Isogenic Cell Lines Derived from Specific Organ Metastases Exhibit Divergent Cytogenomic Aberrations.

Aneuploidy, a deviation in chromosome numbers from the normal diploid set, is now recognized as a fundamental characteristic of all cancer types and is found in 70-90% of all solid tumors. The majority of aneuploidies are generated by chromosomal instability (CIN). CIN/aneuploidy is an independent prognostic marker of cancer survival and is a cause of drug resistance. Hence, ongoing research has been directed towards the development of therapeutics aimed at targeting CIN/aneuploidy. However, there are relatively limited reports on the evolution of CIN/aneuploidies within or across metastatic lesions. In this work, we built on our previous studies using a human xenograft model system of metastatic disease in mice that is based on isogenic cell lines derived from the primary tumor and specific metastatic organs (brain, liver, lung, and spine). As such, these studies were aimed at exploring distinctions and commonalities between the karyotypes; biological processes that have been implicated in CIN; single-nucleotide polymorphisms (SNPs); losses, gains, and amplifications of chromosomal regions; and gene mutation variants across these cell lines. Substantial amounts of inter- and intra-heterogeneity were found across karyotypes, along with distinctions between SNP frequencies across each chromosome of each metastatic cell line relative the primary tumor cell line. There were disconnects between chromosomal gains or amplifications and protein levels of the genes in those regions. However, commonalities across all cell lines provide opportunities to select biological processes as druggable targets that could have efficacy against the primary tumor, as well as metastases.

CD34+ cell of origin for immunoglobulin heavy chain variable region unmutated, but not mutated, chronic lymphocytic leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Immunoglobulin heavy chain variable region (IgHV) mutation status is among the most important prognostic factors, with unmutated IgHV associated with inferior outcomes. CLL presumably arises from mature B cells. However, we hypothesized that IgHV unmutated CLL could arise early in B cell differentiation. We prospectively studied 29 patients with mutated and 88 with unmutated IgHV CLL for the presence of CD34+CD19+ cells harboring CLL chromosomal abnormalities. CD34+CD19+ cells were never detected in mutated CLL. In contrast, a small but distinct population of CD34+CD19+ cells harboring the CLL chromosomal abnormality was present in 86/88 patients with unmutated IgHV across all cytogenetic subtypes. Moreover, the CD34+CD19+ cells generated a 3.8 ± 0.7 fold CLL cell expansion over 3-4 weeks in cultures containing IL-3 and IL-2. Unmutated IgHV CLL appears to arise in CD34+ B cells, which perhaps contributes to its poorer prognosis.

Diagnostic Utility of Gene Fusion Panel to Detect Gene Fusions in Fresh and Formalin-Fixed, Paraffin-Embedded Cancer Specimens.

Somatic gene fusions are common in leukemias/lymphomas and solid tumors. The detection of gene fusions is crucial for diagnosis. NanoString fusion technology is a multiplexed hybridization method that interrogates hundreds of gene fusions in a single reaction. This study's objective was to determine the performance characteristics and diagnostic utility of NanoString fusion assays in a clinical diagnostics laboratory. Validation using 100 positive specimens and 15 negative specimens by a combined reference standard of fluorescence in situ hybridization (FISH)/RT-PCR/next-generation sequencing (NGS) assays achieved 100% sensitivity in leukemias/lymphomas and 95.0% sensitivity and 100% specificity in solid tumors. Subsequently, 214 consecutive clinical cases, including 73 leukemia/lymphoma specimens and 141 formalin-fixed, paraffin-embedded solid tumor specimens, were analyzed by gene fusion panels across 638 unique gene fusion transcripts. A variety of comparator tests, including FISH panels, conventional karyotyping, a DNA-based targeted NGS assay, and custom RT-PCR testing, were performed in parallel. The gene fusion assay detected 31 gene fusions, including 16 in leukemia/lymphoma specimens and 15 in solid tumor specimens. The overall sensitivity, specificity, and accuracy of gene fusions detected by the gene fusion panel in all 329 specimens (validation and consecutive clinical specimens) tested in this study were 94.8%, 100%, and 97.9%, respectively, compared with FISH/RT-PCR/NGS assays. The gene fusion panel is a reliable approach that maximizes molecular detection of fusions among both fresh and formalin-fixed, paraffin-embedded cancer specimens.

