Finding the morphologic clues to etiology

Karen Lusky

February 2021— morphology may contain clues to neutrophilia etiology, and that was the focus of a CAP20 virtual presentation by Olga Pozdnyakova, MD, PhD, associate professor of pathology at Harvard Medical School and medical director of the hematology laboratory at Brigham and Women’s Hospital.

Reactive changes can mimic myeloproliferative neoplasm, but myeloproliferative neoplasm can have reactive morphology, she said. Pathologists can piece together clinical and morphological clues, “especially in concert with the clinical team, that may help them decide whether the changes are more reactive or more neoplastic in nature,” she told CAP TODAY in a follow-up interview.

Neutrophilia is defined as greater than 7.7 × 109/L or two standard deviations above the mean, and it is important to note whether it is present in the context of the left shift.

Dr. Pozdnyakova

Also important to note is whether neutrophilia is accompanied by other cytoses or cytopenias and what the neutrophil morphologic changes are, Dr. Pozdnyakova said. “And as always, it is essential to review the peripheral smear in the context of the clinical picture. Is neutrophilia symptomatic or incidental? How long has it been present? Is the patient taking medications?”

The primary causes of neutrophilia are both constitutional and acquired. Leukocyte adhesion deficiency, familial myeloproliferative neoplasm, and Down syndrome are examples of constitutional etiologies. Acquired primary neutrophilias are usually associated with myeloproliferative neoplasms, CML being one of the most common, followed by chronic neutrophilic leukemia or other Philadelphia-negative myeloproliferative neoplasms, atypical chronic myeloid leukemia, or other myelodysplastic/myeloproliferative neoplasm (MDS/MPN) overlap syndromes.

Secondary neutrophilia signifies reactive changes to various conditions, she said, such as smoking, and , medications (growth factors and corticosteroids are most common), and stress. Neutrophilia can be part of the paraneoplastic syndrome or be seen in patients with a nonfunctioning spleen. Secondary neutrophilias are more common than primary and generally accompanied by count changes and various morphological changes, which Dr. Pozdnyakova reviewed in her presentation.

She described a 36-year-old female who presented with an absolute neutrophil count of 14,000 in the setting of ongoing infection and accompanied by the left shift (Fig. 1). Metamyelocytes, promyelocytes, and myelocytes were reported on the patient’s white blood cell differential. “The presence of anemia and low platelets are likely related to ongoing chemotherapy for primary CNS lymphoma. Review of the smear shows characteristic reactive changes of toxic granulation, cytoplasmic vacuolization, and the presence of Dohle bodies in segmented neutrophils and in bands.” In another case, a 59-year-old male presented with extensive metastatic adrenocortical carcinoma (Fig. 2). “The patient had significant of greater than 20,000,” and bandemia with neutrophilia, Dr. Pozdnyakova said. “And on review of the peripheral blood smear we noticed that some of the neutrophils contained blue-green inclusions indicative of liver failure.” Chemistry results were consistent with multisystem organ failure, with elevated ALT, AST, BUN, and creatinine. These inclusions have been “unfortunately termed ‘death crystals,’” she said, because their emergence is usually associated with imminent death. The patient died soon after presenting.

Dr. Pozdnyakova reported the case of a 64-year-old male admitted with respiratory failure and hypertension after three weeks of low-grade fever, cough, headache, and periodic sweating (Fig. 3). He was a hunter who resided in New England and his wife reported several tick bites. His WBC count of almost 20,000, with 84 percent neutrophils, was associated with cytoplasmic inclusions consistent with Anaplasma morulae, a rickettsial bacterium.

“The morphologic impression, once we reviewed the smear, was confirmed by the PCR test,” Dr. Pozdnyakova said. “Presentation of leukocytosis in this particular case was unusual because most of the patients present with and thrombocytopenia. And presence of leukocytosis may indicate coinfection with babesiosis, since the same tick transmits all three pathogens. (Fig. 3, lower left: Babesia within red blood cells with a characteristic Maltese cross.) She reported that their patient hadn’t contracted other pathogens. Howell-Jolly body-like inclusions (Fig. 4) can imitate intracytoplasmic organisms. These are nuclear fragments that could be seen in connection with antiviral medications, immunosuppression, and sometimes with myelodysplastic syndrome, Dr. Pozdnyakova reported, pointing out the “spherical nuclear fragments where they are usually peripherally placed” in the neutrophils. “In this case, with a patient with end-stage renal disease status post-kidney transplant, those Howell-Jolly body-like inclusions were likely associated with an ongoing infection because you can also see toxic granulation and some vacuolation in neutrophils.”

