Case 4 -

Pleuropulmonary Blastoma D. Ashley Hill, M.D. Mihaela Onciu M.D.

Case History: This five month old female presented acutely with fever, tachypnea and labored respirations. She was diagnosed with Influenza B and admitted to the hospital. Born at term with an uneventful neonatal course, she had no significant illnesses prior to admission, although her mother stated that her breathing had not been "normal" for some time. The family history was notable for two relatives with a history of childhood cancer including the patient's mother who had acute B-cell leukemia as a child and a maternal cousin who was being treated for neuroblastoma. A chest X-ray on admission showed hyperaeration of the left lung. A CT scan showed a markedly emphysematous left upper lobe with compressive atelectasis of the left lower lobe and mediastinal shift. There were multiple, fine septations in the hyperinflated lobe with no visible normal lung tissue. The radiographic differential diagnosis included congenital pulmonary airway malformation (CPAM) and congenital lobar emphysema. The patient recovered from her upper respiratory infection and returned approximately three weeks later for resection of the abnormal left upper lobe. At surgery, the left upper lobe showed massive overinflation with attenuation of the visceral pleura. The upper lobe was dissected off the lower lobe with ligation of upper lobe branches of the pulmonary vascular structures and the left upper lobe bronchus. No other cystic abnormalities involving the pleura, mediastinum or left lower lobe were noted. Gross Examination: The left upper lobe measured 11.0 x 7.0 x 1.3 cm. Sections showed a 6.5 cm diameter cystic lesion in the apex covered by smooth visceral pleura. Multiple incomplete fibrous septae with mural thicknesses of 0.2 cm or less and collapsed air-filled cysts ranging from 1.0 to 2.0 cm in diameter were noted. No nodular thickenings or solid areas were seen. The uninvolved parenchyma showed atelectasis and consolidation. Microscopic Examination: Sections showed pleural-based cysts whose walls were variably cellular and lined by a bland low-cuboidal type epithelium. Focal condensations of primitive stellate, round and spindle-shaped cells were noted beneath the cyst lining cells imparting a cambium layer-like appearance. The primitive cells showed nuclear hyperchromatism and high nuclear-to-cytoplasmic ratios and were mitotically active. Loose fibrovascular tissue containing spindle-shaped cells resembling fibroblasts predominated in the deeper portions of the septae. Small nodules of primitive cartilage were also seen focally in the cyst walls. The multicystic lesion did not involve the margins of excision. Diagnosis: Pleuropumonary blastoma, Type I Clinical Course: Following the pathologic diagnosis, further clinical evaluation included a full body CT scan and head MR. There was no evidence of metastatic disease. However, a hypodense lesion in the lower pole of the left kidney and jejunal "polyps" were seen. A subsequent renal ultrasound was performed and the interpretation was a simple renal cyst. The patient was then followed with serial chest X-rays and CTs. At 4 months post-surgery a CT scan identified three parenchymal cysts in the remaining left lung ranging from 0.24 to 0.62 cm in diameter, which in retrospect were there 1 month post-surgery. Two cysts in the left kidney were also noted. At 6 months post-surgery CT scans showed 4 distinct cystic lesions in the left lung ranging from 0.27 to 2.1 cm in diameter, and two distinct cystic lesions in the right lung, measuring 0.24 and 0.57 cm in diameter. The left kidney contained three distinct cysts, each measuring 1.9 cm in maximum dimension. In addition, small bowel polyps were also seen. A left partial nephrectomy and wedge resections of the left lower lobe lung cysts were performed. Gross examination of the kidney specimen showed a subcapsular multilocular cyst measuring 1 cm. Microscopically this lesion was a multilocular cyst lined by bland, flattened cuboidal epithelium. The cyst walls contained loosely arranged primitive mesenchymal cells with a stellate configuration in a