Diagnostic approach to pediatric malignant small round cell

Role of IHC and histopathology

1. DIAGNOSTIC APPROACH TO PEDIATRIC MALIGNANT SMALL ROUND CELL TUMOURS By Sivaranjini. N Guide: Dr. Manisha Shah

2. MALIGNANT SMALL ROUND CELL TUMOURS A group of highly aggressive malignant tumors composed of relatively small and monotonous undifferentiated cells with increased nuclear‑cytoplasmic ratio.

3. The malignant round cells are: 1) slightly larger or double the size of red blood cells in air-dried smears, 2) measure less than 10 μm in diameter in alcohol fixed smears, 3) have scanty cytoplasm.

4. Small round cell tumours of childhood Neuroblastoma /Ganglioneuroblastomas Wilms’ tumour Retinoblastoma/ Pinealoblastoma Medulloblastoma Hepatoblastoma Pleuropulmonary Blastoma Acute lymphoblastic leukaemia and Lymphoblastic leukemia

5. CLASSIFICATION OF TUMOURS BASED ON ORIGIN Neurogenic origin Mesenchymal origin Hematolymphoid origin Organ specific Uncertain type Ewing’s sarcoma/ PNET Myogenic differentiation: RMS Acute lymphoblastic leukemia Hepatoblastoma Desmoplastic small round cell tumour Neuroblastoma Osteoid differentiation: small cell osteosarcoma Lymphoblastic lymphoma Wilm’s tumour Poorly differentiated synovial sarcoma Retinoblastoma Pleuropulmonary Blastoma Pinealoblastoma Medulloblastoma

6. NEUROBLASTOMA Most common extra cranial solid tumor of childhood Most frequently diagnosed tumor of infancy. Median age at diagnosis is 21 months.

7. CYTOGENETICS Familial Neuroblastoma   Rare (<2%), Due to mutations in genes (PHOX2B, ALK, KIF1B ß ) involved in signaling pathways important for development of sympathoadrenal lineage Congenital  central hypoventilation  syndrome (CCHS )/ Ondine's curse (PHOX2B mutation syndrome) : Respiratory dysfunction, autonomic dysfunction , Hirschsprung disease and neural crest tumors

8. Role of RAS Pathway Mutations Neural crest cells, from which Neuroblastoma originates, migrate and innervate multiple organs and rely on RAS signaling for terminal maturation Recurrent, sporadic tumors have high frequencies of mutations in this pathway These mutations are an indicator of more aggressive disease

9. Other predisposing syndromes Costello syndrome : HRAS Activating missense mutation Noonan syndrome : KRAS Activating sense mutation Neurofibromatosis type I : NF1 Activating mutation Beckwith–Wiedemann syndrome : Abnormal methylation of chromosome 11 or uniparental disomy Li–Fraumeni syndrome : TP53 Missense Familial Paraganglioma syndrome : SDH mutation Fanconi Anemia

10. Sporadic Neuroblastoma ALK amplification associated with poor prognosis MYCN amplification associated with poor prognosis Decreased expression of NBAT-1 has also been correlated with high-risk Neuroblastoma

11. Distribution by site and age

12. Range in size from minute nodules to large masses more than 1 kg. Cut section: soft , gray-tan, tissue. Larger tumors have areas of necrosis, cystic softening, and hemorrhage.

13. Small, primitive-appearing cells with dark nuclei, scant cytoplasm, and poorly defined cell borders growing in solid sheets.

14. The background often demonstrates a faintly eosinophilic fibrillary material (neuropil) that corresponds to neuritic processes of the primitive neuroblasts.

15. NEUROPIL

16. Homer- Wright pseudo rosettes can be found in which the tumor cells are concentrically arranged about a central space filled with neuropil

17. FNAC paraspinal mass. Neuroblastoma. Homer-Wright rosette.

18. Ultra structural demonstration of small, membrane-bound, cytoplasmic catecholamine-containing secretory granules; the latter contain characteristic central dense cores surrounded by a peripheral halo (dense core granules).

19. Larger cells having more abundant cytoplasm, large vesicular nuclei, and a prominent nucleolus, representing ganglion cells in various stages of maturation, may be found in tumors admixed with primitive neuroblasts ( ganglioneuroblastoma ).

20. Even better differentiated lesions contain many more large cells resembling mature ganglion cells with few if any residual neuroblasts; i.e. ganglioneuroma . Maturation of neuroblasts into ganglion cells is usually accompanied by the appearance of Schwann cells .

21. The International Neuroblastoma Pathology Classification STROMA HISTOLOGY DIAGNOSIS Schwannian stroma poor No Schwannian stroma/ no neuropil Requires adjunctive diagnostic tests Neuroblastoma, undifferentiated Neuropil + <5% differentiating neuroblasts Neuroblastoma, poorly differentiated More abundant neuropil >5% differentiating neuroblasts Neuroblastoma, differentiating Schwannian stroma rich (>50%) >50% of tumour has Schwannian stroma Microscopic foci of neuropil with neuroblastic cells, No macroscopic nodules Ganglioneuroblastoma, intermixed Schwannian stroma dominant ( No neuropil) Scattered ganglion cells are mature/ maturing Ganglioneuroblastoma, maturing Scattered ganglion cells are surrounded by satellite cells Ganglioneuroma, mature Composite stroma rich/dominant and stroma poor Grossly visible nodules Background of ganglioneuroma/-Blastoma Ganglioneuroblastoma, Nodular

22. IMMUNOHISTOCHEMISTRY Positive stains Neuron specific enolase (NSE) ,  CD57 ,  CD56 , Synaptophysin ,   Chromogranin ,   Neurofilament protein ,   ALK-1  (>90%), PHOX2B ,  Glial fibrillary acidic protein (GFAP)  (variable ) Negative stains EMA ,  Cytokeratin ,   Vimentin ,   HMB45 , (melanoma) WT1 ,  (Wilms’ tumour) CD99 ,  (PNET) CD45 ,  (Lymphoma) Desmin , (RMS) M yogenic markers: myogenin ,  Myo-D1 (RMS) S100   (Paraganglioma)