HER-2 Amplification in Uterine Serous Carcinoma and Serous Endometrial Intraepithelial Carcinoma.

Human epidermal growth factor receptor 2 (HER-2) targeted therapy shows promising results in HER-2-positive uterine serous carcinoma (USC). HER-2 scoring criteria for USC and its associated noninvasive lesion, serous endometrial intraepithelial carcinoma (SEIC), are not well-established. Here, we compare the breast and gastric (GI) HER-2 immunohistochemistry (IHC) scoring criteria for HER-2 with HER-2/neu fluorescence in situ hybridization (FISH) in 68 tumors (17 USC with SEIC, 30 USC, 18 SEIC, 3 metastatic USC). The majority (97%) of lesions displayed intratumoral HER-2 IHC heterogeneity. Breast or GI IHC scoring criteria were performed equivalently. The breast and GI IHC criteria classified 51% and 47% USC as HER-2 negative (IHC 0/1+), 40% and 45% as equivocal (IHC 2+), and 9% each as HER-2 positive (IHC 3+). A quarter of USC classified as HER-2 negative or positive with the breast (25%, n=7/28) or GI IHC criteria (23%, n=6/26) was discordant by FISH. Specifically, 13% to 14% of IHC 0/1+ USC were FISH amplified; 50% of IHC 3+ USC were FISH negative. The majority (77% to 83%) of SEIC were HER-2 IHC 0/1+, and no SEIC was HER-2 IHC 3+. A minority (4% to 7%) of IHC 0/1+ SEIC were FISH positive. Discordant HER-2 status was observed between half (47%,bn=7/15) of synchronous SEIC and USC. In conclusion, USC displays HER-2 intratumoral heterogeneity, a high IHC/FISH discordance rate, and variation in HER-2 status between the SEIC and invasive components. Caution is required when evaluating HER-2 in small biopsies, which should be repeated on excisions. Both IHC and FISH should be performed on USC until clinical trials correlate HER-2 status with clinical response to HER-2-targeted therapy.

The Decline of Salivary Adenocarcinoma Not Otherwise Specified as a Tumor Entity: Reclassification Using Contemporary Immunohistochemical Profiling and Diagnostic Criteria.

The classification of salivary gland carcinomas has become increasingly specific over the last decade with the definition of new tumor types, documentation of novel molecular and immunohistochemical findings, and development of more refined diagnostic criteria. In this setting, it is unclear how many salivary tumors still cannot be easily categorized-and whether such tumors represent undifferentiated malignancies or include additional definable entities. Relying largely on current classification schemes and contemporary immunohistochemical panels, we reassessed salivary tumors previously diagnosed as adenocarcinoma, not otherwise specified (ACA NOS) from 2 large academic medical centers. Fifty-seven ACA NOS (72%) could be reclassified as more specific entities including 31 salivary duct carcinomas (39%), 7 polymorphous adenocarcinomas (9%), 5 epithelial-myoepithelial carcinomas (6%), 4 myoepithelial carcinomas (5%), 4 secretory carcinomas (5%), 1 acinic cell carcinoma (1%), 1 basal cell adenocarcinoma (1%), 1 intraductal carcinoma (1%), and 1 clear cell carcinoma (1%) as well as 2 metastatic squamous cell carcinomas (3%). Of reclassified cases, 21 (37%) represented variant histologies within these categories. ACA NOS comprised 11% of salivary malignancies before reclassification, but only 4% after reclassification. The remaining 22 ACA NOS demonstrated heterogeneous features, with an association between histologic grade and clinical outcome. In effect, ACA NOS is becoming a bygone entity as modern classification schemes and ancillary techniques now permit more specific typing of a majority of these tumors, potentially facilitating more specific prognostication and treatment. Additional distinctive entities such as mucinous adenocarcinoma may still be definable within the ACA NOS category.

CREBBP and STAT6 co-mutation and 16p13 and 1p36 loss define the t(14;18)-negative diffuse variant of follicular lymphoma.

