Noncanonical activation of GLI signaling in SOX2+ cells drives medulloblastoma relapse.

SRY (sex determining region Y)-box 2 (SOX2)-labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.

Myeloid sarcoma diagnosed on pleural effusion cytology: A case report and literature review.

Myeloid sarcoma (MS) is a mass-forming, extramedullary infiltration of myeloid blasts rarely presenting in cases of acute myeloid leukemia (AML). These tumoral masses rarely occur at any and multiple anatomic sites, precedent or coincident with bone marrow evidence of AML. We report a case of MS that presented as pancreatic and cardiac masses where subsequent evaluation of pleural effusion cytology rendered the diagnosis. Primary MS diagnosed via pleural effusion cytology is not yet reported in literature. Herein, we report the case of a 45-year-old man who presented with abdominal pain. An infiltrative mass was identified in the pancreatic head, suspicious for pancreatic adenocarcinoma. Despite multiple attempts, Fine needle aspiration cytology of the pancreatic mass failed to render a definitive diagnosis. Subsequent thoracentesis of a right pleural effusion revealed cytologically malignant cells, identified as myeloid blasts after immunohistochemical and flow cytometric evaluation. Although rare, MS should be considered as a diagnostic possibility in the evaluation of malignancy with an unknown primary.

Integrated genomic analysis using chromosomal microarray, fluorescence in situ hybridization and mate pair analyses: Characterization of a cryptic t(9;22)(p24.1;q11.2)/BCR-JAK2 in myeloid/lymphoid neoplasm with eosinophilia.

The 2016 World Health Organization entity 'Myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2' encompasses a group of rare neoplasms that result from the formation of a fusion gene that leads to expression of an aberrant tyrosine kinase. This entity also contains variant JAK2 fusion partners, and detection of this defining event can be facilitated by various cytogenetic and molecular methods. Cryptic rearrangements of 9p24/JAK2 can be particularly challenging to identify. We describe the use of chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH) with a probe for JAK2, and genomic mate pair analysis to describe a complex karyotype with a t(9;22) that produced a functional BCR-JAK2 fusion, leading to the appropriate diagnosis for the patient. This case highlights the importance of using an integrated genomic approach to fully define complex aberrations to assign proper diagnoses.

Cryptic and atypical KMT2A-USP2 and KMT2A-USP8 rearrangements identified by mate pair sequencing in infant and childhood leukemia.

Infant leukemias are a rare group of neoplasms that are clinically and biologically distinct from their pediatric and adult counterparts. Unlike leukemia in older children where survival rates are generally favorable, infants with leukemia have a 5-year event-free survival rate of <50%. The majority of infant leukemias are characterized by KMT2A (MLL) rearrangements (~70 to 80% in acute lymphoblastic leukemia), which appear to be drivers of early leukemogenesis. In this report, we describe three cases: a 9-month-old female infant with B-acute lymphoblastic leukemia (B-ALL), an 8-month-old female presenting with B/myeloid mixed phenotype acute leukemia (MPAL), and a 16-month-old male with B-ALL. The first case had a normal karyotype and B-ALL FISH results consistent with an atypical KMT2A rearrangement. The second case had trisomy 10 as the sole chromosomal abnormality and a normal KMT2A FISH result. Case 3 had trisomy 8 and a t(11;15)(q23;q21), an atypical KMT2A rearrangement by FISH studies, and a focal deletion of 15q with a breakpoint within the USP8 gene by chromosomal microarray. Mate pair sequencing was performed on all three cases and identified a KMT2A-USP2 rearrangement (cases 1 and 2) or a KMT2A-USP8 rearrangement (case 3). These recently characterized KMT2A fusions have been described exclusively in infant and pediatric leukemia cases where the incidence varies vary according to leukemia subtype, are considered high-risk, with a high incidence of central nervous system involvement, poor response to initial prednisone treatment, and poor event free survival. Additionally, approximately half of cases are unable to be resolved using standard cytogenetic approaches and are likely under recognized. Therefore, targeted molecular approaches are suggested in genetically unresolved infant leukemia cases to characterize these prognostically relevant clones.

Erythrophagocytosis by leukemic blasts in acute myeloid leukemia with a normal karyotype and no detectable mutations.

The phagocytosis of erythrocytes by leukemic blasts is a rare finding in acute myeloid leukemia and has been reported most commonly in monocytic and megakaryocytic morphologies. In the reported cases, erythrophagocytosis by leukemic blasts has been associated with t(8;16). This translocation is associated with a poor outcome independent of erythrophagocytosis. There has only been one reported case of erythrophagocytosis by leukemic blasts occurring in a patient with a normal karyotype. We report a 62-year-old woman with acute myeloid leukemia with monocytic features and a normal karyotype noted to have erythrophagocytosis by leukemic blasts.