myxoid background. No cambium layer was seen. Within the parenchyma adjacent to the cysts, rare abortive glomeruli and dilated, irregular tubules were seen similar to those seen in embryologic renal dysplasia. No blastema or cartilage was present. The wedge biopsies of lung showed subpleural, thin-walled cysts, ranging from 1.2 and 1.5 cm in greatest dimension. Microscopically the cysts resembled those in the left upper lobe specimen. There were subepithelial condensations of spindle cells showing rare mitotic activity. No cartilage was present. Focally, the subepithelial spindle cells contained abundant pink cytoplasm suggestive of rhabdomyoblastic differentiation. These cells were desmin positive. The patient was started on Vincristine, Actinomycin D, Cyclophosphamide alternating with Carboplatin, Etoposide and Ifosfamide following her second surgery. She is currently well 21 months after her original diagnosis. She has persistent but stable right lung cysts that are being followed radiographically. Final Diagnoses: Lung, left upper and lower lobes, excisions - Pleuropulmonary blastoma, Type I, multifocal Kidney, left, partial nephrectomy - Cystic nephroma of childhood Discussion Pleuropulmonary blastoma is a rare pediatric lung cancer that almost exclusively affects children less than 5 years of age. PPB was initially described as a unique entity in 1988 by L.P. Dehner and colleagues. Approximately 15 to 20 children in the United States and 30 to 40 children worldwide are diagnosed with PPB annually. Despite its rarity, the tumor has been extensively studied and is well characterized from a clinical and pathologic perspective. I have included it for discussion here because of its interesting relationship with the other tumors described above. Prior to the introduction of the PPB as a distinct entity, this tumor had been reported in the earlier literature as pulmonary blastoma, sarcoma arising in mesenchymal cystic hamartoma, embryonal sarcoma, malignant mesenchymoma, primary pulmonary rhabdomyosarcoma and rhabdomyosarcoma arising in congenital adenomatoid malformation or bronchogenic cyst. In today's literature there is still some confusion about the distinction between the early stage PPB and cystic adenomatoid malformation type IV. The establishment of the Pleuropulmonary Blastoma Registry by Jack Priest, M.D. and colleagues has improved our understanding of this unique developmental neoplasm. The registry website serves as an important resource for both physicians and families (http://www.ppbregistry.org). Pathologic Types One of the important pathologic features of the PPB is that this neoplasm manifests itself as one of three morphologic subtypes which correlate with the age at diagnosis and outcome (Table 4.1). In its earliest form, PPB is a multilocular cyst composed of mesenchymal tumor cells beneath an intact, benign epithelium (Type I PPB). In later stages the mesenchymal components overgrow the epithelial cysts into an overtly malignant cystic and solid (Type II) or purely solid neoplasm (Type III). The pathologic categories of PPB appear to be related along a spectrum showing increased biologic aggressiveness as the neoplasm progressively acquires solid features. It is interesting to note that the median ages of the three types show PPB type I occurring in the youngest group and type III occurring in the oldest group with type II tumors in the intermediate group. The clinical progression of a type I to a type II or type III PPB within a given patient has been well documented in those cases in which a previously diagnosed "congenital lung cyst" or "congenital cystic adenomatoid malformation" was followed a year or two later by a type II or III PPB. Wright has documented the progression of a type I PPB, originally interpreted as a cystic hamartoma which recurred as a solid type III PPB. These types of cases have caused some authors to caution against non-operative management of lung cysts in young children. Table 4.2 highlights the progression from "benign cystic disease" to PPB in 21 Registry cases. Type I PPB (27% of all PPB) occurs on average in a younger age group compared to the