OPENING PLENARY SESSION

Invited Presentation Abstracts 1
© 2015 The Authors IJLH © 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015 37 (Suppl. 2) 1-130
OPENING PLENARY SESSION (1) DIAGNOSTIC IMPACT OF GENETICS AND EPIGENETICS IN ACUTE MYELOID LEUKEMIA Daniel A. Arber, MD Stanford University
The diagnostic category of acute myeloid leukemia (AML) is actually a heterogeneous group of diseases that are now partially subclassified based on recurring cytogenetic abnormalities and the presence of specific gene mutations. Other cytogenetic and molec- ular genetic changes in AML impact the prognosis of these specific disease groups and are increasingly used to place patients into risk groups that may drive specific therapies. This presentation will review the more common genetic events in AML, including their impact on the epigenetics of this disease. The 2008 WHO classifica- tion includes a number of specific cytogenetic abnormalities as defi- nitional for AML subtypes and these genetic abnormalities are felt to represent disease-initiating genetic changes. The most common of these include the t(15;17)(q24.1;q21.1), resulting in the fusion of PML and RARA, in acute promyelocytic leukemia, the t(9;11) (p22;q23) involving the KMT2A (previously known as MLL) and translocations that disrupt the core binding factor (CBF). Acute promyelocytic leukemia with PML-RARA now has a favorable prognosis when treated with therapies that include all-trans retinoic acid or arsenic trioxide. The CBF AMLs include AML with t(8;21) (q22;q22) RUNX1-RUNX1T1 and AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) CBFB-MYH11. The RUNX1 and CBFB genes encode different components of the CBF protein, a critical protein in normal hematopoiesis. Disruption of the CBF protein inhibits binding of the protein to DNA targets and perturbs this normal process. Despite the difference in morphology for the two CBF AMLs, they have a similar, favorable prognosis with current therapeutic regimens. Other AML subtypes with recurring genetic abnormalities in the 2008 WHO classification include AML with t(6;9)(p23;q34) DEK-NUP214 and AML with inv(3)(q21q26.1) or t(3;3)(q21;q26.1), now known to result in a GATA2-EVI1 fusion, which are associated with myelodysplastic changes and a generally poor prognosis; and AML with t(1;22)(p13;q13) RBM15-MKL1 which is usually an infant leukemia with megakaryoblastic features. Although not included in the 2008 classification, AML with BCR- ABL1 is now considered a distinct entity if prior CML is excluded and is possibly amenable to therapy with tyrosine kinase inhibitors. Outside of the specific cytogenetic abnormalities listed above, other cytogenetic changes impact prognosis and disease classification in AML. Cytogenetic changes associated with myelodysplastic syndromes (MDS), most commonly complex abnormalities and de- letions of chromosomes 5 and 7, are predictive of a poor prognosis and are used, along with other MDS-associated abnormalities, to support a diagnosis of AML with myelodysplasia-related changes (in the absence of prior cytotoxic therapy). In addition to specific cytogenetic translocations, various gene mutations also frequently occur in AML. Similar to karyotype abnormalities, some of these mutations are considered to be early, disease-defining events in AML and others occur across a spectrum of AML types and are considered prognostic markers. While initially studied as poten- tial prognostic markers in AML with a normal karyotype, gene mutations are now often studied in a variety of settings that include testing in association with specific cytogenetic abnormalities. AML with mutated NPM1, the most common mutation in AML, and AML with mutated CEBPA are provisional entities in the 2008 WHO classification and are generally associated with a favorable prognosis. These mutations do not usually occur with the recurring cytogenetic abnormalities discussed above. While usually asso- ciated with a normal karyotype, approximately 15% of cases will occur with a non-specific cytogenetic abnormality and this finding does not appear to impact the favorable prognosis of this general group. AML with mutated RUNX1 is also felt to represent a dis- tinct disease group when occurring in de novo AML, although this mutation is associated with a generally poor prognosis. Most of the