Polyoma virus-associated carcinomas of the urologic tract: a clinicopathologic and molecular study.

In recent years, there has been increased interest in carcinomas of the urologic tract, that demonstrate association with the polyoma virus BK arising in immunosuppressed individuals, though the nature of this association is uncertain. To begin to understand this phenomenon, we reviewed the clinical, morphological, and immunohistochemical features of 11 carcinomas of the urologic tract, mainly urothelial (N = 9) and collecting duct carcinomas (N = 2), occurring during immunosuppression, and expressing polyoma virus T-antigen by immunohistochemistry. These were compared to a control group of carcinomas (N = 8), also arising during immunosuppression, but without T-antigen expression. A subset of both groups were also studied by hybrid capture-based DNA sequencing, probing not only for 479 cancer-related human genes, but also for polyoma and other viral sequences. Polyoma T-antigen-expressing tumors arose in 7 males and 4 females, at a median age of 66, and were aggressive, high-grade tumors with more than 1 variant morphologic pattern identified in 81% of cases, and a majority (73%) presenting at high stage category (>pT3). Diffuse polyoma T-antigen staining was seen in 91% of cases, with co-localization of aberrant p53 staining in 89%. Sequencing detected a lower number of deleterious mutations among T-antigen-expressing cases (average 1.62; 1/8 with TP53 mutation) compared to control cases (average 3.5, 2/4 with TP53 mutation). Only BK virus was detected with clonal integration and breakpoints randomly distributed across the human and viral genomes in 5/5 of the polyoma T-antigen-expressing carcinomas, and in none of the controls (0/4). In summary, these findings identify aggressive clinicopathologic features of polyoma T-antigen-expressing carcinomas, document BK as the strain involved, and associate BK viral integration with T-antigen expression and p53 aberrancy. While the apparent randomness of viral insertion sites is functionally unclear, the differing rates of mutations between T-antigen-expressing and control cases is intriguing.

Cytology and high risk HPV testing in cervical cancer screening program: Outcome of 3-year follow-up in an academic institute.

BACKGROUND: Combination of cervical cytology and high-risk human papillomavirus (HR-HPV) testing, co-testing, has been increasingly used in screening cervical cancers. The present study summarized the outcome of co-testing by reviewing 3-year clinical and pathological follow-up information. METHODS: Patients were retrospectively identified via computerized search and were grouped based on the cytologic diagnosis and HR-HPV status as negative for intraepithelial lesion or malignancy (NILM)/HPV-, NILM/HPV+, atypical squamous cells of undetermined significance (ASC-US)/HPV-, ASC-US/HPV+, low grade squamous intraepithelial lesion (LSIL)/HPV-, LSIL/HPV+, atypical squamous cells, cannot exclude high grade squamous intraepithelial lesion (ASC-H)/HPV-, ASC-H/HPV+, high grade squamous intraepithelial lesion (HSIL)/HPV-, and HSIL/HPV+. The patients' pertinent past medical history and follow-up information were analyzed. RESULTS: During 3-year follow-up period, histologically proven HSIL was found in 5 of 1565 (0.3%) patients with NILM/HPV-, 7 of 141 (5.0%) with NILM/HPV+, 2 of 502 (0.4%) with ASC-US/HPV-, 30 of 274 (10.9%) with ASC-US/HPV+, 1 of 81 (1.2%) with LSIL/HPV-, 28 of 159 (17.6%) with LSIL/HPV+, 3 of 18 (16.7%) with ASC-H/HPV-, 34 of 69 (49.3%) with ASC-H/HPV+, 7 of 7 (100%) with HSIL/HPV-, and 35 of 56 (62.5%) HSIL/HPV+. In reviewing 12 HSIL cases that were originally diagnosed as NILM, 7 remained as NILM, and the other 5 were reclassified as 1 HSIL, 1 ASC-H, and 3 ASC-US, respectively. In 18 HSIL cases with negative HR-HPV, 12 patients had a prior history of positive HR-HPV testing and/or positive p16 IHC stain in the follow-up cervical biopsy. CONCLUSION: HR-HPV testing plays an important role in cervical cancer screening by identifying HSIL in patients with ASC-US, LSIL, and NILM. Co-testing is an optimal method to identifying the patients with higher risk for developing cervical abnormalities.

Three-year review of gene sequencing analyses of pulmonary non-small cell lung cancers obtained by fine-needle aspiration or surgical biopsy: mutation and failure rates.

