Germline BARD1 variants predispose to mesothelioma by impairing DNA repair and calcium signaling.

We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.

Risk prediction for metastasis of clear cell renal cell carcinoma using digital multiplex ligation-dependent probe amplification.

Precise quantification of copy-number alterations (CNAs) in a tumor genome is difficult. We have applied a comprehensive copy-number analysis method, digital multiplex ligation-dependent probe amplification (digitalMLPA), for targeted gene copy-number analysis in clear cell renal cell carcinoma (ccRCC). Copy-number status of all chromosomal arms and 11 genes was determined in 60 ccRCC samples. Chromosome 3p loss and 5q gain, known as early changes in ccRCC development, as well as losses at 9p and 14q were detected in 56/60 (93.3%), 31/60 (51.7%), 11/60 (18.3%), and 33/60 (55%), respectively. Through gene expression analysis, a significant positive correlation was detected in terms of 14q loss determined using digitalMLPA and downregulation of mRNA expression ratios with HIF1A and L2HGDH (P = .0253 and .0117, respectively). Patients with early metastasis (<1 y) (n = 18) showed CNAs in 6 arms (in median), whereas metastasis-free patients (n = 34) showed those in significantly less arms (3 arms in median) (P = .0289). In particular, biallelic deletion of CDKN2A/2B was associated with multiple CNAs (≥7 arms) in 3 tumors. Together with sequence-level mutations in genes VHL, PBRM1, SETD2, and BAP1, we performed multiple correspondence analysis, which identified the association of 9p loss and 4q loss with early metastasis (both P < .05). This analysis indicated the association of 4p loss and 1p loss with poor survival (both, P < .05). These findings suggest that CNAs have essential roles in aggressiveness of ccRCC. We showed that our approach of measuring CNA through digitalMLPA will facilitate the selection of patients who may develop metastasis.

Expression of ERBB gene family in females with breast cancer and its correlation with clinicopathological characteristics of the disease.

BACKGROUND: Breast cancer (BC) is the most prevalent and fatal cancer in women. Given that there are very few studies investigating the overexpression of four members of ERBB genes, we decided to investigate the correlation between these four genes with clinicopathological characteristics in breast cancer cases. METHODS: Tumoural tissue of 50 patients with sporadic invasive ductal BC was recruited. Also, control samples were provided from adjacent non-cancerous tissues (ANCTs) of the same patients. The expression of four ERBB genes was evaluated by real-time PCR and its correlation with clinicopathological characteristics was assessed. RESULTS: Only ERBB2 (HER2) was overexpressed in tumoural tissue compared with ANCTs. Our data showed a significant relationship between ERBB1 overexpression with triple-negative tumors, ER, and PR negativity (P < 0.05). Also, ERBB2 overexpression indicated a significant correlation with several pathological characteristics such as age < 50, tumor size larger than 2 cm, early and advanced stages, negative involved lymph nodes, luminal B, triple-negative, ERBB2-enrich, estrogen receptor (ER) and progesterone receptor (PR) negative tumors, Ki-67 mutation more than 15%, and finally HER2/neu immunohistochemistry (IHC) positive and intermediate (P < 0.05). Moreover, this study demonstrated that ERBB4 overexpression had a significant correlation with tumor size smaller than 2 cm, grade I and II tumors (early-stage tumors), luminal A, ER and PR positive tumors, HER-2/neu IHC intermediate, and tumors that had a Ki-67 mutation lower than 15% (P < 0.05). Besides, our analysis showed a significant correlation between the expression of ERBB1 with ERBB2 and ERBB3 with ERBB4 (P < 0.05). CONCLUSIONS: Our findings showed a significant relationship between unfavorable clinicopathological characteristics with ERBB1 and ERBB2 overexpression, but overexpression of ERBB4 was correlated with favorable outcomes.

Renal cell carcinoma with smooth muscle stroma lacks chromosome 3p and VHL alterations.

