—  SPECIALTY CONFERENCE  —

Hematopathology

Case 4 - Myeloid Sarcoma, NPM1c positive

Stefano Pileri
Bologna University
Bologna Italy





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Clinical History
A 5 year-old Caucasian child referred to the Department of Paediatrics of Catholic University in Rome because of migrant joint pain and swelling of the right sub- mandibular gland and latero-cervical lymph nodes. Total body CT-scan resulted negative whereas bone scintigraphy showed an increased uptake in the right shoulder, right foot and ribs. A bone marrow (BM) aspirate and a biopsy of the right sub-mandibular gland turned out negative. The patient was discharged with a diagnosis of Parvovirus B19 infection based on a PCR positive test performed on BM, peripheral blood and saliva. Accordingly, a therapy with FANS and intravenous immunoglobulins was administered. For the onset of a deficit of the right detrusor muscle of the sub-mandibular angle, interpreted as iatrogenic, steroid therapy was then started. One month later, a nodule measuring 3 cm across and extending from the left nasal fossa to the homolateral hard palate was detected. Staging procedures were negative. An incisional biopsy was performed: the material was sent in consultation to the Unit of Haemolymphopathology of Bologna University. Following the diagnosis, the patient underwent chemotherapy according to the AIEOP LAM 2002/1 protocol achieving complete remission.

Pertinent Laboratory Data:

Immunomorphologic findings The biopsy consisted of fragments of palatine mucosa whose chorion was infiltrated by large blasts, with round or kidney-shaped nuclei, rather dispersed chromatin, prominent central nucleoli, an a wide rim of cytoplasm, grayish at Giemsa. The growth either dissociated the dermis producing a Indian file appearance or gave rise to cuffs around vessels. Mitotic figures were numerous. At immunohistochemistry, the above described population resulted: CD45+, CD68PGM1+, CD43+, BCL2+, Vimentin+, CD99+, CD56-/+, TdT-, CD34-, MPO-, PAX5-, CD3-, CD1a-, HECA-, CD123-, S100-, MyogeninD1-, Desmin-, Actin-, Synaptophysin-, Ki-67+ (proliferation rate 60-70%). In addition, the monoclonal antibody NPM1c displayed a strong nuclear and cytoplasmic positivity in most if not all neoplastic cells. FISH analysis The probes (from Vysis) for AML1/ETO, CBF-Beta, MLL, EGR1/D5S23, D5S721, D7S486/CEP7, CEP4, CEP8, CEP11, CEP16 provided negative results.


Case 4 - Slide 1
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Case 4 - Figure 1
CD56. Blasts CD56+
Case 4 - Figure 2
CD68. Blasts showing granular positivity for CD68PGM1
Case 4 - Figure 3
CD99. Blasts CD99+
Case 4 - Figure 4
H&E. Large blasts, with round or kidney-shaped nuclei, rather dispersed chromatin, prominent central nucleoli, an a wide rim of cytoplasm

Case 4 - Figure 5
GM1. Giemsa stain: fragments of palatine mucosa whose chorion was infiltrated by large blasts
Case 4 - Figure 6
GM2. Giemsa stain: large blasts, with round or kidney-shaped nuclei, rather dispersed chromatin, prominent central nucleoli, an a wide rim of cytoplasm
Case 4 - Figure 7
LCA. Blasts LCA+
Case 4 - Figure 8
MPO. Blasts were negative for Myeloperoxydase MPO

Case 4 - Figure 9
NPM1 LP. Low power field: the monoclonal antibody NPM1c displayed a strong nuclear and cytoplasmic positivity in most if not all neoplastic cells NPM1 HP
Case 4 - Figure 10
NPM1 HP. High power field: the monoclonal antibody NPM1c displayed a strong nuclear and cytoplasmic positivity in most if not all neoplastic cells NPM1 LP
Case 4 - Figure 11
TdT. Blasts were TdT negative
Case 4 - Figure 12
CD123. Blasts were TdT negative

Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
The biopsy consisted of fragments of palatine mucosa whose chorion was infiltrated by large blasts, with round or kidney-shaped nuclei, rather dispersed chromatin, prominent central nucleoli, an a wide rim of cytoplasm, grayish at Giemsa. The growth either dissociated the dermis producing a Indian file appearance or gave rise to cuffs around vessels. Mitotic figures were numerous. At immunohistochemistry, the above described population resulted: CD45+, CD68PGM1+, CD43+, BCL2+, Vimentin+, CD99+, CD56-/+, TdT-, CD34-, MPO-, PAX5-, CD3-, CD1a-, HECA-, CD123-, S100-, MyogeninD1-, Desmin-, Actin-, Synaptophysin-, Ki-67+ (proliferation rate 60-70%}. In addition, the monoclonal antibody NPM1c displayed a strong nuclear and cytoplasmic positivity in most if not all neoplastic cells. FISH analysis The probes (from Vysis} for AML1/ETO, CBF-Beta, MLL, EGR1/D5S23, D5S721, D7S486/CEP7, CEP4, CEP8, CEP11, CEP16 provided negative results.