23. NSE Synaptophysin PHOX2B Chromogranin

24. Undifferentiated Neuroblastoma Defined in the International Neuroblastoma Classification as: Diagnosed only by supplementary techniques Patients may have elevated serum catecholamine levels. Contain undifferentiated cells with indiscernible-to-thin cytoplasm Contain no neuropil. Cells contain nuclei with salt-and-pepper chromatin. The majority contain prominent nucleoli, a marker of bad prognosis

25. Undifferentiated Neuroblastoma Prognosis and Clinical Outcome: Classification Good prognosis Low or intermediate MKI ,< 1.5 years of age Poor prognosis High MKI , <1.5 years of age Age 1.5–5 years, any MKI Neuroblastoma , any type, age >5 years

26. Undifferentiated Neuroblastoma Ancillary Diagnostic Techniques Tyrosine Hydroxylase N-myc IHC C-myc IHC PHOX2B : Universally expressed in small series of undifferentiated neuroblastomas in one study (6/6) and in no “neuroblastoma-like undifferentiated sarcoma” Negative in panel of 109 other tumors (8 diagnoses)

27. You are asked to see a child who has Hirschsprung disease who, following surgical correction, is noted to have periods of apnea There is a significant family history of childhood malignancy although the parents cannot recall the types of tumors. This patient most likely has which of the following gene mutations and tumor types: Wilms tumor and Loss of imprinting of 11p15 Neuroblastoma and PHOX2B mutation Pheochromocytoma and TP53 (p53) mutation Neuroblastoma and ALK mutation Wilms tumor and 11p13 deletion

28. A 3 year old girl is referred to you for evaluation of an abdominal mass. The patient’s mother detected the mass while she was bathing her child. The child is well appearing, but has a 8 cm, firm mass in the abdomen that crosses the midline. CBC is normal but a urine microscopy reveals 10-15 RBCs. The most likely diagnosis is: N euroblastoma Wilms tumor Hepatoblastoma Burkitt’s lymphoma Mesoblastic Nephroma

29. WILMS’ TUMOUR Age : Infants (primarily), less than 3 years ( 50%) & less than 6 years (90%) Sex: M=F Presentations: Large abdominal mass Hematuria Pain in abdomen Hypertension

30. Syndromes associated with Wilms’ tumour WAGR syndrome :  due to 11p13 deletion (WT1) W ilms tumor,   A niridia ,  G enitourinary anomalies, mental   R etardation Denys-Drash syndrome : gonadal dysgenesis (male pseudohermaphroditism), glomerulosclerosis and Wilms tumor Beckwith-Wiedemann syndrome : exophthalmos, macroglossia and gigantism; also hemihypertrophy, renal medullary cysts, adrenal cytomegaly and hypoglycemia;

31. Grossly: large , solitary, wellcircumscribed mass C ut section: soft , homogeneous, and tan to gray with occasional foci of hemorrhage, cyst formation, and necrosis

32. The classic triphasic combination of epithelial (A), stromal (B) and blastemal (C) cell types is observed in the vast majority of lesions, although the percentage of each component is variable

33. The  blastemal component shows s heets of small blue cells with few distinctive features .

34. The epithelial component shows various stages of abortive tubal and glomeruloid differentiation.

35. Stromal cells are usually fibrocytic or myxoid in nature .

36. IMMUNOHISTOCHEMISTRY Positive stains Blastema :  WT1 , focal  vimentin, desmin  but not other muscle markers, Epithelium :WT1 ,  keratin  and  EMA ; tubules are  CD57 + Stroma : weak WT1; other stains consistent with morphologic appearance ( myogenin  if rhabdomyoblastomatous,  S100  if neural, etc .) Nuclear PAX 8 TTF-I CD 56 Negative stains Blastema negative for  CK7 , CD57 and usually  p53 Epithelium negative for vimentin CD 99

37. WT1 CD 56 EMA

38. Prognostic factors Good prognosis Age under 2 years Extensive tubular/ glomerular differentiation Skeletal muscle differentiation Bad prognosis Age over 2 years Presence of inflammatory pseudo capsule Renal sinus invasion Renal capsular infiltration Infiltration of intrarenal vessels Anaplasia LOH at 1p and 16q TP53 mutation (associated with anaplasia Post chemotherapy morphology Risk Completely necrotic Low Blastemal predominance High Others Intermediate

39. Anaplasia: marker of unfavorable histology . Three histologic criteria have to be met to classify tumor as having unfavorable histology: Nuclei enlarged  to at least 3 times the size of adjacent nuclei of the same cell type Marked hyperchromasia  of the enlarged nuclei; Multipolar mitotic figures .

40. Focal anaplasia: all conditions must be met: (a) no anaplasia in tumor within renal vessels or outside kidney; (b) random biopsies are free of anaplasia; (c) anaplasia confined to sharply localized regions within primary intrarenal tumor site; and (d) each focus of anaplasia must be surrounded on all sides by nonanaplastic tissue, which does not show severe nuclear unrest Severe nuclear unrest : nuclear pleomorphism or atypia approaching, but insufficient for anaplasia

41. Diffuse anaplasia : any conditions met: Anaplasia in tumor in any extrarenal site, including vessels of renal sinus, extracapsular infiltrates, metastases or intrarenal vessels; Anaplasia in random biopsy; or Anaplasia unequivocally present in 1 region of tumor with extreme nuclear unrest elsewhere in the lesion

42. Tumors with anaplasia are said to have  unfavorable histology  since they show a lesser response to chemotherapy. 

43. Differential diagnosis of Wilms tumour

44. Nephrogenic rests Nephrogenic rests (NRs) are foci of embryonal cells which are abnormally persistent after 36 weeks of gestation and which are capable of developing into WT. The term nephroblastomatosis is used to describe the presence of multiple nephrogenic rests.

45. Perilobar nephrogenic rest showing sclerosing and hyperplastic rests

46. Variable Wilms tumor Nephrogenic rest Shape Spherical Oval Fibrous capsule Present Absent Skeletal muscle differentiation Common Uncommon Number Solitary Multifocal

47. Clear cell sarcoma of kidney : Pushing border, more aggressive invasion, nephrogenic rests rule out CCSK; Wilm tumor is WT1+, epithelioid areas are keratin +. Cyclin D1  may be useful (14 / 14 cases of CCSK are diffusely and strongly positive )

48. Congenital mesoblastic nephroma : Well differentiated spindle cell stroma with entrapment of glomeruli. presents at a very young age, often in intrauterine period.