Renal cell carcinoma with prominent smooth muscle stroma is a rare neoplasm composed of an admixture of epithelial cell with clear cytoplasm arranged in small nest and tubular structures and a stroma composed of smooth muscle. In the epithelial component, loss of chromosome 3p detected by fluorescence in situ hybridization (FISH) has been reported and on this basis these neoplasms have been viewed as variants of clear cell renal cell carcinoma. To test the validity of this classification, we have evaluated the chromosome 3 and VHL status of three of these tumors using FISH, array comparative genomic hybridization, gene sequencing, and methylation-specific multiplex ligation-dependent probe amplification analysis. None of the tumors showed deletion of chromosome 3p, VHL mutation, a significant VHL methylation, or changes in VHL copy number and all three tumors demonstrated a flat profile in the comparative genomic hybridization analysis. We conclude that renal cell carcinoma with smooth muscle stroma should be considered as an entity distinct from clear cell renal cell carcinoma.

Hallmarks of a genomically distinct subclass of head and neck cancer.

Cancer is caused by an accumulation of somatic mutations and copy number alterations (CNAs). Besides mutations, these copy number changes are key characteristics of cancer development. Nonetheless, some tumors show hardly any CNAs, a remarkable phenomenon in oncogenesis. Head and neck squamous cell carcinomas (HNSCCs) arise by either exposure to carcinogens, or infection with the human papillomavirus (HPV). HPV-negative HNSCCs are generally characterized by many CNAs and frequent mutations in CDKN2A, TP53, FAT1, and NOTCH1. Here, we present the hallmarks of the distinct subgroup of HPV-negative HNSCC with no or few CNAs (CNA-quiet) by genetic profiling of 802 oral cavity squamous cell carcinomas (OCSCCs). In total, 73 OCSCC (9.1%) are classified as CNA-quiet and 729 as CNA-other. The CNA-quiet group is characterized by wild-type TP53, frequent CASP8 and HRAS mutations, and a less immunosuppressed tumor immune microenvironment with lower density of regulatory T cells. Patients with CNA-quiet OCSCC are older, more often women, less frequently current smokers, and have a better 5-year overall survival compared to CNA-other OCSCC. This study demonstrates that CNA-quiet OCSCC should be considered as a distinct, clinically relevant subclass. Given the clinical characteristics, the patient group with these tumors will rapidly increase in the aging population.

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals.

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural 'linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regulatory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manner.

The Drosophila copper transporter Ctr1C functions in male fertility.

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.

Characterization of MtnE, the fifth metallothionein member in Drosophila.

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor 1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missing.

Copy number evolution and its relationship with patient outcome-an analysis of 178 matched presentation-relapse tumor pairs from the Myeloma XI trial.

Structural chromosomal changes including copy number aberrations (CNAs) are a major feature of multiple myeloma (MM), however their evolution in context of modern biological therapy is not well characterized. To investigate acquisition of CNAs and their prognostic relevance in context of first-line therapy, we profiled tumor diagnosis-relapse pairs from 178 NCRI Myeloma XI (ISRCTN49407852) trial patients using digital multiplex ligation-dependent probe amplification. CNA profiles acquired at relapse differed substantially between MM subtypes: hyperdiploid (HRD) tumors evolved predominantly in branching pattern vs. linear pattern in t(4;14) vs. stable pattern in t(11;14). CNA acquisition also differed between subtypes based on CCND expression, with a marked enrichment of acquired del(17p) in CCND2 over CCND1 tumors. Acquired CNAs were not influenced by high-dose melphalan or lenalidomide maintenance randomization. A branching evolution pattern was significantly associated with inferior overall survival (OS; hazard ratio (HR) 2.61, P = 0.0048). As an individual lesion, acquisition of gain(1q) at relapse was associated with shorter OS, independent of other risk markers or time of relapse (HR = 2.00; P = 0.021). There is an increasing need for rational therapy sequencing in MM. Our data supports the value of repeat molecular profiling to characterize disease evolution and inform management of MM relapse.

High-Throughput Molecular Cancer Cell Line Characterization Using Digital Multiplex Ligation-Dependent Probe Amplification for Improved Standardization of in Vitro Research.