The diffuse variant of follicular lymphoma (dFL) is a rare variant of FL lacking t(14;18) that was first described in 2009. In this study, we use a comprehensive approach to define unifying pathologic and genetic features through gold-standard pathologic review, FISH, SNP-microarray, and next-generation sequencing of 16 cases of dFL. We found unique morphologic features, including interstitial sclerosis, microfollicle formation, and rounded nuclear cytology, confirmed absence of t(14;18) and recurrent deletion of 1p36, and showed a novel association with deletion/CN-LOH of 16p13 (inclusive of CREBBP, CIITA, and SOCS1). Mutational profiling demonstrated near-uniform mutations in CREBBP and STAT6, with clonal dominance of CREBBP, among other mutations typical of germinal-center B-cell lymphomas. Frequent CREBBP and CIITA codeletion/mutation suggested a mechanism for immune evasion, while subclonal STAT6 activating mutations with concurrent SOCS1 loss suggested a mechanism of BCL-xL/BCL2L1 upregulation in the absence of BCL2 rearrangements. A review of the literature showed significant enrichment for 16p13 and 1p36 loss/CN-LOH, STAT6 mutation, and CREBBP and STAT6 comutation in dFL, as compared with conventional FL. With this comprehensive approach, our study demonstrates confirmatory and novel genetic associations that can aid in the diagnosis and subclassification of this rare type of lymphoma.

IGSF3 mutation identified in patient with severe COPD alters cell function and motility.

Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient's risk of severe COPD. We determined that 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in the immunoglobulin superfamily member 3 (IGSF3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs, of which rs1414272 and rs12066192 were directly and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin-enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide and sphingosine-1 phosphate but low glycosphingolipids and ganglioside levels, and they were less apoptotic and more adherent, with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.

Whole-exome sequencing of duodenal neuroendocrine tumors in patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a hereditary cancer predisposition syndrome characterized by frequent cutaneous and nervous system abnormalities. Patients with NF1 also have an increased prevalence of multiple gastrointestinal and peripancreatic neoplasms-neuroendocrine tumors of the ampulla that express somatostatin are particularly characteristic of NF1. In this study, we characterize the genetic alterations of a clinically well-characterized cohort of six NF1-associated duodenal neuroendocrine tumors using whole-exome sequencing. We identified inactivating somatic mutations in the NF1 gene in three of six tumors; the only other gene altered in more than one tumor was IFNB1. Copy number analysis revealed deletion/loss of heterozygosity of chromosome 22 in three of six patients. Analysis of germline variants revealed germline deleterious NF1 variants in four of six patients, as well as deleterious variants in other tumor suppressor genes in two of four patients with deleterious NF1 variants. Taken together, these data confirm the importance of somatic inactivation of the wild-type NF1 allele in the formation of NF1-associated duodenal neuroendocrine tumors and suggest that loss of chromosome 22 is important in at least a subset of cases. However, we did not identify any genes altered in the majority of NF1-associated duodenal neuroendocrine tumors that uniquely characterize the genomic landscape of this tumor. Still, the genetic alterations in these tumors are distinct from sporadic neuroendocrine tumors occurring at these sites, highlighting that unique genetic alterations drive syndromic tumors.

Pancreatic undifferentiated carcinoma with osteoclast-like giant cells is genetically similar to, but clinically distinct from, conventional ductal adenocarcinoma.

Undifferentiated carcinoma of the pancreas with osteoclast-like giant cells (UCOGC) is currently considered a morphologically and clinically distinct variant of pancreatic ductal adenocarcinoma (PDAC). In this study, we report clinical and pathological features of a series of 22 UCOGCs, including the whole exome sequencing of eight UCOGCs. We observed that 60% of the UCOGCs contained a well-defined epithelial component and that patients with pure UCOGC had a significantly better prognosis than did those with an UCOGC with an associated epithelial neoplasm. The genetic alterations in UCOGC are strikingly similar to those known to drive conventional PDAC, including activating mutations in the oncogene KRAS and inactivating mutations in the tumor suppressor genes CDKN2A, TP53, and SMAD4. These results further support the classification of UCOGC as a PDAC variant and suggest that somatic mutations are not the determinants of the unique phenotype of UCOGC. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MYB Labeling by Immunohistochemistry Is More Sensitive and Specific for Breast Adenoid Cystic Carcinoma than MYB Labeling by FISH.