49. Renal rhabdoid tumor : Neoplastic cells have pink cytoplasm with eccentric nuclei due to intracytoplasmic inclusions of eosinophilic hyaline globules. Chromatin is clear / vesicular with central, large nucleoli. S hows loss of nuclear staining of IN1

50. Metanephric adenoma : Older age of presentation,   Well demarcated. WT1 Negative,   CD57 strong and diffuse positive

51. RETINOBLASTOMA Most common intraocular neoplasm of children- 16 months- 2 yrs. It characteristically present as leukocoria/ strabismus . Bilateral in 30% > 90% familial cases .

52. GROSS APPEARANCE: Endophytic type: Protrudes into vitreous . Exophytic type: Grows between retina & pigmented epithelium, resulting in retinal detachment

53. Sheets, trabeculae and nests of small blue cells with scant cytoplasm, hyperchromatic nuclei and scanty stroma

54. Frequent necrosis of tumor cells away from vessels and calcification

55. Flexner-Wintersteiner rosettes (cells line up around empty lumen delineated by a distinct eosinophilic circle composed of terminal bars analogous to outer limiting membrane of normal retina)

56. Frequent Azzopardi phenomena (basophilic deposits around blood vessels)

57. Also fluerettes (tumor cells arranged side by side which show differentiation towards photoreceptors)

58. (a) FNAC smear) showing small round cells in cluster and rosettes formation, suggestive of retinoblastoma . PAS (b) Showing positive granules in the cytoplasm of tumor cells, consistent with diagnosis of retinoblastoma

59. IMMUNOHISTOCHEMISTRY Positive stains Neuron specific enolase ,  Synaptophysin ,   S100 ,  Leu7 ,  GFAP , Myelin basic protein   p53 ; High   Ki-67 Negative stains CD99 (PNET)

60. Differential diagnoses of Rb/ leukocoria Coats’ disease Persistent hyperplastic primary vitreous Vitreous hemorrhage leading to retinal gliosis Ocular toxocariasis Congenital toxoplasmosis Retrolental fibroplasia Retinal astrocytoma

61. A 5 year-old survivor of bilateral retinoblastoma who was successfully treated with chemotherapy presents to your clinic with headaches and vomiting; an MRI shows a midline intracranial mass. What is the most likely diagnosis of this child? Medulloblastoma Pinealoblastoma Atypical Rhabdoid Teratoid Tumor Ependymoma Brainstem glioma

62. PINEALOBLASTOMA Age : Children and adolescents Sex : M>F Presentations : • Obstructive hydrocephalus • Parinaud syndrome • CSF seeding in 15% of patients at the time of diagnosis . • Trilateral retinoblastoma • Association with familial adenomatous colonic polyposis as a possible variant of type 2 Turcot syndrome .

63. Sheets of densely packed cells with high grade (anaplastic / undifferentiated) features including high N/C ratio with minimal cytoplasm and large hyperchromatic nuclei

64. Focal areas of necrosis are seen

65. Numerous atypical mitoses

66. Immunohistochemistry Positive stains Synaptophysin Rhodopsin Retinal S antigen Arrestin Ki-67 NSE Negative stains GFAP (Glial neoplasms) Myoglobin (RMS) HHF35 (RMS)

67. Poor prognostic factors 7 + mitotic figures/10 HPF Presence of necrosis No neurofilament staining

68. DD  Pineocytoma : Similar to normal pineal glands well differentiated cells but hypercellular. May have features of neuronal differentiation (ganglion cells). Noninfiltrative , no / rare mitotic figures, no necrosis, no / minimal atypia

69. MEDULLOBLASTOMA The medulloblastoma has been defined by the World Health Organization (WHO) as a "malignant, invasive embryonal tumor of the cerebellum with preferential manifestation in  children, predominantly neuronal differentiation, and inherent tendency to metastasize via CSF [cerebrospinal fluid] pathways ."

70. SUBTYPE CYTOGENETICS HISTOLOGY PROGNOSIS WNT Type Monosomy of chromosome 6 Nuclear expression of β- catenin. Classical type 90% 5-year survival rate SHH Type Mutations involving the sonic hedgehog signaling pathway MYCN amplification Nodular/ desmoplastic Intermediate Group 3 MYC amplification Isochromosome 17 (i17q) Classical/ Large-cell Worst prognosis Group 4 Isochromosome 17 (i17q) No MYC amplification MYCN amplification may be present Classical/ Large cell Intermediate

71. The tumor is often well circumscribed, gray, and friable, and may be seen extending to the surface of the cerebellar folia and involving the leptomeninges.

72. Densely cellular, with sheets of anaplastic cells. Individual tumor cells are small, with scant cytoplasm and hyperchromatic nuclei.

73. Mitoses are abundant

74. Rosette formation may be seen

75. Intraoperative crush preparation showing a medulloblastoma with poorly differentiated cells with little cytoplasm 

76. Classical variant Desmoplastic/ nodular With extensive nodularity Large cell variant Syncytial arrangement of cells Densely packed, undifferentiated cells Expanded lobular architecture Anaplasia Marked nuclear pleomorphism Mitosis with apoptotic bodies Dense intercellular reticulin fiber network Reticulin free nodular zones are enlarged High mitotic and apoptotic counts Homer Wright rosettes Nodular reticulin free zones Rich in neuropil-like tissue Nuclear molding Cell wrapping

77. Nodular (desmoplastic) medulloblastoma with pale nodules of differentiating neuroblasts (neurocytes ) and prominent inter-nodular area.

78. Extensively nodular medulloblastoma with minimal or no internodular component.

79. Large cell medulloblastoma with large vesicular nuclei, distinct nucleoli, pronounced mitotic and apoptotic activity, imparting a starry-sky appearance

80. Neuroblastic medulloblastoma: typified by the linear streaming of rounded , ‘ neurocytic’ tumor cell nuclei within amassed cytoplasmic processes.

