Diagnostic Workup for Advanced Forms of Mastocytosis

Wolfgang R. Sperr¹, Celalettin Ustun²

¹Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria

²Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Minnesota, USA

Author Contact:

Wolfgang Sperr, MD:  wolfgang.r.sperr@meduniwien.ac.at

Celalettin Ustun, MD:  custun@umn.edu

Mastocytosis denotes a heterogeneous group of disorders characterized by abnormal growth and accumulation of mast cells (MCs) that has a variable prognosis and course of disease. In contrast to adults, childhood mastocytosis often resolves spontaneously before adolescence or young adulthood.^{1, 2} Most systemic mastocytosis (SM) variants diagnosed in adults show a stable clinical course over decades and do not decrease the life span.³ The World Health Organization (WHO) criteria separates cutaneous, indolent and advanced forms. Advanced variants of mastocytosis include aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), and SM with an associated hematologic non mast cell lineage disorder (SM-AHNMD), a term that was shortened in the 2016 revision of the WHO classification for mastocytosis to “SM with an associated hematological neoplasm” (SM-AHN).^{1, 3, 4} The diagnosis of mastocytosis is established in a stepwise approach, using WHO major and minor criteria (with one major and one minor or at least three minor criteria having to be fulfilled).¹ The major criterion is multifocal mast cell infiltration in the bone marrow (BM) or in an extracutaneous organ, as assessed by histology.^{3, 5} Minor SM criteria relate to the morphology of mast cells (histology and/or cytology), their cell surface markers (aberrant expression of CD2 and/or CD25), molecular findings (KIT mutation at codon 816), and serum tryptase level (>20 ng/ml) (Figure 1).⁶ To diagnose an advanced form of mastocytosis, the presence of B-findings, C-findings, or an AHN is of importance (Table 1, Figure 1). In patients with two or more B-findings the diagnosis of smoldering systemic mastocytosis (SSM) can be established.⁷ C-findings (one is enough to diagnose aggressive SM) are indicative of ASM or MCL.^{1, 5, 7} It is of particular importance that organ damage caused by MC infiltration only counts as an SM-related C-finding.^{1, 5, 7, 8} In case of suspected AHN, further markers and criteria to classify the AHN according to WHO criteria have to be applied to fully diagnose both the SM component and the AHN component of the disease and to identify potential drug targets.^{1, 3, 5, 8}

Figure 1. Stepwise approach to diagnosis of systemic mastocytosis variants using World Health Organization Criteria

Table 1. B– and C-findings in advanced SM

*Two or more B-findings, with the absence of C-findings, must be present to diagnose smoldering systemic mastocytosis.

†One or more C-findings must be present to diagnose aggressive systemic mastocytosis.

Clinical findings       

The presence or absence of skin lesions can be an important clinical finding. Patients with ASM or MCL may or may not have skin lesions, with less than 50% of those with MCL presenting with cutaneous involvement.⁹ This is in contrast to patients with ISM or SSM, the majority of whom present with typical skin lesions.⁹ Therefore, the absence of skin lesions may be suggestive of aggressive disease variants. However, the absence of skin lesions is not confined to advanced variants. In bone marrow mastocytosis (BMM), a sub-variant of ISM, no skin lesions are detectable, and the prognosis is excellent.⁵ Indeed, it has been recently estimated that 5% of patients with ISM may also present without skin lesions.⁹

Another observation of importance is weight loss. Together with hypoalbuminemia, it is indicative of mast cell-induced organ damage and thus is a C-finding.⁵

An additional typical finding in SM is symptoms caused by the release of mast cell mediators, including histamine, cytokines, and leukotrienes.⁵ Related symptoms are headache, flushing, pruritus, diarrhea, hypotension, and bone pain. Excessive mast cell mediator release may lead to anaphylaxis in patients with mastocytosis. However, such symptoms can occur in all patients with SM and must not be misinterpreted as signs of advanced SM.⁵

Peripheral blood findings

Elevated serum tryptase is the key finding in patients with SM. Moreover, tryptase may serve as a surrogate marker to estimate the aggressiveness of the disease.⁶ Likewise, very high tryptase levels (>200 ng/ml) represent a B finding necessary to diagnose SSM.^{1, 3, 5, 8} In ASM and MCL the majority of patients present with highly elevated serum tryptase levels.⁵ However, a moderate elevated serum tryptase does not exclude advanced SM.¹⁰ Another important aspect is that tryptase must not be counted as SM criterion when the patient has an AHN, because tryptase can also be expressed in neoplastic myeloid cells or immature basophils.¹¹ Tryptase measurement is also of importance for the monitoring of SM.

