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Biomarkers in Hematological Malignancies: A Review of Molecular Testing in Hematopathology (2)

Biomarkers in Hematological Malignancies: A Review of Molecular Testing in Hematopathology

the Cancer Therapy Advisor take:

Molecular markers are an integral part of determining the diagnosis, prognosis, and therapy of patients with hematologic malignancies and molecular testing has become widely used for the workup of myeloid and lymphoid neoplasms.

In this review, Mohammad Hussaini, MD, from the Department of Hematopathology and Laboratory Medicine at the Moffitt Cancer Center and Research Institute in Tampa, Florida, assessed current practices and trends for molecular testing in hematopathology by disease type, including chronic myelogenous leukemia, myelodysplastic syndrome, acute myeloid leukemia, and lymphoblastic leukemia/lymphoma.

In Philadelphia chromosome-positive chronic myelogenous leukemia, patients possess the BCR-ABL1 fusion gene, which can be detected by conventional cytogenetics the vast majority of the time. This fusion gene is a target for imatinib therapy, a tyrosine kinase inhibitor used to decrease BCR-ABL activity.

In patients with acute myeloid leukemia, genetic abnormalities affect survival rate and influence therapeutic decisions. For example, patients with favorable risk include those with t(15;17), t(8;21), inv(16/t[t16;16]), a normal karyotype and mutated NPM, or a normal karyotype with a biallelic CEBPA mutation, may receive consolidation therapy following 7+3 induction with chemotherapy, while those with adverse risk may undergo transplantation after standard induction.

In lymphoblastic leukemia/lymphoma, 25% of adult cases and 2% to 4% of pediatric cases possess t(9;22)(q34;q11).

These patients can receive adjuvant imatinib, which can improve complete remission rates despite carrying the worst prognosis of all types of lymphoblastic lymphoma. The presence of this translocation can also be used as a marker for minimum residual disease testing.

As technologies for genome sequencing become more affordable and powerful, they will become even more widely used to diagnose hematologic malignancies and play a larger role in therapeutic decisions. 


Background: Molecular interrogation of genetic information has transformed our understanding of disease and is now routinely integrated into the workup and monitoring of hematological malignancies. In this article, a brief but comprehensive review is presented of state-of-the-art testing in hematological disease.

Methods: The primary medical literature and standard textbooks in the field were queried and reviewed to assess current practices and trends for molecular testing in hematopathology by disease.

Results: Pertinent materials were summarized under appropriate disease categories.

Conclusion: Molecular testing is well entrenched in the diagnostic and therapeutic pathways for hematological malignancies, with rapid growth and insights emerging following the integration of next-generation sequencing into the clinical workflow.


Perhaps in no other field of oncology is the routine use of molecular markers more integrated into the diagnostic, prognostic, and therapeutic workup of disease as in the realm of hematological malignancies.

Molecular diagnostics is a burgeoning field in the era of personalized medicine, with high-volume laboratories running 10,000 molecular tests or more every year, many of which are for the workup of leukemia and lymphoma.1,2

Molecular testing has wide applicability in hematopathology, guiding diagnosis (eg, TCR gene rearrangement to establish T-cell clonality), subclassification (eg, recurrent cytogenetic translocations in acute myeloid leukemia [AML]), prognosis (eg, Philadelphia chromosome–positive [Ph+ ] in acute lymphoblastic leukemia [ALL]), and minimal residual disease testing (eg, BCR-ABL transcripts in chronic myelogenous leukemia [CML]).

Myeloproliferative Neoplasms

Chronic Myelogenous Leukemia: The Ph chromosome in CML was discovered in 1960.3,4 t(9;22) (q34;q11) juxtaposes most of ABL1 to 5' regions of BCR, resulting in constitutively increased kinase activity and neoplastic transformation.5

Although the breakpoint for ABL1 is mostly conserved, occurring in the intron preceding exon 2, the breakpoints in BCR are more variable and typically occur in either the major or minor breakpoint regions (M- or m-bpr). M-bpr fusions result in a p210 fusion protein, which is the form typically found in Ph+ CML.

