Pediatric Fibromatosis Lacks the Internal Tandem Duplication of EGFR Seen in Congenital Mesoblastic Nephroma.

Classical and mixed congenital mesoblastic nephroma (CMN) are characterized by an internal tandem duplication (ITD) of the EGFR gene, in contrast to cellular CMN that usually harbors an ETV6::NTRK3 gene fusion. This same fusion occurs in infantile fibrosarcoma, and this tumor can be considered as the soft tissue equivalent of cellular CMN. A soft tissue equivalent of classic/mixed CMN remains undefined at the genetic level. Since classical CMN resembles fibromatosis of soft tissue histologically, we asked whether fibromatosis in children might show EGFR ITD. ITD was investigated using the polymerase chain reaction and primers for exons 18 and 25 of the EGFR gene. Seven of the eight cases of classical or mixed CMN were positive by this approach, but none of the five cellular CMNs. Of 11 cases of fibromatosis (six plantar, two digital, and three desmoid), none were positive for EGFR ITD. Within the limits of this small study, we conclude that pediatric fibromatosis is likely not characterized by EGFR ITD. There are isolated reports of pediatric soft tissue tumors that harbor EGFR ITD, but these have the appearance of infantile fibrosarcoma or mixed CMN rather than fibromatosis. We did not find any such cases, since all 14 cases of infantile fibrosarcoma in our study had an ETV6::NTRK3 fusion. The soft tissue tumors with EGFR ITD are not a morphologic match for the low-grade histology of classical CMN. Whether they have a similar favorable biology or behave more like fibrosarcoma with an ETV6::NTRK3 fusion or an alternative fusion involving other kinases remains to be determined.

Exomic and epigenomic analysis of pulmonary blastoma.

OBJECTIVE: Pulmonary blastoma is a rare, biphasic, adult-onset lung tumor. In this study, we investigate whether DICER1 pathogenic variants are a feature of pulmonary blastomas through in-depth analysis of the molecular events defining them. METHODS: We performed exome-wide sequencing and DNA methylation profiling of 8 pulmonary blastomas from 6 affected persons. RESULTS: We identified biallelic somatic DICER1 pathogenic variants in 7 of 8 cases. The remaining case had a solitary missense pathogenic variant in the RNase IIIb domain of DICER1. Six of 8 cases carried a CTNNB1 hotspot variant and 4 of 8 had a somatic pathogenic variant in TP53. Methylation analysis showed that the pulmonary blastomas clustered with other DICER1-mutated tumors and not with other more common types of lung cancer. CONCLUSION: We conclude somatic DICER1 pathogenic variants are the major driver of pulmonary blastoma and are likely to act in conjunction with CTNNB1 hotspot variants that are often present.

Estrogen Receptor Expression in DICER1-related Lesions is Associated With the Presence of Cystic Components.

DICER1 tumor predisposition syndrome results from pathogenic variants in DICER1 and is associated with a variety of benign and malignant lesions, typically involving kidney, lung, and female reproductive system. Over 70% of sarcomas in DICER1 tumor predisposition syndrome occur in females. Notably, pediatric cystic nephroma (pCN), a classic DICER1 tumor predisposition syndrome lesion, shows estrogen receptor (ER) expression in stromal cells. There are also renal, hepatic, and pancreatic lesions unassociated with DICER1 tumor predisposition syndrome that have an adult female predominance and are characterized/defined by ER-positive stromal cells. Except for pCN, the expression of ER in DICER1-associated lesions remains uninvestigated. In the present study, ER expression was assessed by immunohistochemistry in 89 cases of DICER1-related lesions and 44 lesions lacking DICER1 pathogenic variants. Expression was seen in stromal cells in pCN and pleuropulmonary blastoma (PPB) types I and Ir, whereas anaplastic sarcoma of kidney and PPB types II and III were typically negative, as were other solid tumors of non-Müllerian origin. ER expression was unrelated to the sex or age of the patient. Expression of ER showed an inverse relationship to preferentially expressed antigen in melanoma (PRAME) expression; as lesions progressed from cystic to solid (pCN/anaplastic sarcoma of kidney, and PPB types I to III), ER expression was lost and (PRAME) expression increased. Thus, in DICER1 tumor predisposition syndrome, there is no evidence that non-Müllerian tumors are hormonally driven and antiestrogen therapy is not predicted to be beneficial. Lesions not associated with DICER1 pathogenic variants also showed ER-positive stromal cells, including cystic pulmonary airway malformations, cystic renal dysplasia, and simple renal cysts in adult kidneys. ER expression in stromal cells is not a feature of DICER1 perturbation but rather is related to the presence of cystic components.