other mutations in AML are considered more as prognostic factors and do not define specific disease entities. These mutations may be acquired over time, or may be present very early, in pre-leukemic stem cells, but are often associated with other genetic abnormalities. FLT3 mutations are the second most common mutation in AML and may represent an internal tandem duplication (ITD) or, less com- monly, a point mutation in the tyrosine kinase domain (TKD). In general, FLT3 mutations, especially ITD mutations, confer a worse prognosis in AML, but these mutations occur across a wide spec- trum of AML subtype. While very common in AML with a normal karyotype, FLT3 mutations are also common in acute promyelo- cytic leukemia, AML with t(6;9)(p23;q34) and AML with mutated NPM1. In the latter case, the FLT3 mutation impacts prognosis by moving the case from a good prognostic group to a more intermedi- ate prognosis. Mutations of DNMT3A are the third most common mutation in AML and also occur in conjunction with NPM1 and other mutations and tend to be associated with a worse prognosis. Mutations of KIT are, overall, relatively uncommon in AML, but are present in approximately 25% of CBF-types of AML and are a poor prognostic indicator in these otherwise good prognosis disor- ders. Less common mutations in AML, include mutations of IDH1, IDH2, TET2, WT1, ASXL1, U2AF1 and TP53. While these ab- normalities require further study, most have to date been shown to have an indeterminate or negative impact on prognosis, with TP53 mutations usually associated with complex cytogenetic abnormal- ities and a dismal prognosis. Epigenetic changes in AML include a variety of factors that include protein expression and alteration in DNA methylation. Both global DNA and gene specific methylation are reported to correlate with a poor prognosis, several specific gene mutations (DNMT3A, IDH1, IDH2 and TET2) alter normal gene methylation and hydroxymethylation pathways and these changes may be of more significance than performing complicated meth- ylation studies. Similarly, while detection of increased expression of proteins such as BAALC and ERG are associated with a worse prognosis in AML, such studies are not routinely performed and are of unclear significance when paired with multi-gene panel mutation studies. In summary, the genetics of AML are complicated and the array of gene alterations in the disease may cause confusion on de- termining the optimal approach to the diagnosis of AML. While not all mutation studies are needed in all cases, the rapid development of next generation gene panels may make a step-wise approach to testing unnecessary.
References: 1.Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The 2008 revision of the WHO classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114(5):937-51. 2. Dohner H, Estey EH, Amadori S, et al. Diagnosis and manage- ment of acute myeloid leukemia in adults: Recommendations from an international expert panel, on behalf of the European Leukemi- aNet. Blood. 2010;115(3):453-474. 3.Arber DA, Brunning RD, Le Beau MM, et al. Acute myeloid leukaemia with recurrent genetic abnormalities. In: Swerdlow S, Campo E, Harris NL, et al, eds. WHO classification of tumours of haematopoietic and lymphoid tissue . Geneva, Switzerland: WHO Press; 2008:110-123. 4. Arber DA, Stein AS, Carter NH, Ikle D, Forman SJ, Slovak ML. Prognostic impact of acute myeloid leukemia classification. impor- tance of detection of recurring cytogenetic abnormalities and mul- tilineage dysplasia on survival. Am J Clin Pathol. 2003;119(5):672- 680. 5. Ohgami RS, Ma L, Merker JD, et al. Next-generation sequenc- ing of acute myeloid leukemia identifies the significance of TP53, U2AF1, ASXL1, and TET2 mutations. Mod Pathol. ePub 2014.