other types (median age 9 months; range 0 to 37 months). These patients may present with dyspnea, pneumothorax or compression of intrathoracic structures, with or without mediastinal shift, by large hyperinflated cyst(s). Type I PPB is a potentially deceptive lesion as it is composed of benign-appearing, peripherally-located, thin-walled cyst(s) often submitted to the pathology laboratory as a "congenital lung cyst." There are no grossly observable solid, nodular or plaque-like thickenings in this collapsed multicystic structure. The neoplastic component of the type I PPB is only apparent microscopically with the identification of primitive tumor cells beneath a benign surface epithelium. Classically the tumor cells are arranged similar to a cambium layer of botryoid rhabdomyosarcoma (several layers of primitive, mitotically active tumor cells beneath an intact epithelium). The subepithelial tumor cells contain small, rounded or spindled hyperchromatic nuclei. Elongated strap cells with abundant eosinophilic cytoplasm or large rounded cells with similar appearing cytoplasm like those seen in an embryonal rhabdomyosarcoma can be seen in some but not all cases. Skeletal muscle differentiation can also be demonstrated by immunohistochemical stains for desmin, muscle-specific actin, myogenin or Myo-D1. These stains will highlight both the cells with cytological attributes of rhabdomyoblasts as well as in scattered primitive subepithelial cells. Some tumors also have nodules of immature cartilage in the cyst walls. The present case illustrates one of the challenges in the diagnosing a type I PPB. In our experience many type I PPB do not have an extensive subepithelial zone of tumor cells but rather are characterized by widely scattered and subtle collections of primitive small cells in a subepithelial location within an otherwise bland, hypocellular septal stroma. These diminutive subepithelial buds of primitive small cells and/or nodules of immature cartilage may or may not be accompanied by more compelling foci of dense subepithelial or septal mixed round and spindle cells. Extensive sampling of the cyst walls in these cases is essential. The primitive small and/or spindle cells in these examples may show immunoperoxidase staining for one or another of the muscle markers, however, the absence of myogenic immunophenotype does not preclude the diagnosis of Type I PPB in our experience. Based on a recent unpublished review of 41 Type I PPBs we have observed that early Type I PPBs in neonates have a much more uniform distribution of tumor cells. In these examples, de novo regressive changes such as cyst wall necrosis are common. We hypothesize that these regressive features may be responsible for the variability of tumor burden seen in later stages of Type I disease. The factors that control the balance between continued proliferation (and disease progression) and regression are poorly understood but may be important in predicting which Type I tumors will require adjuvant chemotherapy. Type II PPB occurs in somewhat older children (median 31 months, range 6 to 64) and represents 41% of all PPB. A cystic pattern similar to that seen in the type I PPB is identified either grossly (when this component is substantial) or microscopically with remnants of septa with cambium layers in an otherwise predominantly solid neoplasm. When cystic areas are noted in the gross examination, they are generally characterized as thickened or plaque-like septae. Microscopically the type II PPB is composed of an overgrowth of rhabdomyosarcomatous, spindle cell sarcoma or blastematous elements forming solid confluent areas of tumor with varying amounts of residual septal cystic tissue remaining. Anaplastic tumor cells can be seen in the solid components of type II tumors. Type III PPB accounts for the other 32% of cases and occurs in children with a median age of 42 months (range 1 to 147 months). These patients present with fever, cough, chest and abdominal pain, dyspnea and weight loss. Radiographically, these tumors are typically heterogeneous solid masses with or without involvement of chest wall or mediastinal structures. The