Detection of EGFR T790M mutation using liquid biopsy for non-small cell lung cancer: Utility of droplet digital polymerase chain reaction vs. cobas real-time polymerase chain reaction.

BACKGROUND: Digital platforms for mutation detection yield higher sensitivity than non-digital platforms but lack universal positive cut-off values that correlate with the outcome of osimertinib treatment. This study determined compared droplet digital polymerase chain reaction (ddPCR) to the standard cobas assay for epithelial growth factor receptor (EGFR) T790M mutation detection in patients with non-small cell lung cancer. METHODS: Study patients had EGFR-mutant tumours with disease progression on first/second generation EGFR tyrosine kinase inhibitors, and osimertinib treatment after T790M mutation detection. T790M status was tested by cobas assay using liquid biopsy, and only by ddPCR if an EGFR mutation was identified but T790M was negative. Clinical efficacy of osimertinib was compared between patients with T790M detected by cobas vs. only by ddPCR. A positive cut-off value for ddPCR was determined by assessing efficacy with osimertinib. RESULTS: 61 patients had tumors with an acquired T790M mutation, 38 detected by cobas and an additional 23 only by ddPCR. The median progression-free survival (PFS) for the cobas- and ddPCR-positive groups was 9.5 and 7.8 months, respectively (p=0.43). For ddPCR, a fractional abundance (FA) of 0.1% was used as a cut-off value. The median PFS of patients with FA ≥0.1% and <0.1% was 8.3 and 4.6 months, respectively (p=0.08). FA ≥0.1% was independently associated with a longer PFS. CONCLUSION: Using ddPCR to follow up the cobas assay yielded more cases (38% of total) with a T790M mutation. A cut-off value of FA ≥0.1% identified patients who responded as well to osimertinib as those identified by cobas assay. This sequential approach should detect additional patients who might benefit from osimertinib treatment.

Fine needle aspiration of spindle cell lipoma-Lochkern cells as a clue for diagnosis: A case arising in the parotid gland.

Spindle cell lipoma (SCL) is a rare form of lipoma, typically occurring as a mass in the back, shoulder or posterior neck of adult males. Most cases present little diagnostic difficulty on fine needle aspiration (FNA), but can be problematic when the SCL is in an unusual location. The authors report a case in the parotid gland in a 75-year-old man. FNA was paucicellular and showed loose collections of spindle cells with mild to moderate atypia, admixed with ropy collagen fibers on a myxoid background. The nuclei showed occasional angulation, interpreted on FNA as suspicious for myoepithelial tumor or low-grade sarcoma. The subsequent excisional specimen was diagnosed as SCL. On retrospective review of the FNA, an additional finding was recognized: 'naked' nuclei with intranuclear lipid vacuoles and positive immunostaining for S100 protein, consistent with Lochkern cells of mature adipocytes. This case highlights the challenges of diagnosing SCL on cytology when no fat-containing cells are apparent on the smear, and stresses the significance of Lochkern cells as a clue for diagnosis.

Acantholytic squamous cell carcinoma mimicking epithelioid angiosarcoma: A diagnostic challenge by cytology.