Differential Diagnoses:
Differential diagnosis between Myeloid Sarcoma, Blastic Plasmacytoid Dendritic Cell Tumours and Lymphoma

Final Diagnosis:
Myeloid Sarcoma, NPM1c positive.

Case Discussion:
Myeloid sarcoma (MS} is a rare condition that is characterized by the occurrence of one or more tumour masses, consisting of immature myeloid cells presenting at an extra-medullary site [1]. The latter has been reported more often to correspond to the skin, bone or lymph node, although the process can affect almost every site of the body, It may develop de novo or concurrently with acute myeloid leukaemia (AML}, myeloproliferative neoplasm or myelodysplastic syndrome. Interestingly, MS may be the first evidence of AML or precede it by months or years [2]. Finally, it can represent the initial manifestation of relapse in a previously treated AML in remission. The diagnosis of MS may be difficult since the clinical presentation and morphologic features can mimic another tumour (namely, malignant lymphoma} and/or the size of the bioptic sample may be small, such as in the case of a skin punch biopsy. Since MS is usually treated in the same way as AML, the frequent lack of cytogenetic data in MS represents a significant disadvantage, and the availability of techniques applicable to paraffin samples to detect specific genetic lesions would be of great help both for diagnostic and prognostic purposes. On this respect, FISH does represent a powerful tool to assess the occurrence of the most frequent abnormalities also in the setting of MS [2]. However, the results depend on the optimal preservation of the bioptic sample as well as on the availability of appropriate instruments and skill [2]. Recently, an antibody has been developed that allows the identification of cases carrying nucleophosmin 1 gene (NPM1} mutations [3]. The latter represent the commonest genetic lesion in the setting of de novo AML and is associated with "normal" karyotype, M4/M5 morphology and CD34 negativity [3, 4, 5, 6, 7, 8, 9]. In the absence of FLT3 internal tandem duplication, the patients with AML carrying NPM1 mutations have a more favourable response to therapy and prognosis [3, 4, 5, 6, 7, 8]. In particular, the frameshift caused by NPM1 mutations results in the aberrant cytoplasmic localisation of the nucleophosmin protein that can be easily detected by the NPMc monoclonal antibody [3]. As shown by Falini et al [9] NPMc positivity is recorded in about 15% of MS.

Conclusion(s}:
The case herein presented is prototypic of the difficulties encountered in diagnosing MS. In addition, it highlights the relevance of having easy access to a cheap immunohistochemical test surrogating a complex molecular analysis and fruitfully influencing therapeutic decisions.

References:
  1. Pileri SA, Orazi A, Falini B. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press:Lyon, France, 2008, 140–141.

  2. Pileri SA, Ascani S, Cox MC, Campidelli C, Bacci F, Piccioli M et al. Myeloid sarcoma: clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients.Leukemia 2007; 21:340–350.

  3. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005; 352: 254–266.

  4. Falini B, Nicoletti I, Martelli MF, Mecucci C. Acute myeloidleukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+AML}: biologic and clinical features. Blood 2007; 109: 874–885.

  5. Falini B, Martelli MP, Bolli N, Bonasso R, Ghia E, Pallotta MT et al. Immunohistochemistry predicts nucleophosmin (NPM} mutations in acute myeloid leukemia. Blood 2006; 108: 1999–2005.

  6. Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood 2005; 106: 3733– 3739.

  7. Bolli N, Galimberti S, Martelli MP, Tabarrini A, Roti G, Mecucci C et al. Cytoplasmic nucleophosmin in myeloid sarcoma occurring 20years after diagnosis of acute myeloid leukaemia. Lancet Oncol 2006; 7: 350–352.

  8. Chou WC, Tang JL, Lin LI, Yao M, Tsay W, Chen CY et al. Nucleophosmin mutations in de novo acute myeloid leukemia: the age-dependent incidences and the stability during disease evolution Cancer Res 2006; 66: 3310–3316.

  9. Falini B, Lenze D, Hasserjian R, Coupland S, Jaehne D, Soupir C, Liso A, Martelli MP, Bolli N, Bacci F, Pettirossi V, Santucci A, Martelli MF, Pileri S, Stein H. Cytoplasmic mutated nucleophosmin (NPM} defines the molecular status of a significant fraction of myeloid sarcomas. Leukemia (2007} 21, 1566–1570; Epub 2007 Apr 19.