Ph-negative MPNs are disorders arising in a single clone of multipotent precursor cells in which one or all myeloid lineages are abnormally amplified. Classic and more frequently encountered Ph-negative MPNs include PV, primary myelofibrosis, and essential thrombocytopenia (ET). However, included in this chapter are Fig. 1A and B and 2 in order to illustrate the most frequent abnormalities associated with the Ph-negative MPN. Different 9p chromosomal abnormalities result in JAK2 numerical gain (see Fig. 1) while nine examples of 1q gain, associated with disease progression, are shown in Fig2.
Fig1. TWO DIFFERENT TYPES OF 9P CHROMOSOMAL ABNORMALITIES AND SUBSEQUENT JAK2 COPY NUMBER ALTERATIONS IN Ph-NEGATIVE MYELOPROLIFERATIVE NEOPLASM. (A) Tetrasomy of JAK2 in a newly diagnosed patient with myelofibrosis. The initial full karyo type at diagnosis was 47,XX,+mar. Following metaphase FISH evaluation using JAK2 breakapart probe, the karyotype was revised to 47,XX,+ider(9)(p10)del(9) (p12p21) resulting in tetrasomy of JAK2. The panel on the right shows the results obtained from array CGH+SNP indicating a gain of 24.7 Mb on the short arms of chromosome 9 including the JAK2. (B) Homogenous staining region (HSR) and amplification of JAK2 in a patient with myelofibrosis. The cytogenetic results showed 20% of cells with sole del(20q) and 80% of cells had a complex karyotype including intrachromosomal complexity such as chromothripsis detected within chromosomes 1, 15, and 20 (not shown) and amplification of 9p (right panel), including JAK2 and cn LOH of 17p involving TP53 (not shown). CGH, Comparative genomic hybridization; FISH, fluorescence in situ hybridization; SNP, single-nucleotide polymorphism
Fig2. NINE TYPES OF 1q GAINS IN PATIENTS WITH Ph-NEGATIVE MYELOPROLIFERATIVE NEOPLASM WHO EITHER HAD +1q AT DIAGNOSIS OR PROGRESSED TO MYELOFIBROSIS AND DEVELOPED +1q. (A and B) Shows different sizes of duplicated 1q segment in two patients: 1q24 shown in (A) is a smaller 1q segment and breakpoint at 1q32 shown in (B) a larger duplicated gain of 1q. (C) Almost an entire long arms of chromosome 1 are translocated to the short arms of chromosome 19 resulting in gain of 1q and loss of 19q. (D) This der(6)t(1;6)(q21;p21.3) rearrangement is a recurrent abnormality both in Ph-negative MPN and MDS and most frequently observed in patients characterized with jumping 1q phenomenon. Often the extra copy of 1q is translocated to the short arms of chromosome 6 and very rarely to the long arm. (E) Another recurrent gain of 1q is the translocation to acrocentric chromosomes such as 22 without apparent loss of any genetic material and is described as +1,der((1;22)(q10;q10). (F) der(9)t(1;9)(p24;p21) is rarely observed in Ph-negative MPN and more frequently in MDS. In this unbalanced translocation there is a loss of terminal band from 9p and a gain of distal 1q segment. (G) der(12)(t(1;12)(q21;p13) is a recurrent abnormality in the (H) +1,del(1)(p13p36.1), (I) der(1)t(1;9)(p13;p13), + der(1)t(1;9)(p13;p13) x2 resulting in tetrasomy for 9p and 1q. MDS, Myelodysplastic syndrome.
Less frequently encountered similar disorders include chronic neutrophilic leukemia, hypereosinophilic syndrome/ chronic eosinophilic leukemia, systemic mastocytosis, atypical CML, and unclassifiable MPNs. Rare recurrent balanced abnormalities in atypical Ph-negative MPNs can involve the PDGFRB gene localized at 5q33, FGFR1 gene on 8p11, and PDGFRA gene on 4q12. The best described are t(5;12)(q33;p13), resulting in ETV6-PDGFRB fusion protein, and t(8;13)(p11;q12), resulting in the ZNF198 FGFR1 fusion gene. The disorder known as 8p11 myeloproliferative syndrome has been classified by WHO as belonging to the group of myeloid/lymphoid neoplasms associated with eosinophilia and gene rearrangements. Some of these diseases are related to fusion genes between FGFR1 located on 8p11 and various translocations fusing at least 14 different 5′ partner genes to the 3′ part of the FGFR1 gene that encodes tyrosine kinase domain (Table1). They include TPR (1q25), LRRFIP1 (2q37), SQSTM1 (5q35) FGFR10P (6q27), TRIM24 (7q34), CUX1 (7q22), PLAG1 (8q12), CEP110 (9q33), NUP98 (11p15), FGFR1OP2 (12p11), CPSF6 (12q15), ZMYM2 (13q12), MYO18A (17q23), HERVK (19q13), and BCR (22q11) (Fig. 3). The most frequent translocation is t(8;13), and cases with the submicroscopic deletion of the 5′ part of the FGFR1 gene have also been reported, similar to the formation of t(9;22) with deleted sequences from der(9q) and/or 22q identified in CML. The presence of 8p11 abnormalities in both myeloid and lymphoid cells suggests its origin from a common stem cell.
Table1. Chromosomal Translocations in Ph-Negative Myeloproliferative Neoplasms
Table 1. Chromosomal Translocations in Ph-Negative Myeloproliferative Neoplasms —cont’d
Fig3. (A) A partial karyotype from a patient diagnosed with Ph-negative myeloproliferative neoplasm showing t(8;22)(p11;q11.2). (B) Six months later the patient developed hyperdiploid karyotype, acute lymphoblastic leukemia, and two copies of t(8;22). (C) Interphase FISH confirmed a FGFR1 (red)-BCR (green) fusion (yellow).