81. MEDULLOBLASTOMA WITH MELANOCYTIC DIFFERENTIATION Cells containing brown pigment HMB 45+ S100+

82. MEDULLOBLASTOMA WITH MYOGENIC DIFFERENTIATION DESMIN + SKELETAL MUSCLE FIBRE+

83. Anaplasia of medulloblastomas has been defined as increased nuclear size with more striking nuclear pleomorphism and molding than the classic type, high mitotic activity with atypical forms, frequent apoptotic bodies, and a distinctive wrapping of one tumor cell around another (arrows).

84. Immunohistochemistry Positive stains Synaptophysin   NeuN Nuclear   SMARCB1 / INI1  and  SMARCA4  are retained (AT/RT) TTF1    Negative stains GFAP (Glial neoplasms esp. ependymoma) WT1 CD 99 (PNET)

85. A child diagnosed at 2 years of age with medulloblastoma is noted to have a desmoplastic/nodular medulloblastoma by the neuropathologist. Molecular classification of the tumor is likely to demonstrate that the tumor belongs to which genetically defined medulloblastoma subgroup ? Sonic-hedgehog (SHH) activated Wnt activated Group 3 Group 4

86. HEPATOBLASTOMA Hepatoblastoma is the most common liver tumor of early childhood . Rarely occurs over the age of 3 years .

87. Associated conditions F amilial colonic polyposis Glycogen storage disease Beckwith-Wiedemann syndrome Li-Fraumeni syndrome Premature and low birth weight infants, Trisomy 18 Also associated with adenomatoid transformation of Bowman capsular epithelium in kidney Not associated with cirrhosis

88. Laboratory findings Marked thrombocytosis due to a paraneoplastic effect related to the tumor production of interleukin-6, a potent growth factor for megakaryocytes. Serum AFP level is elevated in at least 70% of children. P atients with low AFP level at diagnosis (< 100 ng/mL) tend to have a more aggressive biological tumor behaviour AFP is also a marker of the tumor response to therapy and in the early detection of tumor recurrence.

89. Well differentiated fetal hepatoblastoma with low mitotic activity comprised of polygonal cells with low nuclear: cytoplasmic ratio, abundant clear cytoplasm and no mitoses

90. The crowded or mitotically active fetal hepatoblastoma shows larger nuclei and amphophilic cytoplasm giving rise to a “crowded” appearance. Scattered mitotic figures are seen (arrows)

91. E mbryonal hepatoblastoma showing large cells with large nuclei containing coarse chromatin. Tubular structures with central lumen are present. (arrow)

92. Pleomorphic fetal hepatoblastoma comprised of large polygonal cells arranged in trabecular pattern and containing abundant eosinophilic cytoplasm, and pleomorphic nuclei with coarse chromatin and prominent nucleoli

93. Macrotrabecular hepatoblastoma showing tumor cells arranged in thick trabeculae. The cells comprising the tumor resemble fetal hepatoblasts .

94. Cholangioblastic hepatoblastoma consisting of incomplete or complete tubulo-glandular structures in direct continuation with the solid part of the tumor and strongly positive for CK-19

95. Mixed epithelial mesenchymal hepatoblastoma comprised of fetal hepatoblastoma intimately mixed with osteoid, which represents the mesenchymal component

96. IMMUNOHISTOCHEMISTRY

97. Alpha-fetoprotein: most likely to be positive in areas of epithelial hepatoblastoma. Frequently results in high-background serum staining, making interpretation difficult .

98. Glypican 3 highlights the epithelial components and is negative in normal liver and benign tumors.

99. Glutamine synthetase: marker for activated Wnt pathway and also a differentiation marker Hep par I INI 1

100. Beta-catenin CTNNB1 mutations are found in over 80% of hepatoblastoma cases. Beta-catenin immunohistochemistry is extremely helpful because of the presence of nuclear-staining pattern in the neoplastic tissue. Diffuse cytoplasmic expression without nuclear staining usually also implies neoplasia, Normal hepatocytes and biliary epithelial cells show only membranous staining.

101. Diffuse membranous positivity Nuclear staining

102. A 8 month old infant presents with a distended abdomen. Diagnostic work-up shows AFP of 50 ng/mL, and imaging studies show a large liver mass with lung metastases. A biopsy of the mass is done. What is the most likely diagnosis? Pure fetal histology hepatoblastoma Hepatocellular carcinoma Fibrolamellar hepatocellular carcinoma Small cell undifferentiated hepatoblastoma

103. PLEUROPULMONARY BLASTOMA Pleuropulmonary Blastoma (PPB) is a rare, primitive primary neoplasm of the thorax in young children The tumor may arise in pulmonary parenchyma, the mediastinum and pleura They are commonly associated with DICER1 mutations

104. PPB-1: peripherally located , multiloculated thin walled cyst filled with scanty clear serous fluid

105. PPB II : Mixed solid and cystic tumor with variable thickened or nodule-like areas. Composed of diffuse proliferation of round to oval cells with hyperchromatic nuclei, prominent nucleoli and scant cytoplasm and c ystic spaces lined with cuboidal and columnar epithelium

106. Type III PPB: Well circumscribed, mucoid, white-tan solid mass attached to the pleura and involves a lobe or entire lung; necrosis and hemorrhage may be present .T umor composed of primitive, undifferentiated blastema cells and sarcomatous elements of different types of differentiation

107. Immunohistochemistry Positive stains Neoplastic cells Vimentin  (diffuse),  CD117  (focally),  A lpha-1-antitrypsin  (focally)   CD99 (weakly ) Surface epithelium :  C ytokeratin Rhabdomyoblasts and primitive cells :  Muscle specific actin Desmin Negative stains EMA Myogenin S100   GFAP N euron specific enolase TTF1 A lpha feto-protein Chromogranin   Synaptophysin

108. 15 year old boy with respiratory distress. Radiology shows cervical and mediastinal lymphadenopathy, which is biopsied. Flow cytometry shows a proliferation of cells that are TdT+, CD10+, CD3+, CD7+, CD4+ and CD8+. Interpretation ? Myeloid sarcoma B lymphoblastic lymphoma T lymphoblastic lymphoma Hodgkin lymphoma Reactive process

109. LYMPHOBLASTIC LYMPHOMA Age : Primarily in children and adolescents, with a second peak after 40 years of age Sex : M>F Site: Mediastinum( Thymus). Presentations: • Typically presents with acute respiratory distress in teenager. • Extremely aggressive, with rapid multisystem dissemination. • Leukemic blood picture (acute lymphoblastic leukemia). • Death after a few months.