In another case, a 62-year-old female with relapsed diffuse large B-cell lymphoma had an ongoing infection (Fig. 5). “The CBC shows neutrophilia with some left shift, six percent myelocytes, microcytic anemia, and very low platelets of 22,000,” Dr. Pozdnyakova reported. “Review of the neutrophils shows some of the changes we have already seen and associated with infection, such as toxic granulation, cytoplasmic vacuolization, Dohle bodies, and Howell-Jolly body-like inclusions.” Yet the cells also display prominent nuclear dysplastic changes, abnormal nuclear lobation in the form of hypolobated nuclei, she said.

Dr. Pozdnyakova noted in Fig. 5 (two left images in top row and one image on bottom left) monolobated nuclei, usually called Stodtmeister cells. She pointed also to bilobed nuclei (lower middle and upper far right), referred to as pseudo Pelger-Huët cells. “Some cells also show cytoplasmic hypergranulation. These morphologic findings are concerning for neoplastic rather than reactive changes,” she said. A molecular analysis identified ASXL1 and DNMT3A mutations consistent with treatment-related MDS. “And in this case, it is possible that hypergranulation and vacuolization are a sign of dysplasia rather than infection.” Dysplastic changes in neutrophils can present in many forms, Dr. Pozdnyakova cautioned, presenting what she later described to CAP TODAY as the most recent difficult case (Fig. 6). The 72-year-old female patient had a known history of clonal hematopoiesis and presented with progressive leukocytosis of greater than 30,000. The WBC count showed neutrophilia and . Ten percent of the cells were called basophils.

“Upon review it was clear that the granules in those cells that were called basophils are much larger than in typical basophils and are reminiscent of encapsulated intracellular yeast, particularly Histoplasma, which corroborated the clinical impression of disseminated fungal infection,” Dr. Pozdnyakova said. “However, Histoplasma urine antigen came back negative.”

Dr. Pozdnyakova then requested a myeloperoxidase stain, which accentuated forms that turned out to be dysplastic primary granules in the setting of progression of the MDS/MPN overlap syndrome. And once a peripheral blood smear was reviewed, it demonstrated other more typical dysplastic changes, she said, noting a pseudo-Pelger-Huët nucleus.

A session attendee noted that few laboratories offer myeloperoxidase. Dr. Pozdnyakova acknowledged that the Dana-Farber Cancer Institute hematology laboratory is likely “the last man standing” in offering the myeloperoxidase cytochemical stain. They use nonspecific esterase, MPO, PAS, and specific esterase for all new cases of acute leukemia. “It’s a very rapid test, and it allows us to put a case into an acute myeloid leukemia with monocytic differentiation category, or just typical AML if it’s MPO positive, very fast,” she said—“or into acute lymphoblastic leukemia if it’s PAS positive.”

Administration of growth factors is another important cause of neutrophilia, presenting with the morphologic finding of a significant degree of left shift that frequently includes blasts, Dr. Pozdnyakova noted. However, occasionally this clinical information isn’t readily accessible. In the case of a 56-year-old male patient with Burkitt lymphoma who was taking Granix for , the blast count was two percent (Fig. 7). She said she has come across cases with blast counts reaching 10 percent. The additional morphologic features to look for in such cases are prominent toxic changes and nuclear- cytoplasmic dyssynchrony. “For example, you can see retention of primary granules with very mature nucleus,” she said. In looking at the promyelocytes, “sometimes you can see prominent perinuclear hoffs. Very often neutrophilia is accompanied by thrombocytosis,” but it wasn’t seen in this case.

Having reviewed the possible causes of neutrophilia and their related morphologic changes, most of which were reactive, Dr. Pozdnyakova introduced a key case: a 69-year-old male who presented with chronic leukocytosis and neutrophilia (Fig. 8). “The CBC showed a WBC count close to 30,000, with an absolute neutrophil count of over 24,000. There was no significant left shift.” The myelocyte count was one percent. The CBC was notable for four percent , a concerning fact in this case, along with the persistent neutrophilia (elevated with a steady increase over 10,000 for about one year).

The neutrophils provided “unremarkable morphology with proper nucleus segmentation and cytoplasmic granulation,” Dr. Pozdnyakova said, noting none of the changes she had reviewed could be seen in this smear.