2 Invited Presentation Abstracts
© 2015 The Authors IJLH © 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015 37 (Suppl. 2) 1-130
(2) HEPCIDIN AND IRON DISORDERS: NEW BIOLOGY AND CLINICAL APPROACHES. Elizabeta Nemeth Department of Medicine, David Geffen School of Medicine at UCLA Hepatic hormone hepcidin is a principal regulator of iron homeo- stasis and a pathogenic factor in common iron disorders. Hepcidin deficiency causes iron overload in hereditary hemochromatosis and iron-loading anemias, whereas hepcidin excess causes or contrib- utes to the development of iron-restricted anemia in inflammatory diseases, infections, some cancers and chronic kidney disease. Be- cause of this, hepcidin may become a useful tool for diagnosis and management of iron disorders. Furthermore, a number of strategies that target hepcidin, its receptor and its regulators are under devel- opment as novel therapeutic approaches for diseases associated with iron dysregulation. References
Ruchala P, Nemeth E. The pathophysiology and pharmacology of hepcidin. Trends Pharmacol Sci. 2014 Mar;35(3):155-61. Ganz T, Nemeth E. Hepcidin and iron homeostasis. Biochim Bio- phys Acta. 2012 Sep;1823(9):1434-43. Kautz L, Jung G, Valore EV, Rivella S, Nemeth E, Ganz T. Identi- fication of erythroferrone as an erythroid regulator of iron metabo- lism. Nat Genet. 2014 Jul;46(7):678-84. Preza GC, Ruchala P, Pinon R, Ramos E, Qiao B, Peralta MA, Sharma S, Waring A, Ganz T, Nemeth E. Minihepcidins are ratio- nally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload. J Clin Invest. 2011 Dec;121(12):4880-8. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004 Dec 17;306(5704):2090-3.
(3) TELOMERE BIOLOGY AND THE NEW TELOMEROPATHIES. N.S. Young Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda MD
Telomeres, repeated short nucleotide sequences and associated shelterin proteins, constitute the termini of linear chromosomes. Telomeres protect chromosomes from recognition as double stranded DNA breaks. Telomeres shorten with every cell division, and attrition is partly abrogated by the telomerase repair complex. Critical telomere shortening accounts for the Hayflick phenomenon, and critically short telomeres cause genomic instability. Telomer- ase mutations are etiologic in the telomeropathies, organ failure syndromes that include bone marrow failure, pulmonary fibrosis, and liver disease. Telomere content of peripheral blood leukocytes can be measured by a variety of assays, some now commercially available. The phenotype and penetrance of telomeropathies is highly variable. Dyskeratosis congenita in childhood is associated with mucocutaneous findings, telomeropathies in adults with early hair graying. Mutations in TERT (the gene encoding the reverse transcriptase) cause liver disease, mutations in TERC (encoding the RNA template), pulmonary fibrosis. Accelerated telomere attrition is the major risk factor for malignant clonal evolution in SAA; chromosomal aberrations can be observed in tissue culture months to years preceding clinical MDS/AML. Sex hormones upregulate TERT expression, and danazol in a prospective NIH clinical proto- col has been effective in improving blood counts in telomeropathy patients and elongating telomeres.
References
Townsley DM1, Dumitriu B1, Young NS1. Bone marrow failure and the telomeropathies. Blood. 2014 Oct 30;124(18):2775-83.
Calado RT1, Regal JA, Kleiner DE, Schrump DS, Peterson NR, Pons V, Chanock SJ, Lansdorp PM, Young NS. A spectrum of severe familial liver disorders associate with telomerase mutations. PLoS One. 2009 Nov 20;4(11):e7926. Calado RT1, Young NS. Telomere diseases. N Engl J Med. 2009 Dec 10;361(24):2353-65.
Gutierrez-Rodrigues F1, Santana-Lemos BA1, Scheucher PS1, Alves-Paiva RM1, Calado RT1. Direct comparison of flow-FISH and qPCR as diagnostic tests for telomere length measurement in humans. PLoS One. 2014 Nov 19;9(11):e113747
Khincha PP1, Savage SA. Genomic characterization of the inherited bone marrow failure syndromes. Semin Hematol. 2013 Oct;50(4):333-47.
Invited Presentation Abstracts 3
© 2015 The Authors IJLH © 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015 37 (Suppl. 2) 1-130
(4) PAST PRESENT AND FUTURE OF QUALITY IN HAEMOSTASIS TESTING Steve Kitchen UK NEQAS Blood Coagulation, Sheffield, United Kingdom
Many tests performed in Coagulation laboratories have a direct impact on patient management and inaccurate results in relation to familial and acquired bleeding and thrombotic disorders could have very serious consequences for patients. Accurate and reliable results are needed when testing is performed in relation to possible familial haemostatic disorders where laboratory error may lead to misdiagnosis. The pre analytical, analytical and post analytical phases of testing are all of great importance. Assessment of ana- lytical quality should include participation in independent external quality assessment/proficiency testing programmes. For a labora- tory testing to reach the quality required for accreditation to ISO standards it is a requirement that the laboratory participates in an inter-laboratory comparison programme appropriate to the exam- ination and interpretation of such examination results (ISO 15189 2012). The ISO 15189 standard requires that the inter-laboratory comparison programme chosen by the laboratory should provide clinically relevant challenges that mimic patient samples and have the effect of checking the entire examination process, including pre-examination and post-examination procedures, where possi- ble. EQA programmes have focussed on the analytical component but studies and articles always show that more errors occur in the pre-analytic phase than any other.
