Modern Genomic Profiling in Myeloid Disorders and Acute Leukemia: A Practical Approach

Although morphologic examination by light microscopy remains an integral part of initial histopathologic assessment for hematolymphoid neoplasms, a variety of laboratory techniques are utilized to optimize diagnostic information in the appropriate clinical context. These techniques include, but are not limited to next generation sequencing, immunohistochemistry, flow cytometry, cytogenetics, fluorescent in situ hybridization (FISH), DNA microarray, and/or single analyte polymerase chain reaction (PCR).¹

Information rendered from these technologies are critical for the molecular diagnostic sub-classification of the genetically defined diseases according to evidence based and standard of care guidelines that are outlined in recent high-impact publications^{2, 3} and the most recent 2017 World Health Organization (WHO) Classification of Hematolymphoid Neoplasms⁴. The WHO book is a peer-reviewed and evidence-based authoritative reference book that provides an international standard for oncologists and pathologists and references more than 4,500 peer reviewed publications.

In addition to providing necessary molecular diagnostic classification, the WHO Classification additionally delineates how genomic information rendered from technologies including but not limited to next generation sequencing, DNA microarray, FISH, IHC, and PCR, can help establish clonality, provide prognostic risk stratification to optimize therapeutic selection, and can be used in predicting or monitoring patient's response to therapy. Diagnostic criteria, pathological features, and associated genetic alterations are detailed in a strictly disease oriented manner ranging from non-Hodgkin lymphoma to acute leukemia.

As a result of numerous high-impact publications and medical society guidelines, genomic profiling in myeloid disorders has become standard of care. Currently, my laboratory utilizes a 65 gene amplicon-based NGS panel and DNA-based cytogenomic microarray in laboratory work-up of myeloid disorders and acute myeloid leukemia (AML). This panel was vetted and designed with input from internal pathologists and oncology clients involved in the management of patients with myeloid disorders. Over the next few paragraphs, I will articulate our clinical, scientific, and economic rationale for this approach.

When designing our assay, we wanted to be able to capture all of the relevant genomic aberrations that affect diagnosis, prognosis, and/or therapy in myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), MDS/MPN, and AML because these diseases are on a continuum⁴. A patient may first develop clonal hematopoiesis of indeterminate potential (CHIP), then develop low-grade MDS, then high-grade MDS, and then AML⁵. Patients with MPN and MDS/MPN have a similar continuum. We attempted to design a 50-gene myeloid panel; however, with the recent and ongoing explosion of literature spanning chronic and acute myeloid disorders, we were not able to do so without leaving off critically important genes that contribute to the biology of myeloid disease. If you review figure 2 from our recently published paper⁶, we identified 72 genes that are important in acute and chronic myeloid disorders. These 72 genes do not include a few genes recently recommended in the 2017 WHO classification of hematologic malignancies. Therefore, we decided to utilize a targeted NGS strategy, complemented by whole genome DNA-based cytogenomic microarray.

It is also critical to ensure that all of the genes on genomic testing panels have clinical utility or are “clinically actionable”. With regard to these terms, the Association of Molecular Pathology (AMP) has published a nice article that succinctly reviews the different facets of clinical utility to include information that enhances molecular based diagnostic stratification, refines prognostic risk-stratification, and informs therapeutic decision-making With regard to the AMP paper on clinical utility⁷, I want to emphasize that diagnosis, prognosis, and/or therapeutic utility are inter-related and not mutually exclusive. That notion has been incorporated into designing our assay and testing strategy.

Diagnosis

In the setting of MDS, MDS/MPN, and MPN, establishing a diagnosis of a myeloid disorder needs to occur prior to initiation of any therapy. In all of these disorders, the diagnostic classification relies on a multi-parameter testing approach in addition to clinical and morphologic findings. Technologies utilized to work up myeloid disorders include light microscopy, immunohistochemistry, PCR, next generation sequencing, DNA cytogenomic microarray, FISH, conventional cytogenetics, and flow cytometry⁹.