A sizeable number of patients with Ph+ B-cell acute lymphoblastic leukemia (B-ALL; 40% of adults and 10% of children6 ) also harbor the p210 product. Conversely, m-bpr translocations result in a p190 fusion found in most Ph+ B-ALL cases but rarely in Ph+ CML.7

Uncommonly, BCR breakpoints fall in the microregion (µ-bcr), resulting in the p230 fusion product associated with chronic neutrophilic leukemia (CNL).6,8

Detection of t(9;22) is most commonly performed by either cytogenetics, fluorescence in situ hybridization (FISH), or reverse transcription–polymerase chain reaction (RT-PCR; amplification of the transcript product); the latter is used for minimal residual disease testing.

At diagnosis, conventional cytogenetics can detect t(9;22) in 95% of cases of CML; however, an additional 2.5% of cases with submicroscopic translocations can be recovered by applying molecular methods.6

If the results of both cytogenetics and FISH are negative, then an alternative diagnosis should be considered. Assaying for t(9;22) can be used to monitor the therapeutic response of imatinib mesylate and for relapse surveillance (using quantitative polymerase chain reaction [qPCR] methods, particularly screening for positive results 6–12 months after transplantation).6

However, the importance of the BCR-ABL1 fusion gene in CML is in its role as a paradigm for targeted cancer therapy; patients with CML may receive imatinib as first-line therapy as established by data from the International Randomized Study of Interferon and STI571 trial.9

Baseline values from this trial are also used as the basis of the international reporting scale (IS). The IS allows for the standardization and comparison between laboratories with regard to BCR-ABL levels.10

Response to therapy can be classified as complete hematological response, complete cytogenetic response, and molecular response based on levels of fusion transcripts by RT-qPCR.

A major molecular response is defined as a 3-log reduction compared with baseline (or ≤ 0.1% IS), and a complete molecular response is defined as a 4.5-log reduction or more from baseline.9,11

A lack of response may indicate acquired resistance. In such cases, Bcr-Abl kinase domain mutations (> 100 types documented) can be found in one-half of refractory cases and are an indication to adjust therapy by integrating a second-generation tyrosine kinase inhibitor into treatment.12

Atypical Chronic Myelogenous Leukemia and Chronic Neutrophilic Leukemia

CNL has been well established given the prior detections of 20q-, 11q-, and JAK2 V617F mutation in this disease, but they are not disease specific.13-15

Deep sequencing has identified CSF3R mutations in CNL and atypical CML in 59% of patients,16 and these findings were subsequently documented in all World Health Organization (WHO)–defined CNL cases, possibly prompting a future revision of the WHO diagnostic criteria.17

Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis

JAK2 codes for an intracellular tyrosine kinase and provides signaling for growth factor receptors, including the erythropoietin receptor. The JAK2 V617F mutation was discovered in 2005 and was shown to be present in 95% of polycythemia vera cases and approximately 50% to 65% of cases of essential thrombocytosis (ET) and primary myelofibrosis (PMF).18

In addition, the JAK2 V617F mutation can also be seen in nearly one-half of cases of refractory anemia with ring sideroblasts associated with marked thrombocytosis.19,20 In cases of polycythemia vera in which the JAK2 V617F mutation is not detected, the remaining 5% of patients may harbor mutations in exon 12 of JAK2.20 

Similarly, in ET and PMF cases lacking the JAK2 V617F mutation, an assessment of MPL is indicated, given that 5% or more of patients with PMF and even fewer patients with ET (1%) will show an aberration in this gene (W515K/L).20-22 Exon 10 c-MPL mutations have also been reported in ET or PMF (5%).23

The JAK2 V617F mutation can be detected via targeted PCR followed by sequencing of the amplicon. Other methods include restriction digest of PCR-amplified products followed by separation by capillary electrophoresis, allele-specific PCR using probe-based gene expression analysis, real-time PCR, pyrosequencing, and melting curve analysis.24,25

Despite the discovery of JAK2 and MPL mutations, until recently many ET and PMF cases did not have a unique genetic basis (ie, JAK2, MPL) until 2 independent groups identified CALR mutations in this patient subset. CALR mutation, which comprises insertions (ins) and deletions (del) leading to a frameshift, are found in 20% to 25% of ET and PMF cases and tend to cluster in exon 9.26-28

