genetic material can be sent to our collaborative centres for additional specialist testing if not currently offered by us. Reporting Laboratory Results Results are reported as an authorised report posted directly to the referrer. Some internal JAK2 results are reported on the internal APEX electronic system. Clinical Scientific Diagnostic Advice and Results Interpretation A key component of the service provided by the laboratory is the availability of expert clinical scientific advice. Advice is provided on the genetics and molecular biology of haematological malignant disorders, the investigation of these and the interpretation of test results. The contact details for staff are indicated on page 3. Discussion on general testing algorithms in this rapidly evolving area, and availability of testing, technologies / methodologies is available from the Principal Clinical Scientist and lead for the service:- Dr Abida Awan Principal Clinical Scientist in Haematological Molecular Oncology Deputy Head MDC (Molecular Oncology) Tel: abida.awan@cmft.nhs.uk Haemato-Oncology Tests Chronic Myeloid Leukaemia Page 8 of 13

9 Directorate of The pre-treatment transcript level of t(9;22) [BCR-ABL] can inform prognosis and determine downstream log reduction levels. In addition, 3 monthly follow up MRD analysis is recommended as per national and international guidelines. Studies have indicated PB monitoring for most follow up time-points is sufficient, with BM monitoring at stipulated time points, according to national and ELN guidelines, where these will help to inform when cytogenetics testing is necessary. The use of donor lymphocyte infusion (DLI) can be indicated in post transplant patients. Therefore if DLI is being used, a clinically urgent test request can be made, so that a shorter turn around time is implemented in the laboratory to support therapeutic decision making. This would also be available when other clinical urgencies require this, such as loss of a response or disease progression. Drug Resistance The availability of 2 nd and 3 rd plus generation drugs etc, their appropriate usage, and the occurrence of resistance to therapy due to the presence of ABL kinase domain mutations, requires additional testing. We are in the process of setting up this work in the MDC laboratory, in addition to the MRD work, however in the interim, we can send appropriate samples for an ABL kinase domain mutation screen to our collaborators, on behalf of referrers. Several mechanisms of resistance have been documented and the current testing would be applied to mutations occurring in the ABL kinase gene. Acute Lymphocytic Leukaemia In ALL however, paired PB and BM samples are required for MRD monitoring in acute phase disease. Refer to the AML sample criteria for PB and BM samples required below. We currently offer BCR-ABL testing but will also be looking into offering the paediatric ALL screen as appropriate in the future as the service develops. Acute Myeloid Leukaemia Minimum residual disease (MRD) monitoring is used to guide therapy and identify those patients at an increased risk of relapse. Paired PB and BM samples are required to accurately determine levels of MRD. This is because there can be large differences in the interpretation of the relapse risk if you are looking at leukaemic gene expression in PB or BM alone. For this reason, it is important to identify the sample type (PB or BM) on the requisition form or request card. The accurate monitoring of MRD requires the samples to be fresh, to reach us ideally within 24 hours of phlebotomy and no later than 48 hours for highly reliable results. When it is not possible to obtain both samples, it is advised to send one sample PB or BM rather than miss the time point of MRD monitoring and the interpretation of the results will take this into account. CBF Leukaemias Page 9 of 13