entire hemi-thorax may be opacified by the mass. Grossly, a well-circumscribed, solid, mucoid, tan-white and friable mass with pleural attachments involving a lobe or entire lung is seen. Hemorrhage and necrosis account in part for the friability of the tumor. If the tumor has extended into the surrounding pleural space, the resection specimen may be submitted in a piecemeal fashion. Microscopically the type III PPB (and solid areas of the type II PPB) shows one or more of four basic histologic patterns which may blend into each other: (1) cohesive aggregates of primitive small cells with hyperchromatic nuclei, high nuclear-to-cytoplasmic ratio and brisk mitoses resembling the blastema of a Wilms; (2) spindled, stellate and small ovoid cells in a variably prominent myxoid stroma resembling rhabdomyosarcoma; (3) spindle cell sarcoma resembling synovial sarcoma or congenital-infantile fibrosarcoma; (4) nodules of immature or overtly malignant cartilage. Individual or groupings of large pleomorphic cells with atypical mitotic figures are present in many cases. Eosinophilic hyaline bodies are often seen in association of anaplastic cells. Within any one tumor, not all histologic patterns are equally represented and one or two patterns may dominate the overall microscopic appearance. Unlike the classic pulmonary blastoma, the PPB does not have a malignant epithelial component. Special Studies Immunohistochemistry and genetic analysis are generally unnecessary to establish the diagnosis of PPB in the majority of the cases. PPB commonly shows staining for desmin and myogenin in areas of obvious rhabdomyosarcomatous differentiation. S100 protein is typically limited to the cartilaginous nodules. When subepithelial foci of primitive cells are identified in a suspected type I PPB, these cells are inconsistently immunoreactive for desmin and/or myogenin in the absence of apparent rhabdomyoblasts. These primitive cells like the other septal stromal cells are positive for vimentin. Cytokeratin highlights the flattened to cuboidal epithelial cells but is negative in the tumor cells. Fluorescence in situ hybridization (FISH) can identify trisomy 8, a common abnormality in PPBs. Trisomy 8 is not specific to the PPB since it has been observed in embryonal rhabdomyosarcoma, granulocytic sarcoma, Ewing sarcoma and congenital-infantile fibrosarcoma. Trisomy 2, and p53 mutations/deletions in a subset of cases have also been described. Differential Diagnosis Because there is significant variability in the morphologic appearance of PPB, the differential diagnosis is broad. For type I PPBs, the distinction from a multilocular pulmonary cyst in the category of congenital pulmonary airway malformations (CPAM) type 1 or 4 is critical to the appropriate management of the patient. Both CPAM and type I PPB both occur in infants and young children and can either be discovered incidentally or present with respiratory distress. The index of suspicion for PPB must be extremely high when encountering a collapsed multilocular cyst from the lung of a young child. We advocate the approach that all lung multilocular cysts in infants and children less than 2 years old are type I PPB until proven otherwise. Proving otherwise is best accomplished by submission of the entire gross specimen for histologic examination (or a substantial portion of it). Many examples of type I PPB have bland histologic features. However, if one notes the presence of dense subepithelial or septal spindle cells with or without nodules of immature cartilage, a diagnosis of type 1 PPB is appropriate. Ancillary studies to differentiate benign cysts from type I PPB are only helpful if they are positive, since the absence of muscle differentiation in a primitive cell component does not preclude the pathologic diagnosis of type I PPB. The utility of FISH in detecting the characteristic trisomy 8 in type I PPB with few neoplastic cells is uncertain. Type II tumors should be distinguished from type I tumors by the presence of a solid component represented by grossly observable thickened, nodular or plaque-like areas containing sarcomatous or blastematous elements