Peripheral blood evaluation: B₁₂ deficiency, anemia, ET, CMML

“In this patient, we could do mutation testing on the peripheral blood before going to bone biopsy,” Dr. Chabot-Richards said. “We sent this for JAK2, CALR, and MPL testing.” The JAK2 V617F testing was negative, and reflex testing for CALR and MPL revealed the patient had a CALR 52 base pair insertion.

“Given what we were seeing in the peripheral blood, the sustained thrombocytosis, the CALR mutation, we said it’s likely essential thrombocythemia, but you cannot make that diagnosis without a bone marrow biopsy to confirm the morphology.”

Essential thrombocythemia is a myeloproliferative neoplasm associated with increased platelets. The blood findings usually consist of platelet anisocytosis with small and large forms, possibly hypogranular platelets (“we think of that as a dysplastic finding, but it can be seen in these cases”), possibly circulating megakaryocytic nuclei appearing as dark, very condensed nuclei, and neutrophilia without significant left shift. “They should not have significant dysplastic features, you shouldn’t be seeing any increase in blasts, and basophils and the red blood cells should be fairly unremarkable,” Dr. Chabot-Richards said.

The 2016 WHO update sets forth the major and minor diagnostic criteria for ET (Fig. 3).

Essential thrombocythemia diagnosis requires either all four major criteria, or the first three major and the minor criteria. “If you can’t find mutation in JAK2, MPL, or CALR, but you can find some other clonal marker, you can still make this diagnosis.”

It’s important to distinguish ET from polycythemia vera, which is commonly associated with increased RBCs as opposed to high platelet count (Fig. 3). Diagnosis requires either all three of the major criteria or the first two and the minor, or a patient with sustained erythrocytosis, JAK2 mutation, and the minor criteria. “You can make this diagnosis without the bone marrow biopsy,” Dr. Chabot-Richards said, but “I would not recommend it because the assessment of myelofibrosis is very important for prognosis.”

Primary myelofibrosis is a third myeloproliferative neoplasm, one associated with leukocytosis with ultimate progression to cytopenias due to fibrosis. There are two stages: pre-fibrotic and overt. The early phase can be similar to ET, and bone marrow is required for diagnosis.

“We usually do reflex testing for these myeloproliferative-associated mutations,” Dr. Chabot-Richards said. “We will start with a JAK2 V617F. It’s the most common mutation we see in a polycythemia vera, but it is also seen in 60 percent of cases of ET and primary myelofibrosis.” If the results are negative, they reflex to additional testing based on the clinical picture.

“Typically, we would either go to JAK2 exon 12, if it looked more consistent with polycythemia vera; that’s positive in an additional four percent of those cases and is not seen in ET and primary myelofibrosis. If it looked more like ET or primary myelofibrosis, we would do CALR and MPL.” Rarely do they do all such tests on any one patient, she said, and initial molecular testing typically consists only of JAK2, CALR, and MPL.

If additional prognostic testing is requested, it can be done on blood, though it is more typically done on bone marrow, and it would be sent for next-generation sequencing using a myeloid gene panel. ASXL1 and SRSF2 are associated with inferior overall and leukemia-free survival. EZH2 mutations are associated with inferior overall survival. IDH1/2 mutations are associated with inferior leukemia-free survival.

“The significance of these is mostly known in primary myelofibrosis. It’s not clear in ET and PV, but it seems reasonable that it would be similar, and preliminary studies have shown that,” she said.

National Comprehensive Cancer Network guidelines recommend that cytogenetic testing be performed on a bone marrow specimen, but peripheral blood testing is acceptable. “This is used to detect clonal abnormalities, and you can see prognostic abnormalities,” Dr. Chabot-Richards said. “Most of the abnormalities here are similar to those we see in any myeloid neoplasm, such as MDS.”

Chronic myeloid leukemia must also be considered with essential thrombocythemia. Patients may show thrombocytosis but should also have absolute basophilia, a leukocytosis with a left shift, and are positive for t(9;22) BCR-ABL1.

Reactive thrombocytosis can have many causes. A patient who has thrombocytosis post-surgery or trauma will resolve quickly, in contrast to patients who have, for example, iron deficiency anemia, chronic inflammation, or post splenectomy. “It’s important to figure that out,” she said.

The initial molecular testing can be performed on peripheral blood specimens, though the NCCN recommends bone marrow, Dr. Chabot-Richards said. Peripheral blood testing results may convince the patient and oncologist that bone marrow testing is necessary. “Bone marrow is required for the ultimate diagnosis because the presence of fibrosis is so important.”

A 68-year-old man with myasthenia gravis was being followed for anemia. His CBC showed a white blood cell count of 7.6 × 10⁹/L, with neutrophils slightly decreased for his age and monocytes slightly increased. His peripheral blood smear revealed only 57 percent neutrophils and many large, mononuclear cells.

A review of the chart found that the patient’s WBC had fluctuated between normal and elevated, and his monocyte count ranged between one and 2.2. His anemia fluctuated between mild and moderate. The patient also had thrombocytopenia, ranging from mild to moderate.

Dr. Chabot-Richards took another look at the blood smear and saw many large cells with convoluted nuclei, consistent with monocytes, and a few neutrophils with nuclear irregularities, including neutrophils with Pelger-Huet-like nuclei. In the lateral edges of the blood smear, there were larger cells with smooth nuclear contours and delicate folds, accounting for less than one percent of cells. She also discovered rare large cells consistent with blasts.