Commercial testing utilizes sequencing and fragment length analysis. Recently, the Dynamic International Prognostic Scoring System Plus listed unfavorable karyotype as a risk factor for predicting survival in primary myelofibrosis.29


Activating point mutations in KIT are highly associated with mastocytosis and can be detected in more than 95% of cases of systemic mastocytosis using real-time qPCR, allele-specific oligonucleotide PCR, or direct sequencing.30,31

KIT mutations result in the ligand-independent activation of the c-kit tyrosine kinase. The most common mutation in systemic mastocytosis is the D816V variant seen in 68% of cases of mastocytosis; however, in certain subsets (eg, aggressive systemic mastocytosis), its incidence may exceed 80%.32

The presence of this variant constitutes a minor criterion for the diagnosis of systemic mastocytosis. Other KIT variants have been described (< 5%) and are more likely to be detected in the context of cutaneous mastocytosis rather than systemic mastocytosis.20 Patients with the D816V variant are resistant to imatinib


Myeloid and Lymphoid Neoplasms With Eosinophilia and Abnormalities of PDGFRA, PDGFRB, or FGFR1

A unique group of myeloid and lymphoid neoplasms are defined by aberrant tyrosine kinase activity due to translocations involving PDGFRA, PDGFRB, or FGFR1, all of which are characteristically associated with eosinophilia.

A workup for abnormalities in these genes should be considered in cases of eosinophilia with end-organ damage or in which secondary reactive eosinophilia has been excluded.

The cellular ontogeny of these disorders may originate from a pluripotent (lymphoid–myeloid) stem cell. PDGFRB or FGFR1 can be detected with conventional cytogenetic analysis (ie, karyotype); however, the FIP1LI-PDGFRA results in an 800-kb cryptic del(4q12) that houses CHIC2.

Typically, it is detected using FISH with a probe spanning CHIC2 or break-apart assay for either of the translocation partners. The translocation can also be detected using RT-PCR.20 FIP1LI–PDGFRA disease commonly manifests as chronic eosinophilic leukemia, and FIP1LI–PDGFRA is detected in 10% to 20% of those with idiopathic hypereosinophilia.34

Patients have a response to imatinib more than 100 times greater than that seen in BCR-ABL rearrangement.20,35 Neoplasms associated with PDGFRB commonly present as chronic myelomonocytic leukemia. ETV6 is the most common translocation partner, but more than 13 others have been described; patients will be responsive to imatinib.34

Neoplasms associated with FGFR1 can manifest as acute leukemias (myeloid or lymphoid) or as chronic eosinophilic leukemia. Translocation partners include ZNF198, CEP110, FGFR10P1, BCR, TRIM24, MYO18A, HERVK, and FGFR10P2.

By contrast to PDGFRA- and PDGFRB-associated neoplasms, these disorders are unresponsive to tyrosine kinase inhibitors.20,34

Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a clonal disorder of myeloid cells characterized by morphological dysplasia and ineffective hematopoiesis that manifests as peripheral cytopenia.36

Cytogenetic abnormalities are seen in one-half of MDS cases, and they most commonly involve del(5q/7q) or monosomies of the same.20,37 TP53 mutations are associated with therapy-related MDS and have a poor prognosis.38

Various cytogenetic abnormalities can be considered presumptive evidence of MDS even in the absence of sufficient dysplasia (ie, -5/del[5q], -7/del[7q], +8,-Y, del[20q], isochromosome [i][17q], -13/del[13q], del[11q], del[12p], del[9q], isodicentric [idic][Xq13], and certain balanced translocations involving chromosomes 1, 2, 3, 9, 11, 16, and 21).20

Various prognostic models are available for MDS. The most widely adopted is the International Prognostic Scoring System (IPSS) and its revised version (IPSS-R), both of which integrate the percentage of blasts in the bone marrow, cytogenetic abnormalities, and number of cytopenias.39