Breast adenoid cystic carcinoma (ACC) is a primary breast carcinoma that, like salivary gland ACC, displays the t(6;9) translocation resulting in the MYB-NFIB gene fusion and immunopositivity for MYB by immunohistochemistry (IHC). However, it is not well established whether MYB immunoreactivity or rearrangement can be used to support a diagnosis of ACC in a malignant basaloid or benign cribriform breast lesion. Whole sections of primary breast ACC (n=11), collagenous spherulosis (CS; n=7), and microglandular adenosis (MGA; n=5) and tissue microarrays containing 16 basal-like, triple-negative breast carcinomas (TNBC) were labeled for MYB by IHC and underwent MYB fluorescence in situ hybridization using a break-apart probe. Strong, diffuse nuclear MYB labeling was seen in 100% ACC compared with no cases of basal-like TNBC, CS, or MGA (P=0.0001). Any degree of nuclear MYB labeling was seen in 100% ACC compared with 54% of all other cases (P=0.007), with any labeling seen in 71% CS, 63% basal-like TNBC, and 0% MGA. MYB rearrangement was detected in 89% (8/9) of evaluable ACC compared with 4% (1/26) of all other evaluable cases (P=0.0001), with a rearrangement detected in 1 (7%; n=1/15) evaluable basal-like TNBC. Strong, diffuse nuclear labeling for MYB is more sensitive than MYB fluorescence in situ hybridization for breast ACC and can be used to support a diagnosis of ACC in a cribriform or basaloid lesion in the breast. However, weak and focal labeling should be interpreted with caution as it can be seen in other benign cribriform and malignant basaloid lesions.

Genetic analyses of isolated high-grade pancreatic intraepithelial neoplasia (HG-PanIN) reveal paucity of alterations in TP53 and SMAD4.

High-grade pancreatic intraepithelial neoplasia (HG-PanIN) is the major precursor of pancreatic ductal adenocarcinoma (PDAC) and is an ideal target for early detection. To characterize pure HG-PanIN, we analysed 23 isolated HG-PanIN lesions occurring in the absence of PDAC. Whole-exome sequencing of five of these HG-PanIN lesions revealed a median of 33 somatic mutations per lesion, with a total of 318 mutated genes. Targeted next-generation sequencing of 17 HG-PanIN lesions identified KRAS mutations in 94% of the lesions. CDKN2A alterations occurred in six HG-PanIN lesions, and RNF43 alterations in five. Mutations in TP53, GNAS, ARID1A, PIK3CA, and TGFBR2 were limited to one or two HG-PanINs. No non-synonymous mutations in SMAD4 were detected. Immunohistochemistry for p53 and SMAD4 proteins in 18 HG-PanINs confirmed the paucity of alterations in these genes, with aberrant p53 labelling noted only in three lesions, two of which were found to be wild type in sequencing analyses. Sixteen adjacent LG-PanIN lesions from ten patients were also sequenced using targeted sequencing. LG-PanIN harboured KRAS mutations in 94% of the lesions; mutations in CDKN2A, TP53, and SMAD4 were not identified. These results suggest that inactivation of TP53 and SMAD4 are late genetic alterations, predominantly occurring in invasive PDAC. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Alterations of type II classical cadherin, cadherin-10 (CDH10), is associated with pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC), either sporadic or familial, has a dismal prognosis and finding candidate genes involved in development of the cancer is crucial for the patient care. First, we identified two patients with germline alterations in or adjacent to CDH10 by chromosome studies and sequencing analyses in 41 familial pancreatic cancer (FPC) cases. One patient had a balanced translocation between chromosome 5 and 20. The breakpoint on chromosome band 5p14.2 was ∼810 Kb upstream of CDH10, while that on chromosome arm 20p was in the pericentromeric region which might result in inactivation of one copy of the gene leading to reduced expression of CDH10. This interpretation was supported by loss of heterozygosity (LOH) seen in this region as determined by short tandem repeat analyses. Another patient had a single nucleotide variant in exon 12 (p.Arg688Gln) of CDH10. This amino acid was conserved among vertebrates and the mutation was predicted to have a pathogenic effect on the protein by several prediction algorithms. Next, we analyzed LOH status in the CDH10 region in sporadic PDAC and at least 24% of tumors had evidence of LOH. Immunohistochemical stains with CDH10 antibody showed a different staining pattern between normal pancreatic ducts and PDAC. Taken together, our data supports the notion that CDH10 is involved in sporadic pancreatic carcinogenesis, and might have a role in rare cases of FPC. Further functional studies are needed to elucidate the tumor suppressive role of CDH10 in pancreatic carcinogenesis.