Detection of specific genomic aberrations are required for diagnosis including mutations in JAK2, CALR, MPL, and CSF3R⁹. In other contexts, having mutations can support a diagnosis when there is a high clinical index of suspicion and morphologic findings are low-grade or subtle. In AML, certain aberrations such as a RUNX1 or NPM1 mutation similarly define a molecular classification of diseases in the 2017 WHO Classification. In the setting of AML, there are a handful of mutations (JAK2, SRSF2, CSF3R, U2AF1, SF3B1, ZRSR2, ASXL1, EZH2, STAG2, and BCOR) that strongly indicate an underlying myeloid stem cell disorder¹⁰. Genomic profiling, thus, provides a molecular-based diagnostic classification of AML. Furthermore, the testing identifies a category of AML patients who typically do not respond to standard induction and consolidation chemotherapy. Rather, these patients are at a very high risk for relapse, disease progression, and poor outcome and will benefit from strategies other than traditional induction and consolidation regimens ¹¹.

Prognosis

Another important purpose of our testing strategy is to provide therapy-driven risk based prognostic classification. According to NCCN guidelines, it is critical to search for certain mutation in genes (i.e. IDH1, IDH2, TP53, ASXL1, RUNX1, and others) to identify patients with high risk chronic or acute myeloid disorders.^12-14 Such patients are at a high risk of disease progression, relapse, and therapy failure; as a result, these patients stand to benefit from aggressive therapeutic regimens described below. Cytogenomic microarray can detect alterations that are missed by next generation sequencing including loss of heterozygosity (LOH) of certain genomic regions that are important to determine prognosis^15,16.

Therapy

As mentioned earlier, some therapeutic decisions require precise molecular-based diagnostic classification and prognostic risk-stratification. Once a diagnosis of myeloid disorder or acute leukemia is established, the appropriate therapy can be implemented and customized according to the molecular aberrations associated with prognosis and also based on the diagnostic context. These include FDA approved chemotherapies and/or hematopoietic cell transplant in high risk patients. FDA approved therapies are now available that are directed against specific molecular aberrations in acute leukemia. Midostaurin is currently FDA approved for acute myeloid leukemia with a FLT3 mutation and Enasidenib has been FDA approved for acute leukemia with an IDH2 mutation^{17, 18}. Over the next few months, we will likely see a therapy approved for AML patients with IDH1 mutation. Currently, clinical trials are enrolling patients with high risk myeloid disorders with the aforementioned mutations (19). For patients with AML and an underlying myeloid stem cell disorder, there are now FDA approved therapies that will improve patient outcome²⁰.

According to evidence-based guidelines from the American Society of Blood and Bone Marrow, hematopoietic stem cell transplant (HSCT) is considered a valuable therapeutic option in high risk myeloid disorders and acute leukemia, provided that patients don’t have certain mutations that are associated with poor outcome in the transplant setting ²¹. Currently, there are several publications that pinpoint certain mutations to be associated with a poor prognosis in patients undergoing HSCT ^22-24. In these situations, molecular testing can help refine who is unlikely to benefit from HSCT, a medical procedure that costs the health care system hundreds of thousands of dollars.

Conclusion

In summary, the management of patients with chronic myeloid stem cell disorders and acute leukemia is challenging, complex, and quickly evolving. Genomic profiling helps to accurately classify disease and provide prognostic risk classification, which, in turn drives therapy management for these spectrum of disorders. The clinical benefit to patients is improved outcomes aligned with optimal and cost-effective utilization of therapeutic strategies. Our oncologists commend us on a regular basis on how the testing has impacted their management of cancer patients and improved outcomes.

In closing, I would like to share a quote from a recently published editorial article from Emory University ²⁵ that details the recent evolution in the practice of pathology resulting from scientific and technological advances in genomics:

“The only constant in pathology is change.”