In the latter scheme, cytogenetics are placed in 5 tiers: very good (-Y, del[11q]), good (normal, del[5q], del[12p], del[20q], and del[5q] + 1 more), intermediate (del[7q], +8, +19, i[17q], and others), poor (-7, inversion [inv][3/t3q/del{3q}], -7/del[7q] + 1 more, and 3 cytogenetic aberrations), and very poor (> 3 abnormalities).40

Although gene-expression profiling and single nucleotide polymorphism arrays are powerful tools, they are not routinely employed in the clinical setting. However, somatic mutation in 40 genes has been found in MDS and analysis for these genes can add prognostic value.41

By contrast to cytogenetic abnormalities, which are seen in one-half of cases, at least 1 of these “driver” mutations can be found in most cases of MDS.42,43 For example, patients with 1 or more mutations in TP53, EZH2, ETV6, RUNX1, or ASXL1 show survival patterns analogous to those in the next higher tier by subgrouping in the IPSS.44

Various epigenetic modifiers (DNA methylation regulators, spliceosome mutations, and histone modifiers TET2, IDH1/2, DNMT3A, EZH2, ASXL1, SF3B1, U2AF1, SRSF2, and ZRSR2), transcription factor genes, and kinase signaling genes have been implicated in MDS, providing a basis for approved therapies and those in development.45

However, these aberrations have limitations because their clinical significance is not always clear given their association with poor prognostic clinical features, our lack of knowledge of their interactions with other markers often concurrently detected, intratumoral clonal heterogeneity, and the wide gamut of mutations in any given gene.42

Next-generation sequencing (NGS) technologies, which garner the power of massively parallel sequence generation, enable laboratories to clinically sequence many genes simultaneously, which was previously untenable by traditional sequencing technologies.

Commercial testing is available for activated signaling genes (KIT, JAK2, NRAS, CBL, MPL), transcription factors (RUNX1, ETV6), epigenetic genes (IDH1/2, TET2, DNMT3A, EZH2, ASXL1, SETBP1), ribonucleic acid splicing genes (SF3B1, U2AF1, ZRSF2, SRSF2), and tumor suppressors (TP53, NPM1, PHF6), among others.

In cases of MDS or MDS/myeloproliferative neoplasms in which the diagnoses are unclear or dysplasia has yet to emerge, detecting a mutation in one of these key genes may be helpful in establishing the diagnosis of a clonal myeloid neoplasm.

Although they are not formally incorporated into prognostic stratification schemas, certain mutations may also carry prognostic implications.46

In addition, robust myeloid testing commercially available (FoundationOne Heme, Cambridge, Massachusetts) can interrogate 405 cancer-related genes, allowing — in theory — for the identification of targetable mutations and patient enrollment in clinical trials.47

Acute Myeloid Leukemia

AML is the most common type of acute leukemia occurring in adults.48 In 2015, an estimated 20,830 new cases of AML will occur in the United States, along with 10,460 deaths.48 AML is a lethal disease and has a 5-year relative survival rate of 24.2%.49

However, outcomes are heterogenous and overall survival rates range from approximately 5% to 70%.50 Thus, a need exists for prognostic markers to predict outcomes and guide therapeutic decision-making. Prognostic markers can be clinical, disease related, and molecular, although the strongest prognostic factor for predicting therapeutic response and survival is cytogenetic subgrouping.

The results of numerous clinical trials across several decades have indicated that overall survival rates can be as long as 11.5 years in favorable patient groups or shorter than 1 year in patients with adverse risk.51-53

Those with favorable risk (5-year survival rate of 50%–80%) include those with t(15;17), t(8;21), inv(16/t[16;16]), a normal karyotype and mutated NPM, or a normal karyotype with biallelic CEBPA mutation.54

These patients may receive consolidation therapy following induction 7 + 3 with chemotherapy (eg, high-dose cytarabine). Those with adverse risk (overall survival rate of 5%–20%), including those with MLL aberrations, inv(3), t(6;9), -7/del(7q), -5/del(5q), TP53 deletions, and a complex karyotype, may undergo transplantation after standard induction.55,56

Determining whether consolidation therapy is appropriate in those with intermediate risk (overall survival rate of 20%–40%55) is not as clear. In this cohort, molecular testing for FLT3, NPM, and CEBPA is informative and has therapeutic implications.