Tumor-Infiltrating Macrophages in Post-Transplant, Relapsed Classical Hodgkin Lymphoma Are Donor-Derived.

Tumor-associated inflammatory cells in classical Hodgkin lymphoma (CHL) typically outnumber the neoplastic Hodgkin/Reed-Sternberg (H/RS) cells. The composition of the inflammatory infiltrate, particularly the fraction of macrophages, has been associated with clinical behavior. Emerging work from animal models demonstrates that most tissue macrophages are maintained by a process of self-renewal under physiologic circumstances and certain inflammatory states, but the contribution from circulating monocytes may be increased in some disease states. This raises the question of the source of macrophages involved in human disease, particularly that of CHL. Patients with relapsed CHL following allogeneic bone marrow transplant (BMT) provide a unique opportunity to begin to address this issue. We identified 4 such patients in our archives. Through molecular chimerism and/or XY FISH studies, we demonstrated the DNA content in the post-BMT recurrent CHL was predominantly donor-derived, while the H/RS cells were derived from the patient. Where possible to evaluate, the cellular composition of the inflammatory infiltrate, including the percentage of macrophages, was similar to that of the original tumor. Our findings suggest that the H/RS cells themselves define the inflammatory environment. In addition, our results demonstrate that tumor-associated macrophages in CHL are predominantly derived from circulating monocytes rather than resident tissue macrophages. Given the association between tumor microenvironment and disease progression, a better understanding of macrophage recruitment to CHL may open new strategies for therapeutic intervention.

For staining of ALK protein, the novel D5F3 antibody demonstrates superior overall performance in terms of intensity and extent of staining in comparison to the currently used ALK1 antibody.

Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm. Approximately 50 % of IMTs show an anaplastic lymphoma kinase (ALK) gene fusion resulting in ALK overexpression on immunohistochemistry (IHC). A novel anti-ALK monoclonal antibody (D5F3) has been suggested to be of superior sensitivity to the ALK1 antibody which is currently used. We compared the performance of D5F3 in detecting ALK protein expression in IMTs from various anatomic sites compared to the currently utilized ALK1. We selected 25 IMTs from our surgical pathology files (2005-2015). The novel rabbit monoclonal anti-human CD246 (clone D5F3) and the currently used mouse monoclonal anti-human CD246 (clone ALK1) were used for immunohistochemical staining (IHC) in an automated slide stainer. The percentage of immunoreactive tumor cells (0, <5 %, 5-50 %, >50 %) and cytoplasmic staining intensity (graded 0-3) were assessed and compared between the two antibodies. Fluorescence in situ hybridization (FISH) studies for ALK gene rearrangement were performed on 11 tumors. D5F3 antibody stained 76 % and ALK1 antibody stained 72 % of IMTs (p = 0.747). Compared to staining with ALK1, D5F3 stained a higher proportion of cases extensively (>50 % cells) (76 vs. 28 %, p < 0.001) and with high intensity (grade 3 76 % vs 0; p < 0.001). FISH and IHC findings (for both antibodies) were concordant in 9/10 (90 %) IMTs, in which results were informative. The novel anti-ALK rabbit monoclonal antibody (D5F3 clone) demonstrates superior overall performance in term of intensity and extent of staining of ALK protein in IMT. We found IHC staining with both antibody clones to correlate equally well with FISH results for detection of ALK rearrangement.