Dr. Pozdnyakova provided a differential diagnosis for persistent non-congenital neutrophilia, which is almost invariably associated with a myeloid neoplasm. Chronic myeloid leukemia, BCR-ABL1 positive, is the most common cause, she said. Other diagnoses associated with persistent neutrophilia are chronic neutrophilic leukemia, Philadelphia-negative myeloproliferative neoplasms, such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis (pre- fibrotic stage).

“Another one is atypical chronic myeloid leukemia, which is an MDS/MPN overlap syndrome. Or chronic myelomonocytic leukemia proliferative type also can present with neutrophilia,” she said. “Acute myeloid leukemia, especially with NPM1 and FLT3 mutations, in addition to blasts, can also present as neutrophilia.”

CML is the most common myeloproliferative neoplasm presenting with neutrophilia, and it has unique treatment, and thus all cases presenting with persistent neutrophilia should undergo testing to look for the presence of BCR-ABL1 translocation, Dr. Pozdnyakova said. Karyotype, FISH, and RT-PCR each have benefits and limitations, and all three may be needed to diagnose CML, she said.

“Karyotype allows us to look for additional abnormalities, in addition to BCR-ABL1 translocation (9;22). And RT-PCR is performed to look for the BCR-ABL1 fusion length, which could be different, and to quantify the levels of the fusion at baseline and for monitoring response to therapy.” FISH is seldom required, she said, though it can detect cryptic BCR-ABL1 rearrangements, observed in about one percent of cases.

Depending on the breakpoints in the BCR or ABL1 genes, three primary types of BCR-ABL1 genes can be formed, which result in fusion proteins of dissimilar lengths: p210, p190, and p230, she said, adding, “It’s important to know the type of BCR-ABL1 fusion for prognostic and diagnostic purposes.”

The most common fusion is p210, seen in more than 95 percent of cases and associated with typical CBC findings of significant leukocytosis with in all stages of maturation, and with increased eosinophils and basophilia. “Those patients demonstrate an excellent response to tyrosine kinase inhibitors,” Dr. Pozdnyakova said.

The p190 fusion is seen in less than five percent of CML cases. “However, it is commonly seen in B lymphoblastic leukemias, but unlike the most typical p210, those patients present with monocytosis rather than ,” she said. “And those patients have a very poor response to imatinib,” and often evolve to accelerated or blast phase.

Also seen in less than five percent of cases is p230, and patients tend to present with a low WBC count, neutrophilia without left shift, mild symptoms, and often an increased platelet count. “Similar to p210, these patients have an excellent response to TKIs and almost never go into accelerated or blast phase.” CML with p230 can imitate other MPNs, she warned, such as CNL, essential thrombocythemia, or primary myelofibrosis.

Returning to the case of the 69-year-old male who presented with chronic leukocytosis and neutrophilia, Dr. Pozdnyakova revealed the first RT-PCR results, which didn’t show BCR-ABL1 mRNA (B3A2, B2A2, or E1A2), or p210 or p190. “Routinely we only test for p210 and p190,” she said, because p210 is most common in CML and p190 is the common fusion seen in B lymphoblastic leukemia. Because they had not tested for p230, they did not rule out CML.

The next steps, she said, are to wait for a karyotype result, perform a next-generation sequencing analysis that would allow them to find somatic mutations in multiple genes associated with other myeloproliferative neoplasm diagnoses, and perform a bone marrow biopsy.

“NGS plays an essential role in an MPN workup, allowing us to distinguish a reactive from a clonal neoplastic condition,” Dr. Pozdnyakova said. “And while there are no official diagnostic mutations, there are several phenotype-associated mutations that allow us to make a correct diagnosis, such as for Ph-negative MPNs.” For those, she said, “We’ll look for JAK2, CALR, MPL mutations. A lot of cases of chronic neutrophilic leukemia are associated with CSF3R mutation. Atypical CML tends to be associated with SETBP1 or ETNK1 mutation.” And chronic myelomonocytic leukemia has a “whole slew of mutations, with ASXL1, SRSF2, RUNX1, and RAS/CBL the most common.”

She and colleagues use NGS to assess for disease progression. “Either we’ll look for an increased allele burden, or for the presence of mutations that we know are associated with a worse prognosis.” They also use NGS to monitor treatment—emergence of the ABL1 resistance mutations, for example, or for targetable mutations, such as FLT3 or IDH1/2.