110. Cytology: intermediate-sized cells ( 9.5–18.5 u m ) with fine nuclear chromatin, small, inconspicuous nucleoli , irregular nuclear contours and scant basophilic cytoplasm .

111. Involved lymph nodes characteristically show total effacement of the normal architecture by a diffuse proliferation of monotonous lymphoid cells with frequent mitoses

112. Primitive-appearing nuclei with nuclear convolutions, finely distributed chromatin and inconspicuous nucleoli.

113. Numerous tingible-body macrophages produce characteristic “starry sky” appearance.

114. The most distinguishing immunohistochemical feature of lymphoblastic lymphoma is strong nuclear TdT positivity, which is present in virtually 100% of cases.

115. Immunohistochemistry Positive stains TdT Cytoplasmic   CD79a   CD10  (most cases ) Weak surface   CD22 (consistent feature), Cytoplasmic CD22 (sensitive marker),  PAX5 Negative stains CD 19, CD 20 (Burkitt's/ DLBCL)   CD30   Myeloid markers (AML/ Myeloid sarcoma) CK   (Thymoma) S100 (Mesenchymal chondrosarcoma) Neuroendocrine markers (neuroblastoma) CD99 (Ewing's)

116. An important differential diagnosis is lymphocyte-rich thymoma with a “starry sky” appearance. Histologically, may closely resemble lymphoblastic lymphoma and also mark positively for T-cell markers, TdT, and CD99 . When in doubt, a cytokeratin stain should be able to highlight the scattered positive thymic epithelial cells in thymoma , as opposed to negative staining in lymphoma.

117. A 14 year old girl was treated for ALL when she was 4 years of age and received cranial prophylaxis with irradiation . She now presents with a two week history of increasing headaches and on the day of presentation has a generalized tonic-clonic seizure. An emergent MRI is obtained which demonstrates an intraparenchymal mass in the parietal lobe. At the time of biopsy, pathology will likely reveal: Glioblastoma multiforme Pilocytic astrocytoma Oligodendroglioma Meningioma Ependymoma  

118. SOFT-TISSUE TUMOURS

119. Pediatric soft tissue tumors Rhabdomyosarcoma Ewing’s Sarcoma/Primitive Neuroectodermal Tumour Desmoplastic small round cell tumor Mesenchymal Chondrosarcoma Small cell osteosarcoma

120. RHABDOMYOSARCOMA Most common soft tissue sarcoma of childhood & adolescence. Usually appears before 20 years of age. Types:- Embryonal (most common) Alveolar Rhabdomyosarcoma Pleomorphic (least common)

121. Embryonal Rhabdomyosarcoma Arise from unsegmented & undifferentiated mesoderm. Common in head & neck region Age :- 3 -12 years, can occur in adults also . Most cases occur sporadically with no genetic predisposition

122. Genetics Seen secondarily in syndromes including:- Costello syndrome ( HRAS ),  N eurofibromatosis type 1  ( NF1 ), Noonan syndrome ( PTPN11  and others),  Beckwith-Wiedemann syndrome (changes to 11p15),  Dicer syndrome ( DICER1 ), Li-Fraumeni syndrome ( TP53 ) Gorlin syndrome ( PTCH1 ) 

123. Grossly: poorly circumscribed, white, soft

124. Sheets of small, stellate, spindled or round cells with scant or deeply eosinophilic cytoplasm and eccentric, small oval nuclei with a light chromatin pattern and inconspicuous nucleoli

125. T umor cells that contain generous amounts of eosinophilic cytoplasm, a feature of rhabdomyoblastic differentiation (so called "strap" cells/ tadpole cells)

126. FNAC: Tadpole cells showing desmin positivity

127. Botryoid variant Clusters of edematous, grape-like masses that protrude into lumen of hollow organs.

128. Polypoid or lobulated masses of cells covered by mucosa, with underlying hypercellular zone of poorly differentiated cells (cambium layer)

129. Anaplasia Anaplasia is an important prognostic feature and is defined as : Significant nuclear variation (cells that are 3x larger than background tumor cells) Presence of atypical multipolar mitotic figures

130. Differential diagnosis Alveolar rhabdomyosarcoma (ARMS) : Diffuse and strong nuclear staining for  myogenin  and  MyoD1 M olecular studies show   PAX-FOXO1  fusion gene product in approx.. 85 % of cases Pleomorphic rhabdomyosarcoma Common in 60-70 years in extremities. Uniformly pleomorphic with diffuse atypia and frequently anaplastic Desmoplastic small round cell tumor : Tumor nodules on serosal surfaces, strongly  keratin + and  EMA +, Muscle specific actin negative. Associated with desmoplasia Ewing / PNET : Often displays Homer Wright rosettes CD99 +,  desmin -,  MyoD1 -,  myogenin - t(11;22 ) or t(12;22 )

131. Large cell lymphoma : CD45 +, B / T cell markers present , Monophasic synovial sarcoma Negative for muscle markers , usually far more spindled and organized (long fascicles) Cytokeratin  and  EMA  expression will show scattered and focal positivity in the spindled cells, Molecular or FISH testing for the t(X;18) fusion Myxoid liposarcoma (MLS) : Signet ring lipoblasts present Neuroblastoma, Undifferentiated Elevated urinary catecholamines, rosettes, granular chromatin, neuropil S100 +   Chromogranin +,   Synaptophysin +,   GFAP + Absence of myogenic markers

132. Alveolar Rhabdomyosarcoma Mostly extremities (forearm, arm), Perirectal & perineal region. Usually manifests as an expanding mass. Hypercalcemia or with elevated parathormone levels in some cases .

133. Cytogenetics Two fusion proteins can be associated with alveolar rhabdomyosarcoma (ARMS): ~ 60% of cases are positive for  PAX3-FOXO1  fusion gene, 20 % for  PAX7-FOXO1  fusion gene,

134. Cellular neoplasm composed of medium to large atypical round cells with scant eosinophilic cytoplasm, which may be eccentric to the nucleus imparting rhabdoid/plasmacytoid appearance and associated with intervening fibrous septa with  dyscohesive areas, reminiscent of alveolar-like spaces.