NUP98-PHF23 fusion is recurrent in acute myeloid leukemia and shares gene expression signature of leukemic stem cells.

Chromosome translocations involving nucleoporin 98 gene (NUP98) have been identified in a wide array of hematologic malignancies, and the resulting NUP98-associated fusions are known to play a critical role in leukemogensis through dysregulation of gene expression. Although NUP98-associated fusions were initially thought to be rare, application of molecular technologies has revealed that cryptic translocations involving NUP98 are more frequent than previously appreciated. We report an additional case of t(11;17)(p15;p13) resulting in the fusion of NUP98 and plant homeodomain finger 23 (PHF23) in a pediatric patient with acute myeloid leukemia (AML). Using RNA sequencing, we determined in-frame fusion points and also analyzed the gene expression profile of NUP98-PHF23 positive AML. Gene set enrichment analysis (GSEA) demonstrates that NUP98-PHF23 fusion shares gene expression signature of NUP98-HOXA9 fusion, the prototype of the NUP98-associated fusions, as well as the signature of leukemic stem cells. To our knowledge this is the first transcriptome analysis of human samples with NUP98-PHF23 positive AML. Our findings are in support of the gene expression study of NUP98-PHF23 mouse model and validate the usefulness of the mouse model in developing therapeutic strategies for the treatment of subsets of AML.

A subset of prostatic basal cell carcinomas harbor the MYB rearrangement of adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a basaloid tumor consisting of myoepithelial and ductal cells typically arranged in a cribriform pattern. Adenoid cystic carcinoma is generally regarded as a form of salivary gland carcinoma, but it can arise from sites unassociated with salivary tissue. A rare form of prostate carcinoma exhibits ACC-like features; it is no longer regarded as a true ACC but rather as prostatic basal cell carcinoma (PBCC) and within the spectrum of basaloid prostatic proliferations. True ACCs often harbor MYB translocations resulting in the MYB-NFIB fusion protein. MYB analysis could clarify the true nature of prostatic carcinomas that exhibit ACC features and thus help refine the classification of prostatic basaloid proliferations. Twelve PBCCs were identified from the pathology consultation files of Johns Hopkins Hospital. The histopathologic features were reviewed, and break-apart fluorescence in situ hybridization for MYB was performed. All 12 cases exhibited prominent basaloid histology. Four were purely solid, 7 exhibited a cribriform pattern reminiscent of salivary ACC, and 1 had a mixed pattern. The MYB rearrangement was detected in 2 (29%) of 7 ACC-like carcinomas but in none (0%) of the 5 PBCCs with a prominent solid pattern. True ACCs can arise in the prostate as is evidenced by the presence of the characteristic MYB rearrangement. When dealing with malignant basaloid proliferations in the prostate, recommendations to consolidate ACCs with other tumor types may need to be reassessed, particularly in light of the rapidly advancing field of biologic therapy where the identification of tumor-specific genetic alterations presents novel therapeutic targets.

MYB rearrangement and clinicopathologic characteristics in head and neck adenoid cystic carcinoma.