Squamous cell carcinoma (SCC) is the most common malignancy of the head and neck region. Most cases present little diagnostic difficulty on fine needle aspiration (FNA), but unusual variants can be problematic. The authors report a case of the acantholytic SCC of the oral cavity in a 36-year-old male. The FNA showed hypercellularity, with malignant cells arranged in isolation, loosely cohesive groups and a linear configuration. Such cells were round to elongated, with vesicular nuclei and prominent nucleoli. Cells possessed occasional intracytoplasmic vacuoles, misinterpreted on FNA to be vasoformative features as seen in malignant endothelial cells. The cytologic diagnosis was "positive for malignancy, suggestive of angiosarcoma". A total excision was performed and by histology, the tumor was diagnosed as acantholytic SCC. The malignant cells were positive by immunostaining for AE1/AE3, p40, p63 and vimentin, but negative for CD31, CD34 and ERG. The intracytoplasmic vacuoles were PAS- and mucin-negative and negative for the above antibodies. Testing for HPV (molecular and p16 immunostaining) was negative. This case highlights the diagnostic challenges on cytology when malignant acantholytic squamous cells show intracytoplasmic vacuoles, and stresses how immunohistochemistry is important for distinguishing acantholytic SCC from other mimics.

Fetal Type Morphologies Suggest the Presence of DICER1 Hotspot Mutations in Non-small Cell Lung Cancer.

Germline and somatic pathogenic variants (PVs) in DICER1 , encoding a miRNA biogenesis protein, are associated with a wide variety of highly specific pathologic entities. The lung tumors pleuropulmonary blastoma, pulmonary blastoma (PB), and well-differentiated fetal lung adenocarcinoma (WDFLAC) are all known to harbor DICER1 biallelic variants (loss of function and/or somatic hotspot missense mutations), and all share pathologic features reminiscent of the immature lung. However, the role of DICER1 PVs in non-small cell lung cancer (NSCLC) is relatively unknown. Here, we aimed to establish the spectrum of lung pathologies associated with DICER1 hotspot PVs and to compare the mutational landscape of DICER1 -mutated NSCLC with and without hotspots. We queried DNA sequencing data from 12,146 NSCLCs featuring somatic DICER1 variants. 235 (1.9%) cases harboring ≥ 1 DICER1 PV were found and 9/235 (3.8%) were DICER1 hotspot-positive cases. Histologic review of DICER1 hotspot-positive cases showed that all but one tumor were classified as within the histologic spectrum of PB/WDFLAC, whereas all the DICER1 non-hotspot double variants were classified as lung adenocarcinomas, not otherwise specified. Comparison between the mutational landscape of DICER1 hotspot-positive and hotspot-negative cases revealed a higher frequency of CTNNB1 mutations in the hotspot-positive cases (5/9 vs. 2/225; P <0.00001). We conclude that DICER1 somatic hotspots are not implicated in the most common forms of NSCLC but rather select for morphologic features of lung tumor types such as PB and WDFLAC. As a corollary, cases showing this tumor morphology should undergo testing for DICER1 variants, and if positive, genetic counseling should be considered.

Glioblastoma with novel EGFR mutations (T790M and exon 20 insertion) yet unresponsive to osimertinib: A case report.

Glioblastoma (GBM) is a high-grade adult-type IDH-wildtype diffuse glioma, commonly harboring epidermal growth factor receptor (EGFR) amplification. Here, we describe a case of a 49-year-old man with a GBM harboring a TERT promoter mutation. Despite surgical and chemoradiation therapy, the tumor recurred. At that time, comprehensive genomic profiling by next-generation sequencing identified two rare mutations in EGFR: T790M and an exon 20 insertion. Based on these findings, the patient elected to undergo off-label therapy with osimertinib, a third-generation EGFR tyrosine kinase inhibitor that has shown promising results in non-small cell lung carcinoma, including metastatic to brain, with exactly the same EGFR mutations. Moreover, the drug has excellent central nervous system penetration. Even so, no clinical response was observed, and the patient succumbed to the disease. The lack of response may be related to the specific nature of the EGFR mutations, and/or other unfavorable tumor biology overriding any benefit from osimertinib.

Placenta-Derived Extracellular Vesicles in Pregnancy Complications and Prospects on a Liquid Biopsy for Hemoglobin Bart's Disease.