135. Differential diagnosis Embryonal rhabdomyosarcoma (ERMS ) Dense ERMS is characterized by variation in cellular and nuclear size and shape within a tumor. Merkel cell carcinoma: negative for muscle specific actin, desmin, myogenin and MyoD1, lacks fusion gene Metastatic neuroendocrine carcinoma:  keratin+, EMA+, desmin-, muscle specific actin-, lacks fusion gene

136. Pleomorphic Rhabdomyosarcoma Least common . Site :- Extremities & thigh. Age :- Adult Grossly :- It is confined within fascial compartment & may have the shape of muscle from which it arises

137. Sheets of large, atypical and frequently multinucleated polygonal eosinophilic cells

138. Immunohistochemistry Positive stains Desmin ,  MyoD1  or  myogenin  are critical to document . Without myogenic differentiation , it is very difficult to diagnose embryonal rhabdomyosarcoma Vimentin Actin ,  myoglobin ,   myosin  and  creatine kinase M  staining in more differentiated cells. PAS  highlights glycogen in most tumors Negative stains CD 99 (Ewing's) EMA, Cytokeratin (Desmoplastic small round cell tumour) CD 45 (Lymphoma) S100, Synaptophysin, Chromogranin, GFAP (Neuroblastoma)

139. ARMSs typically show strong, robust, homogeneous expression with myogenin IHC, as compared with the relatively weak to heterogeneous pattern seen with ERMS

140. A 16-year old presents with osteosarcoma in the proximal fibula. She has a history of treatement for vaginal rhabdomyosarcoma when she was 2 years of age. Which genetic mutation best explains this cancer pattern? A . Germline p53 mutation B. Germline Rb mutation C. Germline RECQL4 mutation D. Germline PAX3 mutation E. Germline LOH at 11p15.5

141. A 10 year old presents with a mass involving the humerus. Frozen section reveals a small round blue cell tumor. Based on H&E and immunohistochemistry, a diagnosis of Ewing’s sarcoma is made. Which of the following will you look for? a. t(2;13)(q35;q14) b. t(X;18)(p11;q11) c. t(12;15)(p13;q25) d. t(12;16)(q13;p11) e. t(11;22)(q24;q12)

142. Ewing sarcoma family of tumours Poorly differentiated Arise in bone and soft tissue (mostly bone) Characteristic genetic fusions of EWS gene with ETS group of genes Mostly adolescents and young adults Slightly more common in boys.

143. Clinical features There is no evidence of familial predisposition or an association with environmental factors. P resents as a rapidly growing, deeply located mass If peripheral nerves or the spinal cord are involved, there may be progressive sensory or motor disturbances. Catecholamine levels are within normal limits.

144. Microscopic features Three major varieties Classical Ewing sarcoma Atypical Ewing sarcoma Peripheral neuroectodermal tumor (PNET)

145. I t is multi-lobulated , soft, and friable; it rarely exceeds 10 cm in greatest diameter. The cut surface has a gray-yellow or graytan appearance, often with large areas of necrosis, cyst formation, or hemorrhage. Despite the extensive necrosis, calcification is rare.

146. Low-power view of Ewing's sarcoma characterized by a lobular round cell pattern of striking uniformity

147. Classical Ewing's sarcoma : The tumor cells have a round or ovoid nucleus with a distinct nuclear membrane, fine powdery chromatin, and inconspicuous nucleoli . There are no multinucleated giant cells.

148. The cytoplasm is ill-defined, scanty, and pale staining; and, in many cases, it is irregularly vacuolated as a result of intracellular deposits of glycogen.

149. Adamantinoma-like variant: prominent peripheral nuclear palisading and contain large polygonal cells with irregularly contoured , hyperchromatic nuclei, prominent nucleoli, and moderate amounts of cytoplasm

150. Strong cytokeratin positivity in adamantinoma -like variant

151. The typical PNET comprising is composed of sheets or lobules of small round cells containing darkly staining, round, or oval nuclei.

152. Numerous rosettes visible even at low power

153. PNET with Rosettes: Most of the rosettes are similar to those seen in neuroblastomas and contain a central solid core of neurofibrillary material (Homer Wright pseudo-rosette ).

154. PNET with spindle cells

155. Immunohistochemistry

156. CD99  is positive in virtually all cases and diffusely expressed in the vast majority of cases in a membranous pattern

157. A lthough CD99 is highly sensitive, it should always be used as part of a panel of immunostains because it lacks specificity. Given the rarity of CD99 negativity, suspected cases should be confirmed by molecular techniques prior to rendering a definitive diagnosis . PNET tend to express one or more of neural markers with greater frequency than typical ES.

158. cytogenetics The defining feature of Ewing’s sarcoma is the presence of nonrandom translocations leading to the fusion of the EWSR1 gene on 22q12 with one of several members of the ETS family of transcription factors

159. Genetics Karyotype t(11;22) ~85%: EWS-FLI1 t(21;22 ) ~10-15%: EWS-ERG Other (rare) t(2;22 ) - EWS-FEV t(7;22 ) – EWS-ETV1 t(17;22 ) – EWS-E1AF

160. NKX2.2 , a new marker Transcription factor for neuronal development Target gene product of EWS-FLI1 fusion protein 80 % sensitivity, 84% specificity for fusion-positive Ewing sarcomas Specificity is improved with concurrent use of CD99 (98%).

161. NKX 2.2 positivity

162. Prognostic factors Favorable prognosis Age : < 10 years and 10 - 17 years Site : extremities Size : < 8 cm greatest diameter Stage : nonmetastatic tumor Unfavorable prognosis Age : ≥ 18 years Site : pelvis Size : ≥ 8 cm in greatest dimension Stage : metastatic tumor

163. Differential diagnosis Neuroblastoma Lymphoma Metastatic pulmonary small cell carcinoma Merkel cell carcinoma Mesenchymal chondrosarcoma Synovial sarcoma S mall cell osteosarcoma : Y ounger patient age. CD 99 negative CD3, CD20, CD45, TdT TTF-1 positive, CD99 negative CK 20 positive, CD 99 negative Cartilage , HPC-like foci. t(11;22) is absent Nuclear TLE1 positive, detection of t(X;18) CD 57, osteoid SALLB4

164. Undifferentiated “Ewing-like” sarcoma Those with EWS fusion with non-ETS family member Those without EWS fusion but with a similar genetic profile The clinical setting and therapeutic responses are heterogeneous and unpredictable.

