Molecular markers

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• Chromosomal aberrations leading to rearrangement of JAK2 define a WHO family of entities comprising many myeloid or lymphoid neoplasms
  • Patients with JAK2 fusions may present with chronic myeloproliferative neoplasms (i.e., chronic eosinophilic leukemia, primary myelofibrosis, etc.), myelodysplastic / myeloproliferative neoplasms with neutrophilia or monocytosis often associated with hypereosinophilia or with B cell acute lymphoblastic leukemia (B ALL) with BCR1::ABL1-like features
  • Many fusion partners are possible but all result in constitutive activation of JAK-STAT pathway
  • Translocation t(8;9)(p22;p24) leading to fusion product PCM1::JAK2 is most common
  • Other fusion partners include ETV6 and BCR
  • PCM1::JAK2 fusion also reported in many mature T cell lymphomas, particularly mycosis fungoides (Virchows Arch 2022;481:967)
• Pathogenic mutations of JAK2 are described in myeloproliferative neoplasms (StatPearls: Myeloproliferative Neoplasms [Accessed 18 July 2024])
  • JAK2 p.V617F (exon 14) detectable in > 95% of patients with polycythemia vera (PV), 50 - 60% of patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF) (Hematol Oncol Clin North Am 2021;35:217)
  • JAK2 mutations in exon 12 (typically small in frame insertion or deletions affecting the pseudokinase domain) present in virtually all patients with PV without JAK2 p.V617F and typically absent in ET (N Engl J Med 2007;356:459)
  • JAK2 mutations are one of the major criteria for diagnosis of PV, ET and PMF according to WHO 2022
  • JAK2 indels in exon 13 described in chronic eosinophilic leukemia (CEL), NOS (Blood 2019;134:2388)
• JAK2 p.V617F allele burden (AB) correlated with
  • Increased risk of thrombosis in PV (Leukemia 2010;24:1574)
  • Evolution in secondary myelofibrosis in PV (Leuk Res 2011;35:177)
  • Reduced survival in PMF (Blood 2009;114:1477)
• Mutations of JAK2 in up to 10% of chronic myelomonocytic leukemia (CMML) (J Clin Oncol 2013;31:2428)
• JAK2 mutations can be found in acute myeloid leukemia (AML) (Br J Haematol 2018;182:78)
  • More frequently in secondary AML transformed from a myeloproliferative neoplasia
  • Can occur in de novo AML
• Mutations (p.V617F or missense / indel in amino acid range p.536 - p.547) typical in clonal hematopoiesis of indeterminate potential (CHIP) (Cells 2019;8:854)
• JAK2 mutations p.R683G, p.H574R and I682T identified in T cell lymphoblastic lymphoma (T LBL) (Leukemia 2016;30:94)
• Alteration (amplifications, fusions, mutations, etc.) of JAK2 found in 2.01% of malignant solid tumors, with JAK2 mutations in 1.38% of all malignant solid tumors (My Cancer Genome: JAK2 Mutation [Accessed 18 July 2024])
  • Most commonly lung adenocarcinoma, colon adenocarcinoma, endometrial carcinoma, bladder urothelial carcinoma and breast carcinoma
• Many JAK inhibitors approved for therapy in hematopoietic neoplasms (PV, PMF) and inflammatory / autoimmune syndromes (e.g., rheumatoid arthritis, ulcerative colitis, severe COVID-19, etc.)
  • Examples: ruxolitinib, baricitinib, fedratinib
  • Can inhibit the activity of 1 or more proteins of JAK family with different specificity
  • Mechanism of action: target the catalytic ATP binding site of JAK proteins
  • Side effects include infections, embolisms, thrombosis and skin cancer (Sci Rep 2022;12:7140)

Contributed by Umberto Maccio, M.D., M.Sc., M.B.A.