References:

1. Li et al. Multimodality Technologies in the Assessment of Hematolymphoid Neoplasms. Arch Patholo Lab Med. 2017 Mar;141(3):341-354.
2. Taylor et al. Diagnosis and classification of hematologic malignancies on the basis of genetics. Blood. 2017 Jul 27;130(4):410-423.
3. Montalban-Bravo et al. Myelodysplastic syndromes: 2018 Update on Diagnosis, Risk-stratification and Management. Am J Hematol. 2018 Jan;93(1):129-147.
4. Flach et al. An accumulation of cytogenetic and molecular genetic events characterizes the progression from MDS to secondary AML: an alysis of 38 paired samples analyzed by cytogenetics, molecular mutation analysis and SNP microarray profiling. Leukemia. 2011 Apr;25(4):713-8.
5. Ganguly et al. Impact of chromosome alterations, genetic mutations and clonal hematopoiesis of indeterminate potential (CHIP) on the classification and risk stratification of MDS. Blood Cells Mol Dis. 2018 Mar;69:90-100.
6. Mukherjee et al., Addition of Chromosomal Microarray and Next Generation Sequencing to FISH and Classical Cytogenetics Enhances Genomic Profiling of Myeloid Malignancies. Cancer Genet. 2017 Oct; 216-217: 128-141.
7. Joseph et al. The Spectrum of Clinical Utilities in Molecular Pathology Testing Procedures for Inherited Conditions and Cancer: A Report of the Association of Molecular Pathology. J Mol Diagn. 2016 Sep;18(5):605-619.
8. Li et al., Multimodality Technologies in the Assessment of Hematolymphoid Neoplasms. Arch Pathol Lab Med. 2017 Mar;141(3):341-354.
9. Swerdlow et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; 2017. World Health Organization Classification of Tumours; vol 2.
10. Lindsley et al. Acute Myeloid Leukemia Ontogeny is Defined by Distinct Somatic Mutations. Blood. 2015 Feb 26;125(9):1367-76.
11. Ikegawa et al. Allogeneic Hematopoietic Stem Cell Transplant Overcomes Poor Prognosis of Acute Myeloid Leukemia with Myelodysplasia-related Changes. Leuk Lymphoma. 2016;57(1):76-80.
12. NCCN Clinical Practice Guidelines in Oncology. Acute Myeloid Leukemia. Version 3.2017. URL: https://www.nccn.org/professio...
13. NCCN Clinical Practice Guidelines in Oncology. Myelodysplastic Syndromes. Version 1.2018. URL: https://www.nccn.org/professio...
14. NCCN Clinical Practice Guidelines in Oncology. Myeloproliferative Neoplasms. Version 2.2018. URL: https://www.nccn.org/professio...
15. Grosneth et al., Prognostic Significance of Acquired Copy-neutral Loss of Heterozygosity in Acute Myeloid Leukemia. Cancer. 2015 Sep 1;121(17):2900-8.
16. Parkin et al., Integrated Genomic Profiling, Therapy Response, and Survival in Adult Acute Myelogenous Leukemia. Clin Can Res. 2015 May 1;21(9):2045-56.
17. FDA package insert for Midostaurin URL: https://www.accessdata.fda.gov...
18. FDA package insert for Enasidenib URL: https://www.accessdata.fda.gov...
19. https://clinicaltrials.gov/
20. Vyxeos: https://www.fda.gov/NewsEvents... FDA Package insert: http://pp.jazzpharma.com/pi/vy...
21. Majhail et al., Indications for Autologous and Allogeneic Hematopoietic Cell Transplantations: Guidelines From the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2015 Nov;21(11):1863-1869.
22. Lindsley et al. Prognostic Mutations in Myelodysplastic Syndrome After Stem Cell Transplantation. N Engl J Med. 2017;376(6):536–547.
23. Middeke et al., TP53 Mutation in Patients with High-Risk Acute Myeloid Leukemia Treated with Allogeneic Haematopoietic Stem Cell Transplantation. Br J Haematol. 2016 Mar;172(6):914-22.
24. Bejar et al. Somatic Mutations Predict Poor Outcome in Patients With Myelodysplastic Syndrome After Hematopoietic Stem-Cell Transplantation. J Clin Oncol. 2014 Sep 1;32(25):2691-8.
25. Tristram et al. Impacts of New Concepts and Technologies on the Practice of Diagnostic Pathology: An Emory University Perspective. Archives of Pathology & Laboratory Medicine: March 2017, Vol. 141, No. 3, pp. 325-328.

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