165. EWS fusion with non-ETS family member EWS-PATZ1 & EWS-SP3: shows polyphenotypia . More aggressive and drug resistant similar to DSRCT EWS-OCT4 : Heterogeneous morphology, with areas containing nested polygonal and spindle cells

166. Ewing-like sarcoma with non-EWS fusions BCOR-CCNB3 fusion- paracentric inv(X) Usually arise in bone Resembles Ewing sarcomas clinically and morphologically Fuses BCOR, an epigenetic repressor, to cyclin B3

167. CIC-DUX4 fusions –t(4;19) and t(10;19 ) Usually extraskeletal. Most often in extremities Aggressive course with early metastasis More like atypical Ewing sarcoma with nucleoli, more abundant cytoplasm, and extensive necrosis and mitoses Variable CD99 positivity. Frequently WT1 positive

168. C entral area of necrosis surrounded by malignant round cells.

169. Desmoplastic Small Round Cell Tumor (DSRCT ) High grade malignancy that predominately occurs in peritoneal-lined cavities. Small cells with prominent stromal desmoplasia Polyphenotypia : epithelial, neural, and mesenchymal (dot-like desmin expression) EWS-WT1 fusion with WT1 expression Aggressive behavior with poor chemotherapy response and peritoneal seeding and metastasis

170. Clinical features 15 to 35 years of age, M:F ratio 4:1 Most present with a large abdominal and/or pelvic mass with extensive peritoneal involvement, usually without an identifiable visceral site of origin The most common complaint is abdominal distension, often associated with pain and constipation.

171. Genetics t(11;22 )(p13;q12) EWS-WT1 fusion The fusion protein appears to induce expression of PDGFA, a potent mitogen and chemo-attractant for fibroblasts and endothelial cells

172. Cords of cells are surrounded by a dense fibrous stroma mimicking lobular carcinoma of the breast.

173. The tumor cells appear undifferentiated with small hyperchromatic nuclei with inconspicuous nucleoli .

174. Additional features Epithelial differentiation Glands Rosettes Rhabdoid cells

175. Focus of cells with a rhabdoid appearance

176. IMMUNOHISTOCHEMISTRY Positive stains Epithelial :  cytokeratin,   EMA Mesenchymal : vimentin, desmin (dot pattern of reactivity) Neural : NSE, CD57 WT1  in > 90 % Negative stains CD99   (Ewing's sarcoma) MyoD1  and  myogenin (RMS)   CK20 (Merkel cell carcinoma),   CK5/6,Thrombomodulin   (mesothelioma) Neurofilament   proteins, S100 (Neuroblastoma) S ynaptophysin ,   chromogranin,

177. Typical perinuclear globular pattern of desmin immunoreactivity

178. Cytokeratin + Desmin +

179. Mesenchymal chondrosarcoma Most common form of chondrosarcoma in children Occurs in Craniofacial bones, chest wall, spine, sacrum Extraosseous locations: Meninges included Highly malignant Prolonged course with late metastases (survival 21-67%)

180. Microscopic features Biphasic appearance Undifferentiated small blue cells or spindle cells Well-differentiated cartilage Other features: Hemangiopericytoma appearance Foci of chondroid ossification Myxoid zones

181. Bimorphic picture : islands of well-differentiated cartilage surrounded by sheets of small, undifferentiated tumor cells.

182. Sharp demarcation between small, undifferentiated tumor cells and well-differentiated cartilage.

183. S mall round cells surrounding hemangiopericytoma-like vessels

184. Hemangiopericytoma like pattern

185. Immunohistochemistry Sox9 : positive in round cells and chondrocytes β-catenin : negative in round cells, positive at cartilage interface Osteocalcin : negative in round cells, positive in bony matrix S100 : positive in only occasional cases in round cells; usually positive in cartilage EMA (30%) and desmin (50%) can be positive FLI1 negative ; CD99 can be positive

186. Small cell osteosarcoma A rare form of osteosarcoma (<2%) Small blue cell tumors with osteoid production Usually in long bones, second decade Slight female predilection May have worse prognosis than other osteosarcomas

187. Histology Has a fleshy appearance. Three histologic patterns have been described: Ewing sarcoma–like (the most common), Lymphoma-like , and S mall spindle cell .

188. Composed of small cells associated with osteoid production.

189. Lace-like osteoid is always present.

190. The cells have scanty amounts of cytoplasm. Nuclei are round to oval and the chromatin may be fine to coarse. Mitoses range from 3 to 5 /HPF.

191. FNAC: Giemsa stain showing malignant osteoblasts and lacy osteoid

192. FNAC: PAP

193. Differential diagnosis Malignant lymphoma Cells have larger nuclei, often with vesicular chromatin, irregular nuclear membranes, prominent nucleoli, a lack of cellular cohesion, and lymphoglandular bodies, CD45 positivity Metastatic small cell carcinoma Older age of presentation. Cytokeratins, synaptophysin and chromogranin will be positive

194. Metastatic neuroblastoma Presence of Homer Wright rosettes or pseudo rosettes Neuroblastomas are immunoreactive for synaptophysin and chromogranin but are virtually never immunoreactive for CD99 Mesenchymal chondrosarcoma H as hemangiopericytoma-like vessels and areas of differentiated cartilage,

195. The most difficult differential diagnosis is between small cell osteosarcoma and Ewing sarcoma/PNET. In the absence of mineralization, true osteoid of small cell osteosarcoma can be very difficult to differentiate from collagen or fibrin that can be seen in Ewing sarcoma/PNET.

