AMP case report: A case of a rare myeloid neoplasm presenting with features mimicking primary myelofibrosis

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The PCM1 gene is located on the short arm of chromosome 8 and encodes a protein with multiple coiled-coil motifs that are essential for proper centrosome assembly. JAK2 is a tyrosine-protein kinase gene located on the short arm of chromosome 9. The fusion of PCM1–JAK2 can result from breakpoints at different exons of both PCM1 and JAK2. This chimeric gene product retains several of the coiled-coil motifs from PCM1 as well as the entire tyrosine kinase encoding domain of JAK2 resulting in constitutive activation of the JAK2 kinase domain (Fig. 3).⁵

Discussion. Molecular testing plays an important role in evaluating MPNs. Characteristic variants in JAK2, CALR, and MPL help confirm the diagnosis. However, this case highlights how coincidental variants in these genes can lead to premature diagnostic closure. The classic CALR exon 9 variants associated with MPNs are a 52 bp deletion (p.L367Tfs*46) and a 5 bp insertion (p.K385Tfs*47) resulting in a pathogenic frameshift in the CALR gene encoding the calreticulin protein.^6,7 These mutations are considered mutually exclusive with JAK2 and MPL genes in MPNs, although rare cases of ET and PMF with concurrent JAK2 p.V617F and CALR mutations have been reported.^8-11 CALR exon 9 mutations are not reported in PV. CALR exon 9 frameshift deletion/insertion has been associated with a favorable prognosis in MPNs as compared with JAK2 and MPL-mutated MPNs.⁷

The variant identified in this patient—CALR exon 9 deletion, c.1142_1144del—is a 3 bp in-frame deletion resulting in the deletion of glutamine (p.E381del). Interpretations of its significance have varied.^6,12 It has been considered as an in-frame pathogenic germline mutation in a patient with a diagnosis of MPN-unclassifiable.⁶ In another study, this variant was identified in JAK2 p.V617F-positive ET patients by high-resolution melting curve analysis, but was wild type by Sanger sequencing.¹² At the time of this analysis, the CALR c.1142_1144del has not been reported in somatic variant cancer databases, such as COSMIC and cBioPortal. In gnomAD, a database of genomic variants in healthy populations, this in-frame deletion has been reported with an overall allele frequency of 0.013 percent, with highest frequency reported in the Ashkenazi Jewish population (0.155 percent).¹³ While there is no definitive consensus, population frequencies above 0.1 percent are typically benign, representing normal genomic variation in the human population.¹⁴ Based on these data and the observed VAF of 50 percent in our case, this 3 bp in-frame deletion (p.E381del) of a single amino acid is likely a benign germline polymorphism. Studies have reported 3 bp, 9 bp, and 12 bp CALR exon 9 in-frame deletions, which are also considered likely germline polymorphisms.^7,15-17 The presence of benign in-frame deletions, while rare, creates a potential diagnostic pitfall for molecular assays using polymerase chain reaction and fragment analysis. Since in-frame germline variants in exon 9 could be confused with pathogenic frameshift deletions by sizing methods, confirmation by MPS or Sanger sequencing is prudent when uncommon variants are detected.

In view of the detection of a PCM1–JAK2 rearrangement, the final diagnosis in this case was revised from CALR-positive myelofibrosis to PCM1–JAK2-fusion–positive myelofibrosis. Patients with PCM1–JAK2-fusion–positive neoplasms often present with hepatosplenomegaly, a leukoerythroblastic picture and neutrophilic precursors in the peripheral blood, and eosinophilia, although this feature may be absent in some cases. Only a mild increase in eosinophils was noted in the bone marrow aspirate smear for our patient. The marrow can also show fibrosis, similar to ET and PMF. Prognosis and survival are variable, and an aggressive clinical course with transformation to acute leukemia has been reported in the literature.⁴ Based on the diagnosis of a CALR-mutated MPN, a lower-risk MPN, the initial plan was to observe the patient with blood monitoring every three months without need for a systemic therapy. However, detection of PCM1–JAK2 rearrangement predicted higher-risk disease and changed her management to include a JAK2 inhibitor (ruxolitinib) as first-line therapy for symptom control. Because of a variable and nondurable response to tyrosine kinase inhibitors and a significant risk of disease progression,¹⁸ an allogeneic bone marrow transplant is planned as soon as possible for this patient.

Conclusion. As this case illustrates, the diagnostic workup of MPNs and related neoplasms requires the synthesis of complex clinical, pathologic, and laboratory data. Molecular diagnostics and interpretation of genomic variants is an important step in this process and may differ among laboratories based on adopted analysis strategies. Efforts are being made to standardize the approach to variant interpretation in cancer genomics.¹⁹ While there was clearly an initial error in the predicted protein consequence resulting from the CALR in-frame deletion described in this case, it serves as a reminder that coincidental, nonpathogenic germline variants do occur in disease-specific genes.²⁰ Proper variant curation must rigorously apply objective data to classify variants and should avoid undue bias based on the patient’s diagnosis as this may lead to suboptimal patient care.

Variant curation is complicated, and having access to molecular pathology expertise can help improve patient care. Consult services are prevalent in multiple other areas of pathology and laboratory medicine; however, they are relatively new in the molecular genomic arena. Seeking a second molecular opinion, as illustrated in this case, can help identify discrepancies and recognize opportunities for additional high-yield testing.

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Dr. Gupta was a molecular genetic pathology fellow at time of writing, Dr. Frank is associate professor of clinical pathology and laboratory medicine, Dr. Roth is assistant professor of clinical pathology and laboratory medicine, and Dr. Priore is assistant professor of clinical pathology and laboratory medicine—all in the Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania Perelman School of Medicine, Philadelphia. Dr. Luger is professor of medicine, Department of Medicine, Division of Hematology and Oncology, Hospital of the University of Pennsylvania Perelman School of Medicine.

Test yourself

Here are three questions taken from the case report.

1. What is the most common type of pathogenic CALR variant in myeloproliferative neoplasms?
a. Frameshift variant.
b. Missense variant.
c. Nonsense variant.
d. In-frame insertion or deletions.

2. The following statement is true about PCM1-JAK2–mutated myeloid neoplasms:
a. These neoplasms have favorable prognosis.
b. These neoplasms have a variable prognosis and require TKIs and allogeneic transplant in most cases.
c. These neoplasms can be observed and managed conservatively as compared with other myeloproliferative neoplasms.
d. Histopathological features are distinct from the more common myeloproliferative neoplasms.

3. Which of the following describes a frameshift variant?
a. CALR c.1142A>T.
b. CALR c.1142_1144del.
c. CALR c.1142_1143del.
d. CALR c.1142_1144dup.

Answers are online now at www.amp.org/casereports.