INTRODUCTION: Mutational analysis is becoming the standard of diagnostic workup. Sufficient amounts of and quality tumor tissue can be challenging when faced with a small biopsy or biopsy by fine-needle aspiration (FNA). MATERIALS AND METHODS: We reviewed the failures of FNA and surgical biopsy to yield sequencing data and causes thereof over a 3-year period. We executed a search of the laboratory information system for requests to perform our targeted 50-gene assay by massively parallel sequencing on surgical biopsies and FNAs and compared the results. RESULTS: Three failure causes were assigned: insufficient tissue as defined by the pathologist, failure to meet quality control indicating library preparation or sequencing failure, and failure of pre-qualifying step for DNA integrity. A total of 327 of 354 cases were successfully sequenced (92%), including 151 FNA cases and 203 biopsies, with 16 (10.6%) and 11 (5.4%) failures, respectively. The Fisher's exact test two-tailed P-value equals 0.050381, making the difference between FNA and biopsy not statistically significant. Insufficient tissue, quality control failure, and DNA integrity were identified as the cause of the failure in 10 (62%), 3 (19%), and 3 (19%) FNA biopsies, and in 5 (45.5%), 1 (9%), and 5 (45.5%) surgical biopsies. The most common cause of failure of FNA was insufficient tissue. For surgical biopsies, DNA integrity and insufficient tissue were equally as likely to be implicated. Both FNA and surgical biopsy have a low failure rate overall without statistical significance between them. CONCLUSIONS: Although surgical biopsy is considered the gold standard, these findings support FNA as an equal modality.

Myeloid sarcoma identified on liquid-based cervical cytology samples: A report of two cases.

The purpose of the Papanicolaou (Pap) smear is to detect primary squamous lesions of the uterine cervix. Although most successful at detection of squamous lesions, the Pap may also detect metastatic carcinomas, sarcomas, and melanomas. We report two rare cases of myeloid sarcoma (MS) of the uterine cervix identified on screening Pap smears with concurrent confirmatory cervical biopsies. The purpose of our study is to identify and report cytologic features of MS on Pap smears utilizing a liquid-based ThinPrep method, which has not been previously documented in literature. Two Pap smears were identified from the pathology laboratory information system, both with positive cervical biopsy findings of MS. Both women, age 40 and 39, presented with ureteral obstruction, hydronephrosis, and past medical histories significant for acute myeloid leukemia (AML). On imaging, cervical masses were identified, and subsequent work-up with Pap smears and biopsies were performed. Cytologic examination of the ThinPrep Pap smears were negative for squamous intraepithelial lesion. Atypical hematologic cells were seen in the background with irregular nuclear contours, increased nuclear to cytoplasmic ratios, variably prominent nucleoli, and variable amounts of agranular cytoplasm. The biopsy confirmed these findings to represent MS. MS is defined as a tumor mass of myeloblasts or immature myeloid cells occurring in an extramedullary site. This rarely involves the female genital tract, about 50 reported cases. Although very rare, MSs in the setting of a history of AML are able to be identified on liquid-based ThinPrep smears.

Young Investigator Challenge: A novel, simple method for cell block preparation, implementation, and use over 2 years.

BACKGROUND: The cell block is an essential adjunct to conventional cytopreparatory techniques. The need for molecular analysis and immunostains will increase the need for successful cell block preparation. Even with this need, to the authors' knowledge very little has changed regarding the way in which cell blocks are produced. METHODS: The authors developed A Formalin-Fixed, paraffin Embedded Cytology cell block Technique (AFFECT) that uses a cytospin centrifuge and funnel to deposit a cell pellet into a well on a piece of open-cell, absorbent foam. The foam and the pellet are then sent through normal processing. Herein, the authors present the implementation of this method and some of their experience with its performance over the course of 2 years. RESULTS: Although a comparison of the methods indicated good correlation for the production of a cell block between AFFECT and the agarose method, the AFFECT blocks demonstrated markedly improved cellular morphology. Over the first 6 months of use, AFFECT produced a successful cell block in 74% of cases overall, and in 65% of cases with a cell pellet measuring ≤0.1 mL. The year preceding the implementation of AFFECT and its first year of use were compared for endoscopic and bronchoscopic ultrasound-guided fine-needle aspiration specimens, and demonstrated an improved success rate. CONCLUSIONS: The authors developed a novel method of cell block preparation that demonstrates improved histology and has increased the success rate of cell block production compared with the agarose method. Cancer Cytopathol 2016;124:885-892. © 2016 American Cancer Society.