10 Directorate of Paired PB and BM samples are required for MRD as per the above specimen requirements table Page 7. Use of DLI or monitoring patients pre and post transplant can be very informative for their clinical management. A repeat sample should always be tested if a significant change in transcript levels is observed to confirm this change, prior to any alteration in therapeutic decision making, which applies to all MRD monitoring. Acute Promyelocytic Leukaemias For APML, BM samples can indicate increased risk of relapse much earlier than PB. Residual transcript detection, following completion of treatment, is highly indicative of an increased risk of relapse. Paired samples are the most informative specimens for MRD monitoring. As with all MRD, a pre-treatment diagnostic level is important to look at log reduction in levels over time. In APL, there is also the advantage of being able to detect the genetic lesion in molecular assays if cytogenetic testing is unable to detect the translocation due to technical issues with the sample. This helps to confirm the diagnosis and continue with the clinical management of the patient. Normal Karyotype AML testing As many patients do not have a specific translocation to facilitate MRD monitoring, we routinely monitor for NPM1 and FLT3 ITD on the diagnostic pre-treatment sample. This is usually done on PB alone for FLT3 ITD and both PB and BM for NPM1. Two separate samples are required for FLT3 ITD and NPM1 because the tests are carried out on DNA and RNA respectively. Therefore to facilitate appropriate testing, 15ml EDTA PB is required for FLT3 ITD testing and a separate 15ml PB plus 2-3ml EDTA BM for NPM1 testing. The risk profiling of AML patients at diagnosis is currently being discussed in working groups. With newer technologies, it is envisaged that a larger panel of genes will be tested to obtain a risk profile. It is anticipated that this will be introduced once technology such as next generation sequencing becomes widely available with appropriate gene panels as per agreements within the working groups. Myeloproliferative Disorders In 2005, the JAK2 V617F mutation was found in almost all patients with PV and about half of those patients with ET and IMF. This test is now part of the clinical work up guidelines for diagnosing these patients. For JAK2 molecular tests, 10ml EDTA PB is required and not a BM aspirate sample. Real time quantitative PCR testing of JAK2 V617F is also available in addition to an end point qualitative test. This identifies transcript numbers for MRD monitoring, usually applied to the use of a JAK2 inhibitor or a transplant scenario. Polycythaemia Vera The JAK2 V617F mutation is found in ~95% of cases with PV, however approximately 5% of patients do not have this SNP mutation. It has been reported that the majority of Page 10 of 13

11 Directorate of the JAK2 V617F negative PV patients have a mutation in an alternative region of the JAK2 gene, within exon 12. Currently we are able to send an appropriate aliquot of the archived DNA sample for screening exon 12 to our collaborators, on behalf of referrers. With the availability of appropriate technology, this work will ultimately be undertaken by us to provide a comprehensive and complete MPD service. Essential Thrombocythaemia The JAK2 V617F mutation is offered for ET patients, but because 3-4% of patients have a mutation in the MPL gene, this testing is also offered by the laboratory. We look at the most commonly occurring mutation of MPL (W515L/K), although 6 different mutations have been identified in the gene. The very recent and exciting reports of the CALR mutations present in ~80% of JAK2 negative ET patients is being investigated and will be offered once suitable technology is available in the laboratory to undertake this work. Idiopathic Myelofibrosis These patients are tested for JAK2 V617F and MPL, where 4-8% of patients harbour the MPL mutation. Real time PCR for MRD monitoring of JAK2 would be advocated if a JAK2 inhibitor is used therapeutically due to the important clinical requirements for these patients. B and T cell lymphomas The current service provision for the assessment of clonality in putative cases of lymphoma predominantly tests DNA extracted from Formalin Fixed Paraffin embedded (FFPE) tissue. The laboratory undertakes Immunoglobulin and T cell receptor clonality analysis as part of a multidisclipinary approach to Lymphoma diagnosis. Fixing should ideally be for 24 hours in buffered formalin (ph containing % formaldehyde) embedded in paraffin. Decalcification of fixed bone marrow trephines should ideally be performed in EDTA rather than acids which significantly damages genetic material. Blocks should be sent to the appropriate histopathology laboratory to be cut for PCR analysis to be performed in the MDC laboratory. Routinely, the block should be examined for the location of the biopsy tissue and 5x10µM and separately 3x10µm sections should be cut and placed in two respective 1.5ml sterile DNase and RNAse free molecular tubes. If the tissue size is small, multiple 5x10µM sections, each in separate 1.5ml tubes should be sent, depending on the demands and requirements of the tissue for other testing and disciplines. For advice, please speak to molecular oncology staff in the MDC laboratory (contact details indicated above on page 3). Occasionally, fresh biopsy material has been received. This must be discussed with the appropriately qualified oncology staff in the MDC laboratory in advance of sending the specimen so that special arrangements can be made to receive the sample. This will need to be processed and stored safely to avoid any tissue degradation. Page 11 of 13