Many patients with advanced SM present with abnormal peripheral blood counts.¹ Peripheral blood counts also help to differentiate between different variants of advanced SM.¹ Whereas in SSM, the blood counts are within the normal range or slightly abnormal (B-finding), patients with ASM and MCL typically have decreased numbers of cells in the peripheral blood, including granulocytes <1.0 G/L, and/or hemoglobin <10 g/dl, and/or platelets <100 x 10⁹/L.¹ The presence of >10% MCs in the peripheral blood denotes MCL.¹ An increase in the numbers of eosinophil granulocytes is another characteristic finding of advanced disease. An abnormal blood count is also a key finding to detect an AHN and to diagnose SM-AHN.¹

With respect to laboratory chemistry, albumin is an important marker. Decreased albumin levels may be a sign of impaired liver function, in particular when observed together with elevated liver enzymes, enlarged liver, or signs of elevated pressure in the portal vein, like fluid in the abdominal cavity.^{1, 3, 5, 7} The combination of these findings can be indicative of progressive destruction of the liver by mast cell infiltrates.^{1, 3, 5} Typically, alkaline phosphatase is elevated in such patients, as well.^{1, 3, 5, 7, 8} On the other hand as stated above, hypoalbuminemia is also a sign for malabsorption. Both progressive destruction of the liver by mast cell infiltrates and malabsorption are C-findings necessary to diagnose ASM or MCL.^{1, 7}

ß2-microglobulin is a well-established prognostic marker in patients with lymphomas. In these disorders, an elevated level reflects a high tumor burden. In patients with SM, the ß2-microglobulin level was found to be of prognostic significance, predicting an unfavorable outcome.¹² In this regard, one could speculate that involvement of the lymphocytic cells in SM might be associated with progression of disease. However, confirming studies are warranted to determine the impact of lymphocytic involvement and the prognostic value of ß2-microglobulin.

Tissue Biopsies

Although the presence of the major criterion for diagnosis of SM can be evaluated with a biopsy from any organ involved in disease other than skin (e.g., liver, gastrointestinal tract),^{13, 14} bone marrow is the most common site.¹⁵ There are many pros of a bone marrow biopsy/aspirate, including providing information on an AHN, rendering differential diagnosis with other possible malignancies, and being easy and safe.

MCs infiltration can be diffuse or multifocal in the bone marrow. MCs can be morphologically round, oval or spindle shaped.¹⁶ Immunohistochemistry and flow cytometric analyses (e.g., looking at cell markers) for CD33, CD43, CD68, CD117 (KIT), and tryptase should be performed in the tissue. Neoplastic MCs also express aberrant CD2¹⁷ and/or CD25.  Evaluation of bone marrow biopsy and aspirate is also critical for minor diagnostic criteria: the presence and increase of spindle shaped/atypical MCs (≥25%), the expression of CD2, CD25, or both, in MCs, and presence of the KIT D816V mutation.¹⁶

Moreover, a bone marrow biopsy is important to make a differential diagnosis between the variants of advanced SM. For example, although in ASM, dense sheets of MCs in the bone marrow biopsy are seen, if MCs constitute ³20% of all nucleated cells in bone marrow aspirate smears, it is defined as MCL.¹ (MCL can be aleukemic if there are no circulating MCs in peripheral blood.¹⁸) In SM-AHN, the AHN component¹ can be a myeloid malignancy [acute myelogenous leukemia (in particular t(8;21), chronic myelomonocytic leukemia, myeloproliferative neoplasias (very rarely chronic myelogenous leukemia), myelodysplastic syndrome] or plasma/lymphoid malignancies including, chronic lymphocytic leukemia, lymphomas, or plasma cell malignancies (myeloma). Sometimes, hematologic malignancies with severe, diffuse bone marrow infiltration (e.g., acute myelogenous leukemia) can obscure MCs that can be more apparent in the remission bone marrow biopsies after chemotherapy (i.e., occult mastocytosis).¹⁹ Osteosclerosis (“thick, tough bones” that can make performing a bone marrow biopsy technically difficult) is also readily detected in bone marrow biopsies.¹⁴