• Many methods can be used based on laboratory experience and oriented toward clinical questions
• Polymerase chain reaction (PCR) amplifies the DNA region of interest of the gene JAK2 (exon 12, exon 14)
  • Mutations are detected through Sanger sequencing (Int J Lab Hematol 2016;38:34)
• Next generation sequencing (NGS) allows the analysis of JAK2 gene simultaneously with other genes in a diagnostic panel appropriate for hematologic neoplasms (Ann Hematol 2019;98:111)
• Allele specific oligonucleotide (ASO) PCR (also known as ARMS PCR, amplification refractory mutation system): highly sensitive method to detect a specific known mutation (e.g., JAK2 p.V617F) in case of minimal residual disease under the limit of detection (LOD) of other tests (J Mol Diagn 2007;9:272)
• Lock nucleic acid (LNA) PCR utilizes LNA that binds more tightly and specifically wild type sequences due to their thermal stability, leading to selective amplifications of mutated sequences and reducing off target amplification (J Mol Diagn 2010;12:512)
• Rearrangements of JAK2 can be analyzed through ribonucleic acid (RNA) based NGS panels designed for myeloid neoplasms including many fusion partners (J Mol Diagn 2023;25:932)
• Alterations of chr 9p24.1 may be visible on routine karyotyping (Mod Pathol 2019;32:490)
  • Subset of cases may be cryptic
  • Fluorescent in situ hybridization (FISH) for JAK2 (break apart probes) or RT PCR for specific fusion products are more sensitive than karyotyping

Contributed by Umberto Maccio, M.D., M.Sc., M.B.A.

A 72 year old patient evaluated for lung embolism showed elevated hemoglobin concentration with reduced erythropoietin level. RT PCR for BCR1::ABL1 on peripheral blood is negative. Bone marrow biopsy is shown above. Which genetic alteration is most likely to be demonstrated in an NGS analysis of marrow?

1. BRAF p.V600E
2. JAK2 p.V617F
3. KIT p.D816V
4. PML::RARA fusion

B. JAK2 p.V617F is the correct answer. Based on laboratory findings and marrow histology, the most likely diagnosis is polycythemia vera. About 95% of cases harbor this mutation. Answer D is incorrect because PML::RARA fusion is diagnostic of acute promyelocytic leukemia. Answer A is incorrect because BRAF p.V600E is typical for hairy cell leukemia but not myeloproliferative neoplasms. Answer C is incorrect because KIT p.D816V is among several diagnostic criteria for systemic mastocytosis but not diagnostic for polycythemia vera.

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Reference: JAK2

A 76 year old patient has been diagnosed with lung adenocarcinoma with metastasis in the liver and bone (stage IV). Peripheral blood analysis was only significant for mild anemia with increased ferritin and mild leukocytosis. NGS analysis performed on liver biopsy with a tumor cell content of 80% demonstrates following alterations: KRAS p.G12C (VAF 40.5%), JAK2 p.V617F (VAF 1.0%), DNMT3A p.R882H (VAF 0.9%), tumor mutational burden (TMB) 12.7 mutations/megabase, microsatellite stable. Which of the following statements is correct?

1. JAK2 is a driver mutation for lung carcinomas that can be targeted with a JAK inhibitor
2. JAK2 p.V617F probably represents a subclonal mutation in carcinoma cells
3. The patient probably has a clonal hematopoiesis of indeterminate potential (CHIP)
4. The patient probably has a myeloproliferative neoplasm and needs a bone marrow biopsy for definitive diagnosis

C. The patient probably has a clonal hematopoiesis of indeterminate potential (CHIP). JAK2 mutations, as well as DNMT3A, are commonly detected in CHIP. Answer A is incorrect because although JAK2 mutations can be detected in lung carcinomas, the KRAS mutation is the driver mutation here. VAF and tumor cell content are also concordant in this sense. Moreover, JAK inhibitors are not approved for therapy for lung carcinoma. Answer D is incorrect because although the presence of JAK2 mutations is a diagnostic criterion for myeloproliferative neoplasms (MPNs), an MPN is unlikely in the absence of other clinical or laboratory features. Answer B is incorrect because although JAK2 mutations can be detected in lung carcinomas, the concomitant presence of a DNMT3A mutation with comparable VAF makes CHIP the most likely interpretation.

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Reference: JAK2