Extracellular vesicles (EVs) are nano-scaled vesicles released from all cell types into extracellular fluids and specifically contain signature molecules of the original cells and tissues, including the placenta. Placenta-derived EVs can be detected in maternal circulation at as early as six weeks of gestation, and their release can be triggered by the oxygen level and glucose concentration. Placental-associated complications such as preeclampsia, fetal growth restriction, and gestational diabetes have alterations in placenta-derived EVs in maternal plasma, and this can be used as a liquid biopsy for the diagnosis, prediction, and monitoring of such pregnancy complications. Alpha-thalassemia major ("homozygous alpha-thalassemia-1") or hemoglobin Bart's disease is the most severe form of thalassemia disease, and this condition is lethal for the fetus. Women with Bart's hydrops fetalis demonstrate signs of placental hypoxia and placentomegaly, thereby placenta-derived EVs provide an opportunity for a non-invasive liquid biopsy of this lethal condition. In this article, we introduced clinical features and current diagnostic markers of Bart's hydrops fetalis, extensively summarize the characteristics and biology of placenta-derived EVs, and discuss the challenges and opportunities of placenta-derived EVs as part of diagnostic tests for placental complications focusing on Bart's hydrop fetalis.

Paediatric BCOR-associated sarcomas with a novel long spliced internal tandem duplication of BCOR exon 15.

Clear cell sarcoma of the kidney (CCSK) and primitive myxoid mesenchymal tumour of infancy (PMMTI) are paediatric sarcomas that most commonly harbour internal tandem duplications (ITDs) of exon 15 of the BCOR gene, in the range of 87-114 base pairs (bp). Some cases, instead, have BCOR-CCNB3 or YWHAE-NUTM2 gene fusions. About 10% of cases lack any of these genetic alterations when tested by standard methods. Two cases of CCSK and one PMMTI lacking the aforementioned mutations were analysed using Archer FusionPlex technology. Two related BCOR exon 15 RNA transcripts with ITDs of lengths 388 and 96 bp were detected in each case; only the 388 bp transcript was identified when genomic DNA was sequenced. In silico analysis of this transcript revealed acceptor and donor splice sites indicating that, at the RNA level, the 388-bp transcript was likely spliced to form the 96-bp transcript. The results were confirmed by Sanger sequencing using primers targeting the ITD breakpoint. This novel and unusually long ITD segment is difficult to identify by DNA sequencing using typical primer design strategies flanking entire duplicated segments because it exceeds the typical read lengths of most sequencing platforms as well as the usual fragment lengths obtained from formalin-fixed paraffin-embedded material. As diagnosis of CCSK and PMMTI may be challenging by morphology and immunohistochemistry alone, it is important to identify mutations in these cases. Knowledge of this novel BCOR ITD is important in relation to primer design for detection by sequencing, and using RNA versus DNA for sequencing.

Pediatric fibromyxoid tumor with PLAG1 fusion: An emerging entity with a novel intracranial location.

Translocations involving PLAG1 occur in several tumors, most commonly pleomorphic adenoma and lipoblastoma. Recently, a distinctive soft tissue tumor with a PLAG1 fusion has been reported in the pediatric age group. These are low grade tumors with a fibroblastic or mixed fibroblastic and myxoid morphology but no other lines of differentiation. They are typically immunopositive for desmin and CD34. The partner genes for these tumors have included YWHAZ, EEF1A1, ZFHX4l, CHCHD7, and PCMTD1. We report another case of this fibromyxoid tumor with a PLAG1 fusion, this time with COL3A1 as the partner gene. The fusion placed expression of a full-length PLAG1 protein under the control of the constitutively active COL3A1 promoter. Overexpression of PLAG1 was confirmed by diffusely positive immunostaining for PLAG1. The most novel aspect of this tumor is the intracranial location. Opinion has been divided over whether these tumors are a specific entity, or related to lipoblastoma, since that tumor also typically occurs in soft tissue in the pediatric age group and shows many of the same gene fusions. However, lipoblastoma has never been reported in an intracranial location and, thus, our case provides compelling evidence that this fibromyxoid tumor is indeed a distinct entity.

PRAME protein expression in DICER1-related tumours.