Other entities: mast cell sarcoma is a rare and aggressive tumor, but almost always involves extramedullary organs.²⁰ There are also tryptase positive aggressive hematologic malignancies that should be differentiated from advanced SM.^{21, 22}

Ultrasound, X-Ray, and Densitometry

In all patients with SM, bone densitometry should be included in the diagnostic work up and repeated periodically to detect early osteoporosis.^{5, 8} In case of suspected advanced mastocytosis, the staging should include imaging of the skeletal system and ultrasound of the abdomen. Ultrasound of the abdomen may reveal enlargement of liver, spleen or lymph nodes, as well as fluid in the abdominal cavity.²³ As already stated, these findings without impairment of organ function are indicative for SSM (B-findings). Together with signs of organ damage, these observations are C-findings. Imaging of the skeleton may disclose signs of osteoporosis, osteosclerosis, or focal areas of osteolyses.^{14, 24} In most cases, the osteolytic lesions are small in size (0.5 cm) and not accompanied by clinical symptoms. In a few cases, however, larger osteolyses with pathologic (spontaneous) fractures are found.^{23, 24}

Molecular markers

The most important molecular marker to show monoclonality in SM is the KIT point mutation D816V that can be analyzed in the bone marrow and the peripheral blood.^{1, 25} In the majority of patients with SM, neoplastic MCs harbor this mutation.¹ In particular, in >80% of patients with indolent systemic mastocytosis, SSM or SM-AHN, KIT D816V is detectable.^{8, 26} In contrast, the prevalence of KIT D816V is lower in ASM (>60%) and MCL.^{8, 26} In this regard, several points have to be discussed. Firstly, if KIT D816V is negative, patients could harbor other mutations at codon 816, including D816H or D816Y. To detect these mutations, further diagnostic work-up (i.e., the amplification of the codon 17 and sequencing) is important. Secondly, mutations can affect other regions of KIT like the juxtamembrane domain, transmembrane domain or extracellular domain. In these cases, next-generation sequencing should be considered.^{8, 26}

Another interesting aspect is the KIT D816V allele burden. Recently is has been shown that the allele burden differs between disease variants. In patients with advanced SM, a high allele burden can be observed, in contrast to indolent disease or a cutaneous variant. Moreover, the allele burden at diagnosis is of importance for prognostication, follow-up, as well as for quantification of response to treatment.²⁷

Of note, in a majority of patients with SM, KIT D816V is detectable and can occur in cutaneous mastocytosis as well.^{1, 3, 5, 8} Likewise, Bodemer et al. reported that a mutation in codon 816 (exon 17) can be detected in cutaneous lesions of pediatric mastocytosis patients in 42%, and outside exon 17 in 44%.²⁶ This suggests that additional genetic alterations, together with KIT D816V, result in neoplastic proliferation of MCs in advanced SM and thus are responsible for consecutive organ destruction by mast cell infiltration. Indeed, as assessed by next-generation sequencing, additional molecular aberrations were identified in the majority of patients with advanced SM.²⁸ The most frequently affected genes were TET2, SRSF2, ASXL1, CBL, RUNX and JAK2 (n = 11). With regard to overall survival, SRSF2 and ASXL1 were found to be independent predictive factors for a poor prognosis, in contrast to TET2, JAK2, or RUNX1. Moreover, the number of mutated genes of the panel SRSF2/ASXL1/RUNX1 was indicative for an inferior overall survival and adverse clinical characteristics.²⁹

Diagnostic Work-up Summary:

Diagnostic work-up should include:

• Careful history and physical exam
• Serum tryptase
• Bone marrow biopsy and aspirate with appropriate analyses, if serum tryptase is high and/or blood counts are abnormal
• Molecular testing from bone marrow
• Abdominal ultrasound
• Dexa scan and/or other skeletal imaging
• Endoscopies with biopsies in cases with gastrointestinal symptoms
• Chest CT, in patients with pulmonary symptoms (very rarely)

Edited by

Denyse Nanan, MD, PhD

Valerie M. Slee, RN, BSN

Susan Jennings, PhD

The Mastocytosis Society, Inc.