Genetic testing can miss rare CALR mutations in myelofibrosis, case shows

An elderly woman with primary myelofibrosis experienced disease worsening despite being on treatment with hydroxyurea and was later found to have a rare mutation in the CALR gene that had been missed by routine genetic testing.

According to clinicians, the woman’s “clinical progression prompted repeat [blood] and molecular evaluation,” with a bone marrow biopsy and, importantly, more advanced genetic testing. That testing, done eight years after her diagnosis, revealed the rare CALR mutation, the researchers reported.

“This case highlights the limitations of routine assays focused on common CALR mutations,” the team wrote, adding that “there is a pressing need to update and expand the coverage of standard diagnostic assays to include a broader spectrum of CALR mutations.”

The woman’s case was described in a brief report titled “Calreticulin Type 26 Mutation in Myelofibrosis: A Rare Variant with Diagnostic Challenges” and published in the Journal of Clinical Laboratory Analysis by the researchers at a university hospital in Messina, Italy.

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Myelofibrosis is a type of myeloproliferative neoplasm, a blood cancer that usually grows slowly. In myelofibrosis, the bone marrow — the soft, spongy tissue found in the center of most bones —starts producing abnormal blood cells, leading to inflammation and scarring, known as fibrosis. This can make it difficult for the bone marrow to produce healthy blood cells.

Individuals with myelofibrosis often have an enlarged spleen, among other symptoms.

Myeloproliferative neoplasms are often linked to mutations in the JAK2, MPL, or CALR genes, with CALR mutations “almost exclusively” associated with two types of blood cancer: essential thrombocythemia and primary myelofibrosis, the most common form of myelofibrosis. The CALR gene encodes calreticulin, a protein that helps maintain calcium homeostasis within cells. When CALR is mutated, it can cause cells to grow abnormally.

Genetic testing at diagnosis came back negative

This case involved a 78-year-old woman diagnosed with a myeloproliferative neoplasm in 2016. She was treated with hydroxyurea, a chemotherapy agent used to reduce the number of blood cells produced by the bone marrow, including the abnormal ones. At the time of diagnosis, an ultrasound showed an enlarged spleen and small kidney stones and cysts, or fluid-filled sacs. Blood tests showed elevated inflammatory markers.

Genetic testing for two types of mutations — JAK2 V617F and CALR exon 9 indels — that commonly occur with such symptoms came back negative. However, the assay that was used at the time is designed primarily to detect the two most common types of mutation in the CALR gene, called Type 1 and Type 2. Very small or rare mutations can be missed, especially when present at low levels, according to the researchers.

In 2024, the woman’s myeloproliferative neoplasm progressed. Blood testing revealed severe thrombocytosis, or very high platelets, which help blood clot, as well as leukocytosis, or a high white blood cell count. The woman also had persistent microcytic hypochromic anemia, in which red blood cells are smaller and contain less hemoglobin than normal. Hemoglobin is the protein that carries oxygen in red blood cells.

A biopsy was performed to remove a small bone marrow sample for microscopic examination. The biopsy showed fibrosis and increased production of granulocytes, a type of white blood cell. Many abnormal megakaryocytes, the cells that produce platelets, were also observed. These findings were consistent with primary myelofibrosis, in which fibrotic tissue gradually replaces normal bone marrow.

More advanced testing showed rare CALR mutation

More advanced genetic testing was then performed using Sanger sequencing and next-generation sequencing. Sanger sequencing reads the DNA sequence directly, while next-generation sequencing reads many DNA fragments simultaneously with greater sensitivity.

These tests identified a rare mutation in CALR, called c.1122delG, also known as Type 26, the researchers noted.

This case underscores critical challenges in diagnosing and managing [myeloproliferative neoplasms in myelofibrosis], primarily due to the limitations of current diagnostic tools.

Further testing showed that the mutation was heterozygous, meaning only one of the two copies of the CALR gene was mutated. This was confirmed by next-generation sequencing, which showed that the mutation was present in 43% of the analyzed DNA. Another mutation in the CBL gene was also identified. Mutations in this gene may contribute to progression and differences in symptoms between patients, the researchers noted.

The study authors noted that the woman’s correct diagnosis was delayed due to the lack of more complete testing.

“This case underscores critical challenges in diagnosing and managing [myeloproliferative neoplasms], primarily due to the limitations of current diagnostic tools,” the researchers wrote, adding that “expanded molecular testing may improve diagnostic accuracy and clinical classification in [myeloproliferative neoplasms].”