EQA programmes are encouraged by ISO to address the pre-analyt- ic phase. This can be done in a number of ways and in future this will increasingly be included in EQA repertoires (ISO 17043). One approach is to use questionnaires about practice with feedback on how an individual centre compares to other centres. Another option could be use of EQA materials that contain interfering substance such as haemoglobin. The third option is to record error rates sup- plied by participants which can be compared to quality standards. All of these will likely increase in future EQA programmes.
Perhaps the most important component of laboratory testing is the post-analytical interpretation since it is the way that results are interpreted that impacts most on patient management. An example of post analytical assessment in EQA programmes is the use of D-dimer to exclude possible VTE. In the UK NEQAS Pint of Care D-dimer programme participants are invited to perform a D-dimer test on an EQA sample and use their local result along with a clin- ical scenario and pre test probability score such as the Wells score to decide whether VTE can be excluded or not. Their interpretation is then assessed. In future EQA programmes will have to address the post analytical phase more in this way.
References: ISO 15189:2012 Medical laboratories requirements for quality and competence
ISO 17043:2010 Conformity assessment – general requirements for proficiency testing
Key References
1. F Eric Preston, Steve Kitchen, John Olson External Quality Assessment in Haemostasis: Its importance and significance in: Quality in laboratory Haemostasis and Thrombosis 2nd edition: Kitchen s, Olson J, Preston FE (Eds) Wiley-Blackwell (Oxford, UK) 2013
2. Kitchen DP, Jennings I, Kitchen S, Woods TAL, Walker ID, Bridging the gap between point-of-care testing and laboratory test- ing Sem Thromb Haemost 2015 (in press)
3. Kitchen S, Kitchen DP, Jennings I, Woods TA, Walker ID, Pres- ton FE Point-of-care INRs: UK NEQAS experience demonstrates necessity for proficiency testing of 3 different monitors Throm haemost 2006: 96: 590-596
4. Jennings I, Kitchen DP, Woods TAL, Kitchen S, Walker ID Emergency technologies and quality assurance the UK National External Quality Assessment Scheme perspective Sem Thromb Haemost 2007: 33: 246-249
5. Olson JD, Preston FE, Nichols WL, External quality assurance in Thrombosis and Haemostasis an international perspective Semin Thromb Haemost 2007: 33: 220-225
4 Invited Presentation Abstracts
© 2015 The Authors IJLH © 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015 37 (Suppl. 2) 1-130
SPECIAL PLENARY 1: EVOLVING REGULATION OF LABORATORY DEVELOPED TESTS
(5) FDA’S INITIATIVE TO REGULATE LAB-DEVELOPED TESTS (LDT) WILL HARM PATIENTS AND ACADEMIC PATHOLOGY Edward R. Ashwood, MD ARUP Laboratories, University of Utah
In October 2014, the FDA issued two draft guidance documents providing a risk-based framework for the regulation of Lab Devel- oped Tests (LDTs).1,2 The proposals would place LDT regulation under the agency’s oversight authority based on the Medical Device Amendments of 1976.3 By issuing these draft two guidances, the FDA acts as if Congress granted it this expansive authority almost 40 years ago. However, the 1976 Act does not mention laboratories or laboratory testing services. Also, the Act specifically denies FDA the authority to regulate the practice of medicine. Thus, the FDA proposes to oversee LDTs as if these services were in vitro diag- nostic devices, a sweeping shift from the agency’s 38-year history of exercising “enforcement discretion” for LDT. FDA lacks the statutory authority to regulate laboratory-developed testing services. While Congress conferred upon FDA the authority to regulate medical devices, they conferred oversight of clinical laboratories to Centers for Medicare and Medicaid Services (“CMS”) through the comprehensive Clinical Laboratory Improvement Amend- ments of 