Tumor cell lines are widely used for cancer research, but challenges regarding quality control of cell line identity, cross contamination, and tumor somatic molecular stability remain, demanding novel approaches beyond conventional short tandem repeat profiling. A total of 21 commonly used multiple myeloma cell lines obtained from public repositories were analyzed by digital multiplex ligation-dependent probe amplification (digitalMLPA) to characterize germline single-nucleotide polymorphisms, insertions/deletions, and somatic copy number aberrations (CNAs). Using generated profiles and an in-house developed analytical pipeline, blinded experiments were performed to determine capability of digitalMLPA to predict cell line identity and potential spike-in DNA contamination in 41 anonymized cell line samples. The dominant cell line was correctly identified in all cases, and cross contamination was correctly detected in 33 of 37 samples with spike-in DNA; there were no false-positive predictions. The four samples in which spike in was not detected all carried low levels of contamination (1%), whereas levels of contamination ≥5% were correctly identified in all cases. Unsupervised clustering of CNA profiles identified shared commonalities that correlated with initiating Ig heavy locus translocation events. Longitudinal CNA assessment of nine cell lines revealed changes under standard culturing conditions not detected by insertion/deletion profiling alone. Results suggest that digitalMLPA can be utilized as a high-throughput tool for advanced quality assurance for in vitro cancer research.

Working together for the family: determination of HER oncogene co-amplifications in breast cancer.

HER2 is a well-studied tyrosine kinase (TK) membrane receptor which functions as a therapeutic target in invasive ductal breast carcinomas (IDC). The standard of care for the treatment of HER2-positive breast is the antibody trastuzumab. Despite specific treatment unfortunately, 20% of primary and 70% of metastatic HER2 tumors develop resistance. HER2 belongs to a gene family, with four members (HER1-4) and these members could be involved in resistance to anti-HER2 therapies. In this study we designed a probemix to detect the amplification of the four HER oncogenes in a single reaction. In addition, we developed a protocol based on the combination of MLPA with ddPCR to detect the tumor proportion of co-amplified HERs. On 111 IDC, the HER2 MLPA results were validated by FISH (Adjusted r 2 = 0,91, p < 0,0001), CISH (Adjusted r 2 = 0,938, p < 0,0001) and IHC (Adjusted r 2 = 0,31, p < 0,0001). HER1-4 MLPA results were validated by RT-qPCR assays (Spearman Rank test p < 0,05). Of the 111 samples, 26% presented at least one HER amplified, of which 23% showed co-amplifications with other HERs. The percentage of cells with HER2 co-amplified varied among the tumors (from 2-72,6%). Independent in-silico findings show that the outcome of HER2+ patients is conditioned by the status of HER3 and HER4. Our results encourage further studies to investigate the relationship with patient's response to single or combined treatment. The approach could serve as proof of principle for other tumors in which the HER oncogenes are involved.

Application of Multiplex Ligation-Dependent Probe Amplification in Determining the Copy Number Alterations of HER Gene Family Members in Invasive Ductal Breast Carcinoma.

BACKGROUND: The aim of this study was to assess the usability of multiplex ligation-dependent probe amplification (MLPA) for copy number determination of HER gene family members (ERBB1-4) in invasive breast carcinoma and to explore the association of ERBB1-4 gene copy numbers with clinicopathological characteristics of breast cancer (BC) patients. METHODS: Clinical and immunohistochemical characteristics were assessed in 104 BC patients and the molecular subtype was determined for each tumor sample. Furthermore, HER-2/neu status was assessed by immunohistochemistry (IHC) and equivocal results were confirmed by Fluorescent in situ hybridization (FISH). The copy numbers of ERBB1-4 genes were determined by MLPA. RESULTS: Twenty-five percent of all patients showed ERBB2 gene-amplification by MLPA, whereas 14.4% of cases showed ERBB-2/neu overproduction at the protein level (IHC). Moreover, only 2.9% and 1.9% of patients showed amplification in ERBB1 and ERBB4, respectively. No copy number changes were observed in ERBB3. Our results indicated a significant association between ERBB2 copy number gain and histological grade (p value= 0.01), stage (p value= 0.02), and tumor subtypes (p value= <0.001). In addition, we found MLPA more accurate in assessing HER2 status with 15.4% and 9.6% gene amplification detection in early stages (1, 2A and 2B) and advanced tumor stages (3A, 3B, and 4), respectively, compared to IHC (early stages= 13.5% and advanced stages= 4.7%). CONCLUSION: According to our findings, MLPA is a fast, precise and low-cost technique to detect ERBB2 amplification, especially in advanced tumor stages. However, due to infrequent amplification found in ERBB1 and ERBB4 as well as the lack of amplification in ERBB3, their importance in the prognostic evaluation of BC patients remains controversial.