For example, detecting FLT3 internal tandem duplication by PCR in patients with normal karyotype AML may lead to consolidation therapy with hematopoietic stem cell transplantation, after which patients may have a 30% likelihood of cure.55

FLT3 codes for a transmembrane signal-transducing protein of the tyrosine receptor kinase family and reveals 2 major abnormalities in AML, ie, internal tandem duplication in the juxtamembrane portion resulting in constitutive activation and a point mutation in Asp835 (the activity loop portion of protein) resulting in dysregulation. FLT3 aberrations are seen in 5% to 10% of AMLs.57,58

NPM1 encodes nucleophosmin, a 37 kDa protein. NPM1 mutations involve 4 to 11 break-point insertions in exon 12 that lead to the mislocalization of normal nucleophosmin to the cytoplasm via dimerization.59,60

This can be detected by PCR followed by sizing via capillary electrophoresis. NPM1-mutated AML has been designated as a provisional entity in the 2008 WHO classification and is associated with unique morphological (blasts with “cup-like” nuclei) features and a favorable prognosis in normal karyotype AML.20 CCAAT/enhancer-binding protein α is a 42 kDa transcription factor whose loss is associated with the oncogenic transformation of myeloid cells due to a loss of differentiation.61

Patients with mutated CEBPA show outcomes similar to those in the favorable cytogenetic subgroup of AML (eg, t[8;21]+ AML).62 The prognostic value of CEBPA is in the double-mutated subset of patients lacking FLT3 and NPM1 mutations.63

Other single gene alterations that may carry important prognostic implications have been identified in AML — many were identified during whole genome sequencing studies — and include DNMT3A, IDH1/2, TET2, WT1, ERG expression, BAALC expression, and MN1 expression, among others. IDH2 is associated with a good prognosis and TET2, ASXL1, and PHF6 confer poor prognoses.

However, in the absence of prospective trials and the present controversy regarding them, many of these single genes have not been formally integrated into accepted risk-stratification models.64 In general, investigating the mutation status of these genes is simultaneously obtained using NGS technologies.

Biomarkers are important for subclassifying AML types, and several categories of AML are defined based on the presence or absence of recurrent genetic abnormalities alone, in particular t(8;21)(q22;q22), inv(16), t(15;17)(q22;q12), t(9;11)(p22;q23), t(6;9)(p23;q34), inv(3), and t(1;22)(p13;q13).13

In some cases, the detection of 1 of these aberrations alone is enough to diagnose AML, even in the absence of the conventional criteria of 20% blasts in the marrow or peripheral blood, such as in the case of t(15;17) and core-binding factor-related leukemias (eg, t[8;21][q22;q22], inv[16]) and possibly inv(3)/t(3;3).65-67

These translocations can be detected by conventional cytogenetics, FISH, and more novel technologies, including single molecule imaging and NGS. One such type of sequencing uses color-coded barcodes directly hybridized to individual target molecules and then digitally detects them in a multiplexed manner.68

A comprehensively targeted clinical panel currently on the market uses NGS to interrogate the exons of 405 genes and examines the intronic regions of 31 genes involved in rearrangements as well as complementary DNA (ribonucleic acid) to sequence 265 genes to detect translocations.69

In some cases, the detection of a translocation carries both diagnostic and therapeutic importance. Namely, t(15;17)(q22;q21), which is diagnostic for acute promyelocytic leukemia (AML M3), juxtaposes the 17(q21) retinoic acid receptor α (the receptor for vitamin A involved in cell proliferation and differentiation) to the 15(q22) promyelocytic leukemia zinc finger protein (involved in transcriptional regulation and apoptosis). The chimeric protein blocks differentiation beyond the promyelocytic stage, resulting in acute leukemia.

However, treatment with all transretinoic acid allows for differentiation and, in combination with cytotoxic chemotherapy, can result in complete remission. Variant translocations involving RARA are seen in fewer than 2% of cases, but they are important given that some patients may not respond to all transretinoic acid therapy.20,70

Another example of molecular testing informing therapeutic management involves KIT testing in AML. C-kit mutations are associated with core-binding factor AMLs and may abrogate the favorable prognosis generally associated with this group.

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