12 Directorate of EDTA PB and EDTA BM aspirates for lymphoma genetics testing must be received as per all other samples indicated for leukaemia testing for the avoidance of spoilage. This is specified in the specimen requirements table above. Clinical Trials Samples We are the regional centre for haemato-oncology molecular testing. The clinical trial protocol will advise on the appropriateness of referral to designated centres and when it is appropriate to use regional centres. When the MDC laboratory participates in a clinical trial as the designated UK centre for testing, this will be indicated in the trial protocol. It is also appropriate to refer non trial patients for molecular testing that are not participants in a clinical trial. Therefore these can be referred directly to the MDC laboratory and any advice required can be sought by contacting appropriately qualified staff in the oncology team, with contact details indicated on page 3. Future Testing under development Haemato-oncology is under significant development. It is a paradigm for other cancer testing such as solid tumours and leads the way in how technologies are used to diagnose, risk profile and treatment assess patients. The specialist area and availability of targeted therapy, within personalised medicine, renders a requirement to keep pace with testing requirements. This work stream is ongoing, and a partnership with our users, to implement key testing that is nationally advised, is indicated. The milestones of deep remission, which may indicate removal of drug for a variety of reasons, which can include pregnancy, is becoming a reality. Future work on circulating cell free tumour DNA will require monitoring in the PB. Although currently a research tool, it may translate into the clinic in the near future. Future testing platforms and technologies need to be appraised and implemented, if appropriate, to provide the most comprehensive service for our patients and their referrers to be able to access best practise testing in the field. Tests to be implemented and under evaluation - BCR-ABL mutation screen for the ABL kinase domain to provide information on drug resistance and to indicate changes that may be required in therapy based upon the mutation indicated. - FLT3 ITD with increased sensitivity of detection. Tests to be offered as part of future envisaged developments - JAK2 exon 12 - CALR mutations - CEBP-alpha in AML as well as a consortia of panel genes to risk profile at diagnosis Page 12 of 13

13 Directorate of - ALL mutation screening for paediatric patients - Other new and established target genes as required by haemato-oncology patients for the molecular work up indicated by standard and best practise - Next generation sequencing to assess increased panels of genes for diagnostic profiling to risk stratify patients for specific treatment options. In addition to increasing the sensitivity of mutation detection in clonal disorders. This involves utilisation of advanced technologies to be applied to the clinical setting to provide increased information to effectively treat our patients Service development and user engagement The service is offered to users and is a developing service. It is envisaged to be responsive to technological advances and the availability of new therapeutics to offer advanced personalised medicine. The haemato-oncology area is a front runner for use of these techniques, and the resulting information will help to inform other areas of cancer diagnosis and clinical management. The service relies on engagement with referrers to offer the repertoire of tests required, once novel molecular targets are identified and can be exploited. These targets can be used to diagnose and identify the clinical course of the disease, and are used by the pharmaceutical industry to treat the pathological condition. Profiling patients at diagnosis will help to identify those that require increased therapy, stem cell transplant or newer clinical trials drugs. Conversely, those who require reduced therapy due to being in a good responder group, can be spared unnecessary toxicity. Future work on cell free circulating tumour DNA may identify genetic abnormalities less invasively, and extensive work on new technological platforms will offer great promise. Partnership with referrers will allow triage of patients along pathways of testing if initial testing is negative to provide a comprehensive screen of all available genetic markers. Some markers will still be within a research setting and part of clinical trials, whereas others will be validated for use in a clinic setting going forwards. This is a fast moving time for multidisciplinary working to establish services of the future with developing and accessible genomics becoming readily available and interpretable. Page 13 of 13