Cytological diagnosis of metastatic malignant melanoma by fine-needle aspiration biopsy.

Despite increased surveillance and public awareness, the incidence of melanoma is increasing. Frequently, fine-needle aspiration is employed for the diagnosis of metastatic disease, and aspirated material is used for cytogenetic and molecular studies to guide treatment options. The pairing of a significant diagnosis with the numerous morphologic variants of melanoma can make the cytologic evaluation disquieting. We present selected examples of our experiences and a brief review of the literature to provide cytodiagnostic clues for this malignancy. The clinical history is foremost, although the fine-needle aspiration (FNA) of metastatic melanoma can provide a diagnosis before identification of the primary lesion in up to 20% of cases. If a history of melanoma is assured, negative results in sampling of pulmonary and subcutaneous nodules should be suspected as false negatives. The smearing pattern usually features poorly cohesive cells. Frankly malignant, spindled, and epithelioid cell shapes are most common, and cytoplasmic vacuoles, if sought on Romanowsky-stained specimens, can usually be found. The telltale feature of melanin production, although diagnostic, is only present in 50% of cases. Finally, eccentric placement of nuclei, nucleoli, and nuclear pseudoinclusions are accessory features for the cytologic interpretation of melanoma. Numerous morphologic patterns of melanoma are potentially seen, but a stepwise approach to diagnosis usually produces a successful result.

Cytopathologic features of clear cell papillary renal cell carcinoma: A recently described variant to be considered in the differential diagnosis of clear cell renal epithelial neoplasms.

BACKGROUND: Clear cell papillary renal cell carcinoma (CCPRCC) is a distinctive variant of renal cell carcinoma that has been formally adopted by the new Word Health Organization classification. An emerging consensus has documented its particularly indolent course and emphasized its separation from conventional clear cell renal cell carcinoma (CCRCC) for treatment planning. CCPRCC features in cytologic preparations have not been studied. METHODS: This study retrospectively identified a series of CCPRCCs that had cytology samples before the histopathologic diagnosis and reviewed corresponding cytologic materials, including aspirate smears, cell block materials, touch preparations, and core biopsy samples. The identified clinicopathologic and cytologic features were tabulated. RESULTS: Five cases of CCPRCC with cytopathologic materials were identified from 4 women and 1 man aged 34 to 70 years (2 with end-stage renal disease), and the sampled lesions were 1.8 to 11.0 cm. The original cytopathologic diagnostic considerations ranged from atypical cyst-lining cells to angiomyolipoma to CCRCC and CCPRCC. The aspirate and touch preparation samples showed scant cellularity with scattered sheets and clusters of small, bland epithelial cells (much smaller than admixed renal tubular cells) with optically clear cytoplasm (lacking conspicuous cytoplasmic vacuolization) and small, grade 1 nuclei. The cell block materials and the core biopsy samples showed cyst walls with prominent myomatous stroma lined by low-grade epithelium with optically clear cytoplasm, inverse nuclear polarization, and a characteristic cytokeratin 7-positive/carbonic anhydrase IX-positive phenotype. Three cases were treated with resection, 1 case was treated with ablation, and 1 case was under surveillance. CONCLUSIONS: CCPRCC demonstrates recognizable cytomorphologic features and merits consideration in the cytologic differential diagnosis for kidney lesions. With increasing experience, more conservative management may be contemplated. Cancer Cytopathol 2016;124:565-72. © 2016 American Cancer Society.

Bronchogenic squamous cell carcinoma with soft-tissue metastasis to the hand: an unusual case presentation and review of the literature.

Carcinoma of the lung is the most common lethal form of cancer in both men and women worldwide. Orthopedic manifestations of lung cancer frequently include bony metastasis, most commonly the vertebrae (42%), ribs (20%), and pelvis (18%). Acral metastatic disease is defined as metastasis distal to the elbow or the knee. Metastases to the bones of the hand are extremely rare. Only 0.1% of metastatic disease resulting from any type of carcinoma or sarcoma manifests as metastasis in the hand. There are only a few reports in the literature of soft-tissue or muscular metastasis to the hand from a carcinoma. Of these cases, the majority are caused by metastatic lung carcinoma. However, there are no reports in the literature of metastatic disease of squamous cell origin affecting the soft tissues of the hand. We present a case of a man with known metastatic squamous cell carcinoma of the lung who presented with acral soft-tissue metastatic disease. This report highlights a rare clinical scenario that has not been reported in the literature. This report also highlights a rare but important consideration for clinicians who encounter acral soft-tissue lesions in patients with a history of a primary carcinoma.