The patient’s blood was sent for flow cytometry. “Based on our side scatter and CD45, we were able to get 23.4 percent monocytes,” Dr. Chabot-Richards said. “Monocytes showed normal expression of CD36, CD64, CD13, and CD14.” There was aberrant expression of CD56 and abnormally dim HLA-DR.

The diagnosis: chronic myelomonocytic leukemia (CMML), an overlap myelodysplastic/myeloproliferative neoplasm. These neoplasms typically have dysplastic features and a combination of cytopenias and cytoses. “In this disease, patients often have thrombocytopenia. A monocytosis is required for diagnosis,” she said. The WHO criteria consist of a persistent peripheral blood monocytosis of >1 × 10⁹/L and >10 percent of the WBC count. Patients should have less than 20 percent blasts and equivalents in the blood and bone marrow; a higher percentage would give them a diagnosis of leukemia.

“One thing that can trip people up is this idea of blast equivalents,” Dr. Chabot-Richards said. While in most diseases, a myeloblast is a blast, “in this disease, we’re going to include some other things.”

Promonocytes are blast equivalents in CMML. “It’s important to recognize these because while they are blast equivalents, atypical monocytes that are mature are not. It can be difficult to distinguish between the two,” she said (Fig. 4). Dr. Chabot-Richards typically performs her differential twice in these cases.

“You need to do bone marrows on these patients because often the blast count can be much higher in the bone marrow than it is in the peripheral blood,” she said (Fig. 5).

Flow cytometry can help to quantify the immature cells. Most of the monocytes in CMML show classical immunophenotype: CD14 positive and CD16 negative. An aberrant phenotype, such as increased CD56 or aberrant expression of CD2, is also common.

The morphologic blast count takes priority, she said. “No matter what your immature blast count is on flow, if it doesn’t correlate with your morphology, you should trust your morphology.”

Most cases of CMML will have a normal karyotype; when abnormalities are seen they’re similar to those seen in MDS. BCR-ABL1 should be excluded in all cases, and PDGFRA, PDGFRB, FGFR1, and PCM1-JAK2 should be excluded if eosinophilia is present. “The new WHO update recommends that if you have a patient you think has chronic myelomonocytic leukemia, you should do FISH or molecular testing for t(9;22) to completely exclude chronic myeloid leukemia, because it is important to treat those patients with tyrosine kinase inhibitors,” Dr. Chabot-Richards said.

Most cases of CMML have mutations on gene panel testing, commonly TET2, SRSF2, and ASXL1 (the latter is predictive of aggressive disease). SETBP1 is strongly associated with a CMML diagnosis but is less common. “If you do find it, it can be strongly supportive of this specific diagnosis,” she said.

Other mutations seen are RUNX1, NRAS/KRAS, CBL, and EZH2. The NPM1 mutation is rare but associated with increased progression to acute leukemia. “Finding a mutation in any gene is not sufficient for diagnosis of CMML.”

CMML is subdivided into proliferative and dysplastic types. A WBC >13 × 10⁹/L is proliferative, which has a higher monocyte count, higher circulating immature myeloid cells and blasts, and higher LDH, and is more likely to have cytogenetic abnormalities and mutations in ASXL1, JAK2, and CBL. Dysplastic type (WBC <13 × 10⁹/L) has lower monocyte and blast counts, lower LDH, and normal cytogenetics, and is more likely to have a mutation in SF3B1.

Myelodysplastic syndromes, which share the same cytogenetic abnormalities and mutations with CMML, can be distinguished by a predominance of cytopenias and a lack of monocytosis. “These patients have dysplastic findings in one or more of the lineages, in greater than 10 percent of cells of that lineage,” she said. Promonocytes should not be used as blast equivalents in MDS.

Of the myeloproliferative neoplasms, chronic myeloid leukemia must be excluded, and FISH or molecular testing is recommended. “In particular, CML-P190 variant is associated with a monocytosis, so these patients can have similar peripheral blood findings,” Dr. Chabot-Richards said. Dysplasia or thrombocytopenia is not typically seen in CML.

Other myeloproliferative syndromes may present with monocytosis. JAK2, CALR, and MPL mutations may support a diagnosis of MPN. “They’re not specific, so you can see JAK2 mutations in CMML. But if you find them, it might be good to take another look to see if there’s anything else abnormal about the patient presentation,” Dr. Chabot-Richards said.

All MPNs require bone marrow for correct classification and assessment of fibrosis. “This can help you distinguish between MPN and CMML based on the bone marrow morphology.”

Other MDS/MPN overlap syndromes may be confused with CMML. Atypical chronic myeloid leukemia usually has more prominent dysplasia, marked left shift, a lack of significant monocytosis, and SETBP1 mutations. There is some overlap in diagnostic criteria with juvenile myelomonocytic leukemia, which is rare and usually fairly distinct clinically because most patients are under age three.

In summarizing this case, Dr. Chabot-Richards said, “We always suspect CMML in an adult with monocytosis.” The accurate blast count is key for CMML classification, and bone marrow biopsy is required for final classification. Gene mutations are helpful but not sufficient in the absence of other criteria.

Amy Carpenter Aquino is CAP TODAY senior editor. Part one was published in the March issue of CAP TODAY.