or microscopic evidence of a confluent overgrowth of overtly malignant elements widely expanding the septae. Anaplastic cells are typically limited to solid components of type II and III PPB and have only rarely been seen in Type I tumors. The importance of this distinction between type I and type II tumors is the difference in prognosis and treatment regimen. In type II and type III PPB, there is generally little question about the pathologic process being benign or malignant. Depending on the predominance of one or more sarcomatous or blastemal elements, the differential diagnosis includes primary and metastatic rhabdomyosarcoma, malignant teratoma, synovial sarcoma, congenital-infantile fibrosarcoma and other spindle cell or undifferentiated sarcomas. In solid tumors with a predominant blastemal pattern, metastatic Wilms tumor may be considered. Cytokeratin immunohistochemistry (PPB is negative for cytokeratin) and renal ultrasound would be helpful in making this latter distinction. Biological Behavior, Treatment and Outcome Type II and type III PPB are bulky tumors that can spread through direct extension into adjacent uninvolved lung, pleura, diaphragm, chest wall and mediastinal structures or by hematogenous dissemination. The most common sites for distant metastases are the brain (26/172, 15%), bone (10/172, 6%), liver (7/172, 4%) and less commonly adrenal, eye, spinal cord, ovary and pancreas. Because of the predilection for brain metastasis, a head MR is recommended as part of the initial staging evaluation and routine follow-up. Treatment for PPB is based on a combined, multimodality approach similar to other pediatric sarcoma regimens. Additional details and dose regimens is given on the PPB Registry website (http://www.ppbregistry.org). Type I PPB is treated with complete surgical removal and adjuvant chemotherapy. With chemotherapy, the 5-year overall survival for this subgroup is over 90%. There continues to be some debate regarding the need for adjuvant therapy in type I PPB. A recent analysis of Registry patients has shown that there is a statistically significant difference in recurrence free survival in patients who receive adjuvant chemotherapy (p=0.01). When patients with Type I PPB have a recurrence, it has a Type II or Type III pattern. The salvage rate for recurrent PPB is poor. Only 33% of patients with recurrence following Type I PPB survive. The alternative to chemotherapy is a rigorous radiographic follow-up schedule. Hopefully future studies of Type I PPB will allow for more accurate prediction of who is most at risk for recurrence. Despite the fact that children with Types II and III PPB are subject to an aggressive chemotherapy regimen, the 5-year overall survival rates for Type II and Type III PPB are only 60% and 45%, respectively (PPB Registry unpublished data). The decision to give radiation therapy is individualized. PPB Family Cancer Syndrome One of the key observations in children with PPB was the recognition that PPB was a feature of a unique cancer predisposition syndrome. An estimated 25 to 35% of patients with PPB have multifocal disease and/or other rare benign or malignant neoplasms, dysplasias and/or hyperplasias in themselves or their close relatives. These entities include other PPBs, lung cysts, embryonal rhabdomyosarcoma, Wilms tumor and variants (cystic nephroma, cystic partially differentiated nephroblastoma, nephroblastomatosis), medulloblastoma, gonadal germ cell and stromal tumors, primitive retinal tumor, thyroid cancers and nodular hyperplasia, juvenile intestinal polyps, sarcomas and hematopoietic malignancies. This observation is remarkable not only for the rarity of these neoplasms in the general population, but also for these entities having a common association with organ development. Key Points PPB is divided into three clinicopathologic types that represent a continuum of histologic and biologic progression Type I PPB is a neoplasm whose diagnostic features can be subtle and tumor cells may be focally present; extensive sampling of the cyst walls is critical Type II PPB can be distinguished from Type I PPB by the presence of grossly