In patients with chronic eosinophilic leukemia/hypereosinophilic syndrome (Chapter 74) the most frequent recurrent abnormality is the cryptic deletion on 4q12 as a result of FIP1L1-PDGFRA fusion gene reported to occur in from 3% to 56% of cases. The disparity in frequencies reflects differing levels of stringency criteria in the diagnosis of disorders with hypereosinophilia as well as different technologies used for detection of FIP1L1-PDGFRA fusion. An investigation of 376 patients with persistent unexplained hypereosinophilia revealed an 11% incidence of the FIP1L1-PDGFRA fusion gene detected using highly sensitive RQ-PCR. Patients with FIP1L1-PDGFRA fusion are characterized by a male predominance, marrow fibrosis, increased number of mast cells, elevated serum tryptase levels, and a favorable response to low doses of imatinib. Most of these patients have a nor mal karyotype because a cryptic deletion of CHIC2 locus on 4q12 which is only 800 kb in size. This abnormality is detectable using a more sensitive FISH technology and RQ-PCR in the majority of cases. Moreover, the commercially available tricolor FISH probe will detect not only deletion 4q12 as a result of FIP1L1-PDGFRA but also a rare BCR-PDGFRA fusion resulting from the t(4;22)(q12;q11.2) rearrangement (Fig. 4). Serial monitoring with RQ-PCR demonstrates exquisite sensitivity of FIP1L1-PDGFRA-positive patients to low-dose imatinib treatment (Chapter 74). The FIP1L1-PDGFRA has also been detected in histopathologically defined cases of systemic mastocytosis with associated eosinophilia, and in cases of AML and T-cell lymphoblastic lymphoma associated with eosinophilia. In an Italian prospective cohort of 27 patients with FIP1L1-PDGFRA who were treated with imatinib, a complete hematologic response was achieved within 1 month and all patients became PCR-negative for FIP1L1-PDGFRA after a median of 3 months (range 1 to 10 months). In contrast to CML, very few cases of acquired imatinib resistance have been reported. Those rare reported cases had a mutation in T674I within the ATP-binding domain of PDGFRA, analogous to the T315I mutation in CML.
Fig4. BONE MARROW INTERPHASE NUCLEI AFTER FLUORESCENCE IN SITU HYBRIDIZATION STUDIES USING TRICOLOR PROBE FOR CHROMOSOME 4, BAND REGION q12. The green color covers an approximately 750-kb region centromeric from FIP1L1. The red probe is telomeric of the FIP1L1 gene. The aqua color probe begins between exons 15 and 16 of the PDGFRA gene and extends toward the 4q telomere. In normal nuclei, as shown here, the probe appears as two tricolor fusions because of close proximity of probes in interphase DNA. Patients with hypereosinophilic syndrome have fusion of FIP1L1 and PDGFRA genes by interstitial deletion and produce one signal with green-aqua fusion and a missing orange signal. If the translocation involves the PDGFRA gene with loci on other chromosomes, the expected signal pattern is one orange–green fusion and one separate aqua signal.
Although translocations involving PDGFRB at 5q32 region have been identified with 22 different fusion partners, they are indeed very rare. Any patient with a 5q31–q33 chromosomal abnormality with or without eosinophilia should be investigated for PDGFRB rearrangements by FISH testing (Fig. 5).
Fig5. A partial karyotype from a patient with Ph-negative MPN showing in (A) panel an inversion of chromosome 5 (red arrow) identified cytogenetically in 90% of marrow cells. In panel (B) both chromosomes 5 are shown, after metaphase FISH: the left chromosome 5 has a normal PDGFRB while the other homolog 5, with an invasion (yellow arrow), shows the split of the 5′ and 3′ part of the PDGFRB probe, consistent with the rearrangement. The panel (C) shows a bone marrow interphase cell and the yellow arrow points to the split of the red and green signals that reflect rearrangement of the PDGFRB gene detected in 28% of non-dividing cells, indicating that this rearrangement in Ph-negative MPN has a higher proliferative capacity than nondividing cells. FISH, fluorescence in situ hybridization; MPN, myeloproliferative neoplasm
Once the PDGFRA, PDGFRB, and FGFR1 rearrangements are excluded, the only other recurrent secondary abnormalities include trisomy 8 and trisomy 21 (see box on Genetic Testing for Ph-Negative Myeloproliferative Neoplasms).
In the WHO 2016 classification, mastocytosis is no longer listed as a Ph-negative MPN but rather as a distinct category, “Mastocytosis.” Mastocytosis includes a group of rare and heterogeneous diseases characterized by accumulation of clonal mast cells in one or multiple organs. Over 80% of patients with systemic mastocytosis are characterized by KIT mutations, specifically in the activation loop of KIT, KIT D816V. KIT is encoded by a 21-exon-containing gene located on the long arms of chromosome 4, 4q12. Other rare KIT mutations have been described in adult and pediatric patients. The knowledge of the type and structure of KIT mutation is important because the A-loop mutations D816V/H/Y/N disrupt the structure of the receptor, leading permanently to an active conformation and resistance to imatinib. In patients with advanced systemic mastocytosis additional mutations have been observed including TET2 (up to 39%), SRSF2 (up to 36%), ASXL1 (up to 21%), and RUNX1 (23%). At diagnosis of mastocytosis patients testing for KIT D816V in peripheral blood leukocytes, using highly sensitive and specific PCR-based assays, is necessary to determine the KIT allelic burden rapidly, which is now used as a gold standard for assessing disease burden.
