Based on clinical features, ET, PMF, and PV are closely related MPNs, distinguished primarily by their dominant presenting features and clinical course. This close clinical relationship was explained in 2005 when several groups independently identified a point mutation in the JAK2 tyrosine kinase gene as a recurrent abnormality in these three MPNs, and some cases of chronic myelomonocytic leukemia (CMML). The activating JAK2 mutation, V617F, is detected in up to 95 % of cases of PV, and approximately 50 % of ET and PMF cases (Table 44.4). A subset of PV, but not PMF or ET, has activating point mutations or in-frame duplications in exon 12 of JAK2. Among PMF and ET without V617F, activating mutations in the thrombopoietin receptor gene (MPL) are seen in approximately 10 % of cases. Because the JAK2 kinase links multiple cytokine receptors, including the erythropoietin receptor, the thrombopoietin receptor (MPL) and the granulocyte-monocyte cytokine colony stimulating factor receptor (CD116), these mutations define this group of MPNs as having JAK-linked dysregulated cytokine growth factor receptor pathways.

Differences in the features and behavior of JAK2–MPL-mutated MPNs are related to the spectrum of other coexisting somatic mutations in any particular case [5], to genetic polymorphisms in the cytokine genes of the affected individual [6], and to the varying gene dosage and level of expression of the mutated JAK2 or MPL. As a result of loss of heterozygosity or mutation of both alleles, JAK2 V617F can be present in up to several copies per cell, and cases with a higher V617F allele burden often manifest as PV or PMF rather than as ET. In contrast to patients with the V617F mutation, PV patients with JAK2 exon 12 mutations tend to present at a younger age, and with higher hematocrit, lower platelet count, and lower white blood cell (WBC) count. ET and PMF patients with MPL mutations tend to show different patterns of clonal evolution than JAK2-mutated cases, with the common karyotypic findings of gains of chromosome 1 in ET with MPL mutations and chromosome 9 in PMF with MPL mutations [7]. Germline JAK2 and MPL mutations continue to be discovered in families with predisposition to the development of MPNs [8].

Calreticulin gene (CALR) mutations occur in 50–71 % of ET cases, and 56–88 % of PMF cases that are negative for JAK2 and MPL mutations [9–12]. These mutations include a variety of insertion and deletions located in exon 9 of CALR that shift the translational reading frame and produce a common novel C-terminal peptide sequence. Mutated CALR may differentially influence signaling through the JAK-STAT pathway. CALR mutations are largely mutually exclusive with JAK2 and MPL mutations and are not found in PV, CML, AML, or most cases of MDS. CALR-mutated PMF is prognostically distinct from MPL– and JAK2-mutated MPNs and from those cases that lack all three mutations [9–14]. Therefore, testing for CALR mutations can assist in the diagnosis of MPN, and provide a genetic marker for monitoring response to therapy.

In CML, most of the BCR–ABL1 translocations involve the major breakpoint cluster region (M-bcr) adjacent to exons 12–16 of BCR (formerly called exons b1–b5) and result in either the e13a2 or e14a2 BCR–ABL1 gene fusions (or both transcripts), which encode closely related 210 kD fusion proteins (p210). In contrast, in Ph chromosome-positive (Ph+) B-ALL, 50–65 % of the BCR breakpoints arise at the minor breakpoint cluster region (m-bcr), adjacent to exons 1 and 2 of BCR, with the BCR–ABL1 transcript containing the e1a2 junction, which is translated into the p190 BCR-ABL1 fusion protein. Rarely, usually in CML with monocytosis and neutrophilia, the breakpoint occurs in the μ-BCR region (exons 17–20), resulting in a larger p230 fusion BCR-ABL1 protein.

The BCR–ABL1 fusion gene can be detected by several methods. The Ph chromosome can be visualized by conventional G-banded karyotyping with a false-negative rate of < 10 %, which is usually related to more complex fusions that obscure the breakpoints involved. However, karyotyping has a low sensitivity that is not acceptable for minimal residual disease (MRD) monitoring given that only 20 metaphases are usually analyzed (i.e., a maximal sensitivity of 5 %).

Fluorescence in situ hybridization (FISH) on interphase cells is another method to detect the BCR–ABL1 fusion (Fig. 44.1). Dual-color, dual-fusion probes are typically used and can identify variant breakpoints or three-way translocations that do not result in recognizable Ph+ metaphases. Given an adequate sample, the false-negative rate for FISH is much less than 1 %. The use of FISH for MRD monitoring after completion of therapy is more limited given that the maximal sensitivity is approximately 1 % Ph+ cells, given that only several hundred cells are typically counted [16].

Other molecular methods can be used for detecting BCR–ABL1 fusions, including PCR from genomic DNA, Southern blotting, or northern blotting. However, the vast majority of clinical molecular laboratories detect the BCR–ABL1 fusion gene by reverse transcription polymerase chain reaction (RT-PCR). Qualitative RT-PCR has been largely replaced by quantitative/real-time PCR (RT-qPCR) methods using an allele-specific PCR approach with primers directed separately against the M-bcr and m-bcr regions (p210 and p190 isoforms, respectively).

Essentially all RT-qPCR BCR–ABL1 assays normalize the amount of fusion transcript to the level of a control transcript to allow comparison across different levels of input positive cells. This approach also permits assessment of RNA quality, because low levels of the control gene likely indicate a degraded sample. Commonly used genes for normalization include GAPDH, ACTB, BCR, GUSB, and ABL1 [17]. These control genes are expressed at very different levels resulting in highly variable BCR–ABL1 fusion to control transcript values. This variability between assays using different control transcripts makes comparison of results from different laboratories difficult. Additionally, laboratories use a variety of RNA extraction methods, different reverse transcription protocols, and have different reporting criteria.

Standardization of RT-qPCR assays has become a high priority because molecular milestones of BCR–ABL1 transcript levels are now the basis of treatment decision-making for CML. The first advance in standardization, led by the Europe Against Cancer (EAC) initiative, was the development of well-validated RT-qPCR primers and probes, as well as standardized PCR conditions for BCR–ABL1 assays [18]. The second effort, beginning with the European LeukemiaNet collaborative, developed consensus on measurement of therapeutic milestones in CML (Table 44.5) [19]. This effort reached fruition in 2012 and established the International Scale (IS) whereby individual laboratories could adjust their internal BCR–ABL1/control transcript ratios to the molecular milestones based on standard calibrators [20, 21].

With an understanding of the molecular basis of CML, the design and synthesis of small molecules that could inhibit the kinase activity of ABL1 was attempted. This resulted in US Food and Drug Administration (FDA) approval of the kinase inhibitor imatinib mesylate (also called ST1571) in 1999. Before the discovery of imatinib, the widely used therapeutic options for CML were interferon-α with or without cytarabine, hydroxyurea, or allogeneic stem cell transplant (ASCT). With results first published in 2003, the pivotal International Randomized Interferon vs ST1571 (IRIS) study established imatinib as the frontline therapy for newly diagnosed CML, with superior responses and compliance rates compared to interferon/cytarabine [22, 23]. Subsequently, second generation tyrosine kinase inhibitors (TKI) that successfully target BCR-ABL1 were introduced, particularly nilotinib and dasatinib that were both approved as frontline therapy by the FDA in 2010.