Before reporting the patient’s remaining test results, Dr. Pozdnyakova reviewed two more diagnoses that present with peripheral blood neutrophilia that she said are in the differential diagnosis for other patients. The first is chronic neutrophilic leukemia, defined as a persistent neutrophilia in peripheral blood and bone marrow with organomegaly and absence of BCR-ABL1 translocation. Among the clinical features is neutrophilia greater than 25,000 without left shift. “Neutrophils could have hypergranular cytoplasm. Sometimes those cases could be accompanied by increased platelets. Hemoglobin levels could be normal. We tend to associate those cases with CSF3R mutations,” she said. “However, other mutations, such as ASXL1, SETBP1, or JAK2, have been described. There is no specific therapy, but some patients respond to JAK inhibitors and tyrosine kinase inhibitors.” CNL closely resembles CML with p230 or atypical CML. “And we always need to exclude non-neoplastic neutrophilia because sometimes the morphologic findings could be very subtle.” The second diagnosis in the differential is atypical CML (Fig. 9). “Similar to typical CML, patients present with left-shifted granulocytosis,” she said, pointing to metamyelocytes, myelocytes, and bands on the smear. “However, when you look closely at the morphology of those cells, there is significant dysplasia,” she added, noting hypergranular cytoplasm and abnormal nuclear lobation. “Also, since there is an MDS component to that neoplasm, these patients tend to present with anemia and lower platelet counts.”

Atypical CML is an MDS/MPN overlap syndrome with persistent leukocytosis with left shift, prominent dysplasia, organomegaly, and absence of BCR-ABL1 translocation, Dr. Pozdnyakova said. “This is an extremely rare diagnosis, presenting almost exclusively in an older patient population,” with a WBC count of greater than 13,000 with a significant left shift, defined as greater than 10 percent of the WBCs, and significant dysplasia. There is no standard therapy, she said, and patient survival is poor. One-quarter to one-third of cases are associated with the SETBP1 mutation. “However, similar to other MPN/MDS diagnoses,” she said, “you can see nonspecific ASXL1, RAS, SRSF2, TET2, CBL, JAK2, or other mutations. And it closely resembles chronic myeloid leukemia or chronic neutrophilic leukemia, and you have to carefully examine a peripheral blood smear to look for morphologic dysplasia, which will allow you to differentiate this diagnosis from CML and CNL.”

In the case of the 69-year-old male patient, the karyotype came back positive for t(9;22), signifying the presence of the Philadelphia chromosome diagnostic of chronic myeloid leukemia. There were no pathogenic somatic mutations. “Taken together, these genetic findings allowed us to make a diagnosis of CML, BCR-ABL1 positive, chronic phase. So this is likely a case of CML with p230 or neutrophilic CML,” Dr. Pozdnyakova said. The patient was likely to have carried the diagnosis since the elevated WBC count was detected a year earlier. The patient responded well to TKIs.

“p230 is very rare and low yield and that is the main reason why we do not test for it,” she told CAP TODAY. “In addition, regardless of the fusion, t(9;22) is readily picked up by conventional karyotype and/or FISH. If p210 and p190 are negative and t(9;22) is present, we assume p230.” While p190 is rare in CML, she added, it is common in B lymphoblastic leukemia, which is why their assay includes p190.

“We need to always perform BCR-ABL1 testing by different modalities to exclude CML in cases presenting with persistent neutrophilia,” she emphasized. “And next-generation sequencing analysis can help exclude a clonal process or confirm the presence of another myeloproliferative neoplasm, or MDS/MPN overlap.”

Dr. Pozdnyakova offers the following algorithm for workup of a CBC presenting with neutrophilia:

Confirm the neutrophilia. Exclude spurious cases, such as platelet clumping. If true neutrophilia is present, review the clinical presentation (symptomatic neutrophilia that could be explained by ongoing infection, recent surgery, G-CSF, etc.? or unexplained neutrophilia? duration of neutrophilia?) and remainder of the CBC for other abnormalities (other cytoses or cytopenia?) and ask the laboratory to prepare a well-made peripheral smear for assessment of red cell, white cell, and platelet morphology (reactive/toxic/dysplastic morphologic changes? inclusions—initiate microbiology workup?). In an asymptomatic patient with mild neutrophilia and no associated CBC or peripheral smear abnormalities, the laboratory evaluation could end at this point with a plan to repeat testing if symptoms develop and/or in several weeks to determine if the neutrophilia is persistent. If there is suspicion for neoplastic neutrophilia (differential diagnosis: CML, CNL, Ph- MPN, atypical CML, other MDS/MPN), then: BCR-ABL1 (PCR or karyotype); cytogenetics (could be normal, unless CML); NGS-based molecular testing to look for somatic mutations associated with myeloid malignancies; bone marrow biopsy.  Karen Lusky is a writer in Brentwood, Tenn.