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila.

BACKGROUND: Metal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans. Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans. RESULTS: Constitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females. CONCLUSION: Our studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility.

Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA).

This chapter describes a method for the rapid assessment of promoter hypermethylation levels or methylation of imprinted regions in human genomic DNA extracted from various sources using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Multiplex ligation-dependent probe amplification (MLPA) is a powerful and easy-to-perform PCR-based technique that can identify gains, amplifications, losses, deletions, methylation and mutations of up to 55 targets in a single reaction, while requiring only minute quantities of DNA (about 50 ng) extracted from blood, fresh frozen or formalin-fixed paraffin-embedded materials. Methylation-specific MLPA (MS-MLPA) is a variant of MLPA, which does not require sodium bisulfite conversion of unmethylated cytosine residues, but instead makes use of the methylation-sensitive endonuclease HhaI. MS-MLPA probes are designed to contain a HhaI recognition site (GCGC) and thus target one CpG dinucleotide within a CpG island. If the HhaI recognition site is not methylated, HhaI will cut the probe-sample DNA hybrid and no PCR product will be formed. If the target DNA is methylated, HhaI is not able to cut, and the fragment will be amplified during subsequent PCR. For data analysis, MS-MLPA peak patterns of the HhaI-treated and -untreated reactions are compared, leading to calculation of a methylation percentage. The methylation profile of a test sample is assessed by comparing the probe methylation percentages obtained on the test sample to the percentages of the reference samples. MS-MLPA can be combined with copy number and point mutation detection in the same reaction.

High-Throughput Copy Number Profiling by Digital Multiplex Ligation-Dependent Probe Amplification in Multiple Myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease with a diverse clinical outcome. Copy number alterations (CNAs), including whole chromosome and subchromosomal gains and losses, are common contributors of the pathogenesis and have demonstrated prognostic impact in MM. We tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA), a novel technique combining MLPA and next-generation sequencing, to detect disease-related CNAs. Copy number status at 371 genomic loci was simultaneously analyzed in 56 diagnostic bone marrow samples, which were also examined by conventional MLPA and interphase fluorescence in situ hybridization (iFISH). On average, digitalMLPA identified 4.4 subchromosomal CNAs per patient. The increased number of probes compared with conventional MLPA allowed a detailed mapping of CNAs, especially on chromosome 1, where 24 different patterns were observed in 38 patients harboring loss(1p) and/or gain(1q). iFISH, MLPA, and digitalMLPA results at loci investigated by multiple methods showed a congruency of 95%. Besides precise characterization of hyperdiploid karyotypes not efficiently achievable by iFISH or MLPA, digitalMLPA unraveled 156 CNAs not detected by the other two methods in 45 patients (80%). In addition, we provide proof of principle that digitalMLPA can detect known point mutations, in this case the BRAFV600E. Our study demonstrates the robustness of digitalMLPA to profile CNAs and to screen point mutations in MM, which could efficiently be used in myeloma diagnostics.

Accurate detection and quantification of epigenetic and genetic second hits in BRCA1 and BRCA2-associated hereditary breast and ovarian cancer reveals multiple co-acting second hits.

BACKGROUND: This study characterizes the second hit spectrum in BRCA1 and BRCA2-associated breast and ovarian cancers at both gene loci to investigate if second hit mechanisms are mutually exclusive or able to coincide within the same tumor. METHODS: Loss of heterozygosity, somatic point mutations and copy number alterations along with promoter methylation were studied in 56 breast and 15 ovarian cancers from BRCA1 and BRCA2 germline mutation carriers. A mathematical methodology was introduced to quantify the tumor cell population carrying a second hit. RESULTS: Copy neutral LOH was the most prevalent LOH mechanism in this cohort (BC 69%, OC 67%). However, only 36% of BC and 47% of OC showed LOH in all cancerous cells. Somatic intragenic deletions and methylated subclones were also found in combination with (partial) loss of heterozygosity. Unequivocal deleterious somatic point mutations were not identified in this cohort. CONCLUSION: Different mechanisms inactivating the wild type allele are present within the same tumor sample at various extents. Results indicate that BRCA1/2-linked breast and ovarian cancer cells are predominantly characterized by LOH, but harbor a complex combination of second hits at various frequencies.

Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

OBJECTIVES: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. METHODS: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. RESULTS: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. CONCLUSIONS: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

"Atypical" Pleomorphic Lipomatous Tumor: A Clinicopathologic, Immunohistochemical and Molecular Study of 21 Cases, Emphasizing its Relationship to Atypical Spindle Cell Lipomatous Tumor and Suggesting a Morphologic Spectrum (Atypical Spindle Cell/Pleomorphic Lipomatous Tumor).

The classification of the until recently poorly explored group of atypical adipocytic neoplasms with spindle cell features, for which recently the term atypical spindle cell lipomatous tumor (ASLT) has been proposed, remains challenging. Recent studies have proposed ASLT as a unique entity with (in at least a significant subset of cases) a specific genetic background, namely deletions/losses of 13q14, including RB1 and its flanking genes RCBTB2, DLEU1, and ITM2B. Similar genetic aberrations have been reported in pleomorphic liposarcomas (PLSs). This prompted us to investigate a series of 21 low-grade adipocytic neoplasms with a pleomorphic lipoma-like appearance, but with atypical morphologic features (including atypical spindle cells, pleomorphic [multinucleated] cells, pleomorphic lipoblasts and poor circumscription), for which we propose the term "atypical" pleomorphic lipomatous tumor (APLT). Five cases of PLS were also included in this study. We used multiplex ligation-dependent probe amplification to evaluate genetic changes of 13q14. In addition, array-based comparative genomic hybridization was performed on 4 APLTs and all PLSs. Multiplex ligation-dependent probe amplification showed consistent loss of RB1 and its flanking gene RCBTB2 in all cases of APLT. This genetic alteration was also present in all PLSs, suggesting genetic overlap, in addition to morphologic overlap, with APLTs. However, array-based comparative genomic hybridization demonstrated more complex genetic alterations with more losses and gains in PLSs compared with APLTs. APLTs arose in the subcutis (67%) more frequently than in the deep (subfascial) soft tissues (33%). With a median follow-up of 42 months, recurrences were documented in 2 of 12 APLTs for which a long follow-up was available. Herein, we also demonstrate that APLTs share obvious overlapping morphologic, immunohistochemical, genetic and clinical characteristics with the recently defined ASLT, suggesting that they are related lesions that form a spectrum (atypical spindle cell/pleomorphic lipomatous tumor).

Validation of 34betaE12 immunoexpression in clear cell papillary renal cell carcinoma as a sensitive biomarker.

Clear cell papillary renal cell carcinoma (CCPRCC) is a recently recognised neoplasm with a broad spectrum of morphological characteristics, thus representing a challenging differential diagnosis, especially with the low malignant potential multicystic renal cell neoplasms and clear cell renal cell carcinoma. We selected 14 cases of CCPRCC with a wide spectrum of morphological features diagnosed on morphology and CK7 immunoreactivity and analysed them using a panel of immunohistochemical markers, focusing on 34ßE12 and related CKs 1,5,10 and 14 and several molecular analyses such as fluorescence in situ hybridisation (FISH), array comparative genomic hybridisation (aCGH), VHL methylation, VHL and TCEB1 sequencing and multiplex ligation-dependent probe amplification (MLPA). Twelve of 13 (92%) CCPRCC tumours were positive for 34ßE12. One tumour without 3p alteration by FISH revealed VHL mutation and 3p deletion at aCGH; thus, it was re-classified as clear cell RCC. We concluded that: (1) immunohistochemical expression of CK7 is necessary for diagnostic purposes, but may not be sufficient to identify CCPRCC, while 34ßE12, in part due to the presence of CK14 antigen expression, can be extremely useful for the recognition of this tumour; and (2) further molecular analysis of chromosome 3p should be considered to support of CCPRCC diagnosis, when FISH analysis does not evidence the common loss of chromosome 3p.