1988 (“CLIA”).4 This law was specifically designed for clinical laboratories and their tests. The proposed changes will increase costs for laboratories and healthcare.5 While it is possible that increased regulation may increase patient safety, there is little evidence that LDTs have been a significant source of safety issues. Although substandard tests are sometimes marketed, these events are rare and are quickly corrected. The costs of complying with FDA regulation are real. For example, ARUP offers approximately 584 Class II tests and 61 Class III tests on our current test menu. The cost of pre-market submission for class 2 is $50,000-$250,000 per test, and for class 3 is $2,500,000-$5,000,000 per test. The total cost would be about $316 million. Many of these tests are low volume and do not provide sufficient revenue to justify the approval cost. We would abandon about 410 tests. In addition, the financial constraints resulting from approval costs will stifle the improvement of older tests. More than half of ARUP’s R&D resource is used currently for test improvement. If the guidances are enforced, that improvement effort will be curtailed. Healthcare has benefited from innovative efforts within laboratories to advance and update testing methods in a timely and medically relevant way. The need for sub- missions and review will seriously reduce the innovation that drives laboratory medicine. The potential benefits of increased regulation are speculative. It is not clear that FDA regulation of diagnostic testing would lead to significant increases in patient safety. On the other hand, the impacts of regulation are very clear: less innovation, increased costs, reduced competition, and fewer choices for doctors and patients.
References
1. Framework for Regulatory Oversight of Laboratory Developed Tests (LDTs), DRAFT GUIDANCE, October 3, 2014, Docket num- ber: FDA-2011-D-0360. 79 FR 59776 (Oct 3, 2014) 2. FDA Notification and Medical Device Reporting for Laboratory Developed Tests (LDTs); Draft Guidance, Docket number: FDA- 2011-D-0357. 79 FR 59779 (Oct 3, 2014) 3. Medical Device Amendments of 1976. May 28, 1976. http:// www.gpo.gov/fdsys/pkg/STATUTE-90/pdf/STATUTE-90-Pg539. pdf 4. 102 Stat. 2903 - Clinical Laboratory Improvement Amendments of 1988. Oct 31, 1988. http://www.gpo.gov/fdsys/pkg/STAT- UTE-102/pdf/STATUTE-102-Pg2903.pdf
5. Scott MG, Ashwood ER, Annesley TM, Leonard DG, Burgess MC. FDA oversight of laboratory-developed tests: is it neces- sary, and how would it impact clinical laboratories? Clin Chem 2013:59(7), 1017-22.
Invited Presentation Abstracts 5
© 2015 The Authors IJLH © 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015 37 (Suppl. 2) 1-130
CONCURRENT 1: MONITORING OF FVIII AND FIX REPLACEMENT THERAPIES
(7) CHALLENGES IN LABORATORY MEASUREMENT OF NEXT GENERATION RECOMBINANT FVIII AND FIX HEMOPHILIA REPLACEMENT PRODUCTS Stefan Tiefenbacher, PhD Colorado Coagulation, Laboratory Corporation of America® Holdings, Englewood, CO 80112, USA
A number of recombinant factor VIII and IX replacement prod- ucts are currently in late stage development or have recently been approved in both US and/or Europe. One-stage factor assays based on the activated partial thromboplastin time (APTT) are the most commonly used assays for measuring FVIII and FIX activities in clinical laboratories. A wide variety of instruments and APTT reagents for factor testing are currently in use. In addition, reagent dependent responses for some of the new factor replacement prod- ucts in factor assays have been described. As a result, laboratory monitoring of these new products post-market approval has been subject of intense discussion among manufacturers, regulators and the clinical community. This presentation will review available data for novel recombinant factor VIII and IX replacement products in commonly used APTT reagent systems, as well as select chromo- genic assays and summarize approaches…