DICER1 syndrome is an autosomal dominant tumour predisposition syndrome usually affecting persons under 30 years of age. Many of the associated benign and malignant lesions occur almost exclusively in DICER1 syndrome. One such tumour, pituitary blastoma (pitB), overexpresses PRAME 500x above control levels. PRAME (PReferentially expressed Antigen in MElanoma) is expressed in malignancies that are not DICER1-related (e.g. melanoma). To address whether PRAME expression is part of the DICER1 phenotype, or simply a feature of pitB, a series of 75 DICER1-mutated specimens and 33 non-mutated specimens was surveyed using immunohistochemistry for PRAME, together with EZH2, which complexes with PRAME. In DICER1-mutated specimens, positive staining for PRAME was only seen in malignant tumours; 7 of 11 histological types and 34/62 individual tumours were positive, while non-tumourous lesions were always negative. Pleuropulmonary blastoma (PPB) showed a continuum in staining, with type I lesions being PRAME negative (n = 7) but all type II and type III lesions PRAME positive (n = 7). Similarly, cystic nephroma (CN) was negative (n = 8), with anaplastic sarcoma of the kidney being positive (n = 2). However, one atypical CN with mesenchymal cell proliferation was PRAME-positive. Embryonal rhabdomyosarcoma (RMS) with DICER1 pathogenic variants (PVs) was positive for PRAME (5/6), but the same tumour type without DICER1 PVs was also positive (9/15). Staining for EZH2 corresponded to that seen with PRAME, validating the latter. This study leads us to conclude that (1) PRAME expression occurs in two-thirds of DICER1-related malignancies; (2) PRAME may be a marker for the progression that certain DICER1-related lesions are thought to undergo, such as PPB and CN; and (3) PRAME expression in some tumours, such as RMS, appears to be an intrinsic feature of the tumour, rather than specifically related to DICER1 PVs. Therapy directed against PRAME may offer novel treatment options in patients with the DICER1 syndrome.

Simplified approach for pathological diagnosis of diffuse gliomas in adult patients.

The most recent WHO classification (2016) for gliomas introduced integrated diagnoses requiring both phenotypic and genotypic data. This approach presents difficulties for countries with limited resources for laboratory testing. The present study describes a series of 118 adult Thai patients with diffuse gliomas, classified by the WHO 2016 classification. The purpose was to demonstrate how a diagnosis can still be achieved using a simplified approach that combines clinical, morphological, immunohistochemical, and fewer molecular assays than typically performed. This algorithm starts with tumor location (midline vs. non-midline) with diffuse midline glioma identified by H3 K27M immunostaining. All other tumors are placed into one of 6 categories, based on morphologic features rather than specific diagnoses. Molecular testing is limited to IDH1/IDH2 mutations, plus co-deletion of 1p/19q for cases with oligodendroglial features and TERT promoter mutation for cases without such features. Additional testing for co-deletion of 1p/19q, TERT promoter mutation and BRAF mutations are only used in selected cases to refine diagnosis and prognosis. With this approach, we were able to reach the integrated diagnosis in 117/118 cases, saving 50 % of the costs of a more inclusive testing panel. The demographic data and tumor subtypes were found to be similar to series from other regions of the world. To the best of our knowledge, this is to the first reported series of diffuse gliomas in South-East Asia categorized by the WHO 2016 classification system.

Molecular characterization of DICER1-mutated pituitary blastoma.

Pituitary blastoma (PitB) has recently been identified as a rare and potentially lethal pediatric intracranial tumor. All cases that have been studied molecularly possess at least one DICER1 pathogenic variant. Here, we characterized nine pituitary samples, including three fresh frozen PitBs, three normal fetal pituitary glands and three normal postnatal pituitary glands using small-RNA-Seq, RNA-Seq, methylation profiling, whole genome sequencing and Nanostring® miRNA analyses; an extended series of 21 pituitary samples was used for validation purposes. These analyses demonstrated that DICER1 RNase IIIb hotspot mutations in PitBs induced improper processing of miRNA precursors, resulting in aberrant 5p-derived miRNA products and a skewed distribution of miRNAs favoring mature 3p over 5p miRNAs. This led to dysregulation of hundreds of 5p and 3p miRNAs and concomitant dysregulation of numerous mRNA targets. Gene expression analysis revealed PRAME as the most significantly upregulated gene (500-fold increase). PRAME is a member of the Retinoic Acid Receptor (RAR) signaling pathway and in PitBs, the RAR, WNT and NOTCH pathways are dysregulated. Cancer Hallmarks analysis showed that PI3K pathway is activated in the tumors. Whole genome sequencing demonstrated a quiet genome with very few somatic alterations. The comparison of methylation profiles to publicly available data from ~ 3000 other central nervous system tumors revealed that PitBs have a distinct methylation profile compared to all other tumors, including pituitary adenomas. In conclusion, this comprehensive characterization of DICER1-related PitB revealed key molecular underpinnings of PitB and identified pathways that could potentially be exploited in the treatment of this tumor.