OBJECTIVES: Salivary gland adenoid cystic carcinoma (ACC) is rare, aggressive, and challenging to treat. Many ACCs have a t(6;9) chromosomal translocation resulting in a MYB-NFIB fusion gene, but the clinical significance is unclear. The purposes of this study were to describe the clinicopathologic factors impacting survival and to determine the prevalence and clinical significance of MYB-NFIB fusion. STUDY DESIGN: Case series. METHODS: Medical records of patients treated for ACC of the head and neck from 1974 to 2011 were reviewed and clinicopathologic data recorded. Fluorescence in situ hybridization (FISH) was used to detect MYB rearrangement in archival tumor tissue as a marker of MYB-NFIB fusion. RESULTS: One hundred fifty-eight patients were included, with median follow-up 75.1 months. Median overall survival was 171.5 months (95% confidence interval [CI] = 131.9-191.6), and median disease-free survival was 112.0 months (95% CI = 88.7-180.4). Advanced stage was associated with decreased overall survival (adjusted ptrend < 0.001), and positive margins were associated with decreased disease-free survival (adjusted hazard ratio [aHR] = 8.80, 95% CI = 1.25-62.12, P = 0.029). Ninety-one tumors were evaluable using FISH, and 59 (65%) had evidence of a MYB-NFIB fusion. MYB-NFIB positive tumors were more likely than MYB-NFIB negative tumors to originate in minor salivary glands (adjusted prevalence ratios = 1.51, 95% CI = 1.07-2.12, P = 0.019). MYB-NFIB tumor status was not significantly associated with disease-free or overall survival (hazard ratio [HR] = 1.53, 95% CI = 0.77-3.02, P = 0.22 and HR = 0.91, 95% CI = 0.46-1.83, P = 0.80, respectively, for MYB-NFIB positive compared with MYB-NFIB negative tumors). CONCLUSION: Stage and margin status were important prognostic factors for ACC. Tumors with evidence of MYB-NFIB fusion were more likely to originate in minor salivary glands, but MYB-NFIB tumor status was not significantly associated with prognosis. LEVEL OF EVIDENCE: 4.

Mucoepidermoid carcinoma does not harbor transcriptionally active high risk human papillomavirus even in the absence of the MAML2 translocation.

High risk human papillomavirus (HPV) is firmly established as an important cause of oropharyngeal carcinoma. Recent studies have also implicated HPV as a cause of mucoepidermoid carcinoma (MEC)-a tumor of salivary gland origin that frequently harbors MAML2 translocations. The purpose of this study was to determine the prevalence of transcriptionally active HPV in a large group of MECs and to determine whether HPV infection and the MAML2 translocation are mutually exclusive events. Break-apart fluorescence in situ hybridization for MAML2 was performed on a tissue microarray containing 92 MECs. HPV testing was performed using RNA in situ hybridization targeting high risk HPV mRNA E6/E7 transcripts. Of the 71 MECs that could be evaluated by FISH, 57 (80 %) harbored the MAML2 rearrangement. HPV was not detected in any of the 57 MECs that contained a MAML2 rearrangement, in any of the 14 MECs that did not contain the rearrangement, or in any of the 21 MECs where MAML2 status was unknown. High risk HPV does not appear to play any significant role in the development of MEC. It neither complements nor replaces MAML2 translocation in the tumorigenesis of MEC.

Glandular odontogenic cysts (GOCs) lack MAML2 rearrangements: a finding to discredit the putative nature of GOC as a precursor to central mucoepidermoid carcinoma.

Glandular odontogenic cyst (GOC) is a cyst of the gnathic bones that is characterized by squamous and glandular differentiation. The histopathologic features of GOC overlap considerably with central mucoepidermoid carcinoma (MEC), suggesting that GOC could be a precursor lesion to, or even a low-grade form of, central MEC. Differentiating the two lesions may be difficult or impossible on a limited biopsy. MAML2 rearrangements have been recently found to be specific for MEC, even those arising in the jaws. An analysis of MAML2 in GOCs could help clarify its relationship with central MEC. Tissue blocks from 21 GOCs and 5 central MECs were retrieved from the surgical pathology archives of The Johns Hopkins Hospital. Each MEC exhibited solid areas and clear-cut stromal invasion. In addition, 4 of the MECs demonstrated cystic areas that were histologically similar to GOC. Break-apart fluorescence in situ hybridization for MAML2 was performed. For the MECs, analysis was performed on both the solid components and the cystic areas that resembled GOC. MAML2 rearrangements were identified in all 5 of the MECs, but in none of the 21 GOCs (100 vs. 0 %; p < 0.0001, Fisher's Exact). In the MECs, the rearrangement was present in both the solid and GOC-like cystic areas. While central MECs consistently harbor the MAML2 rearrangement, even in low-grade cystic areas that resemble a pre-existing GOC, true GOCs do not. Accordingly, GOC does not appear to represent an early or low-grade form of central MEC, but rather an unrelated lesion. The high sensitivity and specificity of MAML2 rearrangement for MECs points to its utility as a diagnostic adjunct in separating mucinous cystic lesions of the gnathic bones.

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.