identifiable thickened plaque-like, nodular or solid areas containing rhabdomyosarcomatous, blastematous or spindle cell sarcomatous elements or by microscopically evident overgrowth of malignant elements in septa Type II and III PPB have a predilection for metastasizing to brain PPB appears to be part of a familial cancer syndrome which can manifest as multifocal PPB or in association with other neoplasms, dysplasia and/or hyperplasias in patients or their close relatives Association with cystic nephroma is common Table 4.1 Age at Presentation for the Three Clinicopathologic Subtypes of PPB PPB Type Median Age at Diagnosis Survival I 9 months 90% II 31 months 60% III 42 months 45% Overall Survival estimates by Kaplan-Meier at 5 years post-diagnosis Age at presentation data courtesy of Jack Priest M.D., PPB Registry http://www.ppbregistry.org Table 4.2: Progression of "benign cystic disease" to PPB in 21 Registry cases DOD = dead of disease, NED = no evidence of disease; Data courtesy of Jack Priest, M.D., PPB Registry, 5/2003 Recommended Reading: Manivel JC, Priest JR, Watterson J et al. Pleuropulmonary blastoma. The so-called pulmonary blastoma of childhood. Cancer 1988;62:1516-26. Dehner LP. Pleuropulmonary blastoma is THE pulmonary blastoma of childhood. Semin Diagn Pathol 1994;11:144-51. Priest JR, McDermott MB, Bhatia S, Watterson J, Manivel JC, Dehner LP. Pleuropulmonary blastoma: a clinicopathologic study of 50 cases. Cancer 1997;80:147-61. Wright JR, Jr. Pleuropulmonary blastoma: A case report documenting transition from type I (cystic) to type III (solid). Cancer 2000;88:2853-8. Priest JR, Watterson J, Strong L et al. Pleuropulmonary blastoma: a marker for familial disease. J Pediatr 1996;128:220-4. Delahunt B, Thomson KJ, Ferguson AF, Neale TJ, Meffan PJ, Nacey JN. Familial cystic nephroma and pleuropulmonary blastoma. Cancer 1993;71:1338-42. Eble JN, Bonsib SM. Extensively cystic renal neoplasms: cystic nephroma, cystic partially differentiated nephroblastoma, multilocular cystic renal cell carcinoma, and cystic hamartoma of renal pelvis. Semin Diagn Pathol 1998;15:2-20. Joshi VV, Beckwith JB. Multilocular cyst of the kidney (cystic nephroma) and cystic, partially differentiated nephroblastoma. Terminology and criteria for diagnosis. Cancer 1989;64:466-79. Kusafuka T, Kuroda S, Inoue M et al. P53 gene mutations in pleuropulmonary blastomas. Pediatr Hematol Oncol 2002;19:117-28. Novak R, Dasu S, Agamanolis D, Herold W, Malone J, Waterson J. Trisomy 8 is a characteristic finding in pleuropulmonary blastoma. Pediatr Pathol Lab Med 1997;17:99-103. Sebire NJ, Rampling D, Malone M, Ramsay A, Sheppard M. Gains of chromosome 8 in pleuropulmonary blastomas of childhood. Pediatr Dev Pathol 2002;5:221-2. Tagge EP, Mulvihill D, Chandler JC, Richardson M, Uflacker R, Othersen HD. Childhood pleuropulmonary blastoma: caution against nonoperative management of congenital lung cysts. J Pediatr Surg 1996;31:187-9. Yang P, Hasegawa T, Hirose T et al. Pleuropulmonary blastoma: fluorescence in situ hybridization analysis indicating trisomy 2. Am J Surg Pathol 1997;21:854-9. Priest JR, Magnuson J, Williams GM, Abromowitch M, Byrd R, Sprinz P, Finkelstein M, Moertel CL, Hill DA. Cerebral metastasis and other central nervous system complications of pleuropulmonary blastoma. Pediatr Blood Cancer 2006 [E pub ahead of print] Priest JR, Hill DA, Williams GM, Moertel CL, Messinger Y, Finkelstein MJ, Dehner LP. Type I Pleuropulmonary Blastoma: A Report from The International Pleuropulmonary Blastoma Registry. J Clin Oncol. 