Pediatric fibromyxoid soft tissue tumor with PLAG1 fusion: A novel entity?

The classification of undifferentiated soft tissue tumors continues to evolve with the expanded application of molecular analysis in clinical practice. We report three cases of a unique soft tissue tumor in young children (5 months to 2 years old) displaying a purely fibromyxoid histology, with positive staining for desmin and CD34. In two cases, RNA sequencing detected a YWHAZ-PLAG1 gene fusion, while in the third case, a previously unreported EEF1A1-PLAG1 fusion was identified. PLAG1 fusions have been reported in several pathologic entities including pleomorphic adenoma, myoepithelial tumors of skin and soft tissue, and lipoblastoma, the latter occurring preferentially in young children. In these tumors, expression of a full length PLAG1 protein comes under the control of the constitutively active promoter of the partner gene in the fusion, and the current cases conform to that model. Overexpression of PLAG1 was confirmed by diffusely positive immunostaining for PLAG1 in all three cases. Our findings raise the possibility of a novel fibromyxoid neoplasm in childhood associated with these rare PLAG1 fusion variants. The only other report of a PLAG1-YWHAZ fusion occurred in a pediatric tumor diagnosed as a "fibroblastic lipoblastoma." This finding raises the possibility of a relationship with our three cases, even though our cases lacked any fat component. Further studies with regard to a shared pathogenesis are required.

DICER1 screening in 15 paediatric paratesticular sarcomas unveils an unusual DICER1-associated sarcoma.

Individuals with DICER1 syndrome, a genetic disorder caused by pathogenic germline variants in DICER1, are at increased risk of developing a wide array of predominantly childhood onset conditions, including genitourinary sarcomas. However, data on DICER1 involvement in paratesticular sarcomas have not been published. Herein, we analyse a series of 15 paediatric paratesticular sarcomas and describe in detail the case of a male infant with a paratesticular myxoid tumour, considered to be a low-grade sarcoma, who also manifested a cystic nephroma, a classic DICER1 syndrome phenotype. He harboured a pathogenic germline DICER1 variant and different somatic hot-spot mutations in each tumour. The paratesticular tumour showed strong and diffuse expression for WT1 and CD10, an unusual immunophenotype in paediatric sarcomas, but typical of tumours of Müllerian origin. The tumour was postulated to arise from the appendix testis, a Müllerian remnant located in the paratestis. Such an origin would be analogous to other DICER1-associated non-epithelial gynaecological tumours, thought to arise from Müllerian derivatives. These findings point towards a key role of DICER1 in Müllerian-derived structures. Supporting this hypothesis is the fact that the other paratesticular sarcomas from the series were either negative or focally positive for WT1 and for CD10, and none had any DICER1 mutations. In summary, we present the first case of a paratesticular sarcoma associated with DICER1 syndrome, emphasising that paratesticular tumours with an unusual histological appearance may suggest an underlying DICER1 mutation, especially in the presence of a personal or family history of DICER1-associated disease. In this context, DICER1 mutation testing could lead to changes in clinical care including implementation of cancer care surveillance strategies.

Simplified Molecular Subtyping of Medulloblastoma for Reduced Cost and Improved Turnaround Time.