196. Sclerosing PNET

197. Ewing's sarcoma/ PNET vs. Small cell osteosarcoma Ewing's sarcoma Small cell osteosarcoma Presence of Homer Wright rosettes and pseudo rosettes The t(11;22 ) ( q24;q12) translocation is diagnostic for Ewing sarcoma/ PNET Cellular spindling may be present CD 99 negativity

198. Synovial sarcoma Most prevalent in adolescents and young adults M:F ratio: 1.2 to 1.0 Presenting complaints: The most common presentation is that of a palpable, deep-seated swelling or mass associated with pain or tenderness Primary or secondary involvement of nerves may cause projected pain, numbness, and paraesthesia . T hese cases are incorrectly diagnosed initially as arthritis, synovitis , or bursitis.

199. T hey tend to arise in the vicinity of large joints, especially the knee region. They are intimately related to tendons, tendon sheaths, and bursal structures,

200. The tumor has a fleshy gray-tan appearance with focal hemorrhage.

201. T he tumor is composed of two morphologically different types of cells E pithelial cells , resembling those of carcinoma, Fibrosarcoma -like spindle cells . Depending on the relative prominence of the two cellular elements and the degree of differentiation, synovial sarcomas can be broadly classified into the B iphasic type, with distinct epithelial and spindle cell components Monophasic fibrous type M onophasic epithelial type (rare) P oorly differentiated ( round cell type)

202. Biphasic synovial sarcoma showing close apposition of epithelial structures with malignant spindle cells.

203. Epithelial component showing intraluminal eosinophilic secretions .

204. Fibrosarcoma -like area in a synovial sarcoma showing the alternating darkly staining and lightly staining regions, imparting a marbled appearance .

205. The spindle cells are arranged in distinct fascicles.

206. Calcification is a very common feature and is detected radiologically in 30% of cases

207. Mast cells (arrows) are yet another conspicuous feature of synovial sarcoma ; they show no particular distribution but are more numerous in the spindle cell than in the epithelial portions of the neoplasm.

208. Monophasic fibrous synovial sarcoma : spindle-shaped cells of uniform appearance with small amounts of indistinct cytoplasm and oval dark-staining nuclei, forming solid, compact sheets

209. Thick collagen bands separate malignant spindle cells in a monophasic synovial sarcoma. This pattern of hyalinization is characteristic of this tumor.

210. Predominantly epithelial-type synovial sarcoma : Composed of sheets of cohesive epithelioid cells with only small foci of spindle-cell differentiation.

211. Which of the following features excludes a monophasic synovial sarcoma from the differential diagnosis in a mediastinal needle biopsy of a spindle cell tumor? Focal CD99 expression Focal S100 expression Lack of keratin expression Marked nuclear pleomorphism Small round blue cell morphology

212. Poorly differentiated synovial sarcoma C an be considered as a form of tumor progression that can be superimposed on any of the other synovial sarcoma subtypes. It behaves more aggressively and metastasizes in a significantly higher percentage of cases

213. Large-cell or epithelioid pattern : variably sized rounded nuclei with prominent nucleoli and hemiangiopericytoma like vasculature

214. Small-cell pattern with nuclear features similar to other small round cell tumors

215. High-grade spindle-cell pattern : spindle-shaped cells with high-grade nuclear features and a high mitotic rate, often accompanied by necrosis

216. Immunohistochemistry Positive stains TLE1 : 80 - 90%, relatively specific and sensitive marker , Cytokeratins : CK7 , CK8, CK14, CK18,  CK19 EMA BCL2 : 79 - 100% CD99 CD56   CD57   Calretinin SYT : strong nuclear staining, may lack specificity Negative stains CD34 Desmin H- Caldesmon Myogenin   MyoD1   FLI1 WT1 SOX10

217. Vimentin CD99 BCL 2

218. Strong nuclear TLE I positivity

219. Cytogenetics t(X;18 )(p11;q11), is found in virtually all synovial sarcomas, regardless of subtype. This translocation involves the fusion of the SYT gene on chromosome 18 and either the SSX1 or SSX2 gene on the X chromosome Another consistent finding is up-regulation of genes involved with the Wnt signaling pathway, including TLE1 .

220. Ewing sarcoma PAS positive CD99+ Translocation EWSR1 Alveolar RMS Myogenin+ Desmin+ Translocation (FOX01A) Synovial sarcoma TLE I+, BCL 2+ Translocation (SYT) Small cell osteosarcoma Osteoid present Mesenchymal chondrosarcoma Cartilage present DSRCT CK+, Desmin+ Trans lo cation EWSR1-WT1

221. Classification of tumours based on histological pattern Diffuse round cell pattern Septate or lobulated pattern Alveolar/ pseudoalveolar pattern Round cells with rosettes Hemangiopericytomatous vascular pattern Round cell pattern with other components Ewing’s sarcoma Ewing’s sarcoma Alveolar RMS Flexner- Wintersteiner rosettes: PNET, retinoblastoma Poorly differentiated synovial sarcoma, Pseudo glandular: Poorly differentiated synovial sarcoma PNET Alveolar RMS PNET Homer- Wright rosettes: Neuroblastoma Mesenchymal chondrosarcoma Cartilage: Mesenchymal chondrosarcoma Embryonal RMS Lymphoblastic lymphoma

222. A mass on the left arm of a 14-year old girl has been biopsied. On IHC, the mass is desmin positive. Which of the following additional immunostains would be most helpful to establish the diagnosis? A. S100/HMB45 B. Vimentin C. S100/Synaptophysin D. Cytokeratin/Myogenin

223. NEED FOR IHC IN DIAGNOSIS In spite of a similar light microscopic morphology , SRCTs include pathologic entities from vastly different lineages, including:- Epithelial tumors , Mesenchymal tumors T umors with overlapping features, such as lymphoma and melanoma. Because of similar routine light microscopic features of these tumors, immunohistochemistry is often mandated for a definitive diagnosis.

224. Algorithmic approach to identification of SBRCTs

225. Secondary panel CD45+ : Lymphoma S100/HMB45+ : Melanoma All three negative CD99+ : EWS/PNET, Mesenchymal chondrosarcoma CD99- NE+ : Neuroblastoma NE - : small cell osteosarcoma

226. A mass on the left arm of a 14-year old girl has been biopsied. On IHC, the mass is desmin positive. Which of the following additional immunostains would be most helpful to establish the diagnosis? A. S100/HMB45 B. Vimentin C. S100/Synaptophysin D . Cytokeratin/Myogenin

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228. Thank you