2006 Sep 20;24(27):4492-8 Boman F, Hill DAc, Williams GM, Chauvenet A, Fournet JC, Bouron-Dal Soglio D, Messinger Y, Priest JR. Familial Association of Pleuropulmonary Blastoma with Cystic Nephroma and Other Renal Tumors: A Report from the International Pleuropulmonary Blastoma Registry. J Pediatr Dec 2006 HEMATOPOIETIC NEOPLASMS (NON-HODGKIN LYMPHOMA) Hematopoietic (CD45-Positive) Tumors Encountered/Reported in Childhood (* Neoplasms most commonly encountered) Lymphoid Acute lymphoblastic leukemia/ lymphoblastic lymphoma* Non-Hodgkin lymphoma ALCL (ALK positive)* Burkitt lymphoma* Diffuse large B-cell lymphoma (including primary mediastinal variant)* Peripheral T-cell lymphomas Low-grade B-cell lymphoma Follicular lymphoma Marginal zone lymphoma Chronic lymphocytic leukemia Hodgkin lymphoma* Classical type Nodular sclerosis Mixed cellularity Nodular lymphocyte predominance Myeloid Acute myeloid leukemias/ myeloid sarcomas* Chronic myeloproliferative disorders Chronic myeloid leukemia Myeloproliferative/myelodysplastic syndromes Juvenile myelomonocytic leukemia* Myelodysplastic syndromes (association with myeloid sarcoma) Histiocytic Monocyte/macrophage lineage Acute monoblastic/monocytic/myelomonocytic leukemia/ monoblastic sarcoma* Histiocytic sarcoma Rosai-Dorfman disease (nodal/extranodal)* Solitary histiocytoma of macrophage lineage Dendritic cell lineage Langerhans cell histiocytosis* Langerhans cell sarcoma Juvenile xanthogranuloma (dermal dendritic cell origin)* Dendritic reticulum cell tumor/sarcoma Follicular dendritic cell (FDRC) (nodal/extranodal) Interdigitating reticulum cell (IDRC) Immunophenotypic Algorythm for the Differential Diagnosis of the Most Common Pediatric Non-Hodgkin Lymphomas Materials Required for the Diagnostic Work-up of a Hematopoietic Neoplasm Formalin-fixed or B5-fixed tissue - may be used for : Morphologic examination (tumor cell cytology, tumor growth pattern) Immunohistochemical staining (for most antigens critical for a diagnostic work-up). In situ hybridization (including light microscopy and interphase FISH) This test cannot be performed in tissue that has been decalcified using acid or fixed in B5 fixative. Molecular testing (nucleic acid yield considerably reduced by B5 fixation) - the recovery rate for nucleic acids depends of length of fixation in formalin (one day of fixation is ideal). DNA-based (PCR, Southern blotting) RNA-based (RT-PCR) - 60-70% success in good laboratories Fresh tissue (hold in sterile saline, RPMI or under saline-soaked gauze) - may be used for: Flow cytometry Conventional cytogenetics Metaphase FISH Molecular analysis (fresh or snap frozen) DNA-based (PCR, Southern blotting). RNA-based (RT-PCR) - ideal sample is fresh or frozen 3. Touch imprints, smears or cytospins (air dried): a. Morphologic examination (tumor cell cytology) b. Interphase FISH (requires recently prepared smears or smears maintained in freezer) c. Immunohistochemical examination (cold-acetone fixed smears or cytospins prefered) d. Molecular analysis i. DNA based - PCR (limited number of laboratories) The minimum information required to diagnose and classify any lymphoma includes morphologic and immunophenotypic data. For the diagnosis of some neoplasms (e.g. posttransplant lymphoproliferative disorders, T/NK lymphoma of nasal type) documentation of EBV positivity in the tumor cells is critical for diagnosis. This can be most reliably examined used an in situ hybridization assay for EBER. Molecular analysis for immunoglobulin or T-cell receptor genes is less commonly required in the pediatric setting, where most lymphomas are of high-grade and therefore easy to diagnose by morphologic examination. However, a small subset of low-grade lymphomas may require such analysis. Lastly, demonstration of the t(8;14)(q24;q32) chromosomal translocation or of one of its variants is highly desirable in Burkitt lymphoma (although the diagnosis can and is often made without that information). Therefore, if only a small amount of tumor tissue is available, formalin fixation followed by paraffin-embedding will provide the broadest coverage for all these needs. If plenty of tissue is available and a preliminary touch imprint raises the suspicion for lymphoma, portions of the material should be fixed in formalin, snap frozen and stored for any possible molecular analysis, and submitted fresh to the cytogenetics and flow cytometry laboratories (depending on the facilities available in each institution). If there is no clear-cut lymphoma in the preliminary assessment, material should be sent to Microbiology as well. The tissue should be handled under sterile conditions until the triage is completed (at a minimum, samples for Microbiology and Cytogenetics require sterile handling).