Molecular subtyping of medulloblastoma (MB) has become increasingly important for prognosis and management. Typically this involves detailed molecular genetic testing which may not be available in all centers. The purpose of the present study was to find a simplified approach to assign molecular subtypes of MB for routine use in centers with more limited resources. The molecular subtypes of MBs from 32 Thai patients, aged 0.5 to 35 years, were first determined by NanoString. These results were then compared with those obtained using a combination of limited immunohistochemistry (IHC) (ß-catenin, GAB-1, YAP-1, p75-NGFR, OTX2) and CTNNTB exon 3 mutation analysis. By NanoString assay, there were 6 MBs (19%) in the wingless (WNT) group, 8 (25%) in the sonic hedgehog (SHH) group, 7 (22%) in group 3, and 11 (34%) in group 4. Although ß-catenin immunostaining missed 4/6 WNT MBs, CTNNTB mutation analysis confirmed all WNT MB cases with amplifiable DNA. The IHC panel correctly assigned all the other molecular subtypes, except for 1 MB in group 4. Thus, our protocol was able to correctly categorized 31/32 cases or 97% of cases. Our study is the first to report molecular subtypes of MB in Southeast Asia. We found that molecular subgroups of MBs can be reliably assigned using a limited IHC panel of ß-catenin, GAB-1, YAP-1, p75-NGFR, OTX2, together with CTNNTB exon 3 mutation analysis. This simplified approach incurs lower cost and faster turnaround time compared with more elaborate molecular methodologies and should be beneficial to centers with reduced laboratory resources.

Adult-onset neuroblastoma: Report of seven cases with molecular genetic characterization.

Whereas neuroblastoma is the most common extracranial solid tumor of childhood, less than 5% of cases occur in adults. Pediatric neuroblastoma shows marked heterogeneity of histology and molecular biology. Information about this tumor in adults is limited, especially regarding molecular biology. We report a series of nine neuroblastoma cases diagnosed in adulthood (18 to 40 years old) with molecular biologic characterization in seven. All tumors were Schwannian stroma-poor, and mostly poorly differentiated. Tumors expressed neural markers including PHOX2B, NB84, synaptophysin, chromogranin, CD56, neuron-specific enolase, and PGP9.5. Two out of six cases expressed ALK and one had the F1174 L mutation reported in childhood neuroblastoma. Fluorescent in situ hybridization (FISH) revealed MYCN amplification in 2/7 cases, chromosome 1p deletion in 1/5 cases and 17q gain in 4/4 cases. One in five cases showed loss of ATRX expression by immunohistochemistry and alternate lengthening of telomeres by FISH. Zero out of five cases showed rearrangement of the TERT gene by FISH, but one case showed high level amplification. In conclusion, the morphology and immunophenotype of adult-onset neuroblastoma are similar to pediatric cases although less differentiated than some childhood tumors. Similarly, molecular genetic alterations in adult-onset neuroblastoma are not unique to this age group. However, 80% of cases tested showed genetic changes that would promote maintenance of telomeres, which is a molecular marker of high risk cases. This may help explain the poor response in adults to pediatric treatment protocols. Additional studies to characterize the biology of this tumor in the adult age group will facilitate the design of more personalized therapeutic approaches.

Pleomorphic adenoma with bizarre myoepithelial cells: A diagnostic challenge on cytologic smear.

The diagnosis on fine needle aspiration of salivary gland tumors with a myoepithelial component is challenging because myoepithelial cells can have a wide cytomorphologic spectrum. The authors report a case of a pleomorphic adenoma of the parotid gland that expands the spectrum of appearances that myoepithelial cells can show with this tumor. A 55-year-old female was found to have a right parotid gland mass. FNA showed hypercellularity, with loosely cohesive fragments of spindle-shaped myoepithelial cells admixed with small nests of epithelial cells. Interspersed may occasional bizarre cells possessing severely pleomorphic nuclei with hyperchromasia. The cytologic diagnosis was "suspicious for carcinoma ex pleomorphic adenoma." A total parotidectomy was performed with complete resection of the tumor that was confirmed to be a pleomorphic adenoma. The pleomorphic cells noted on FNA were scattered throughout the tumor, and were positive by immunostaining for keratin, S-100 protein and p63, identifying them as myoepithelial cells. These cells did not show mitotic activity and were negative for Ki67. The pleomorphic adenoma showed extensive degenerative changes including central cyst formation, stromal hyalinization and hemosiderin deposition. On the basis of the combined light microscopic and immunohistochemical findings, there was no evidence to support a malignant change in the pleomorphic adenoma. It was concluded the pleomorphic myoepithelial cells were a degenerative change, reminiscent of what is seen in "ancient" schwannoma and some uterine leiomyomata. Our case expands the spectrum of appearances that can be seen in myoepithelial cells in the salivary gland.