Chromatin Pattern Research Articles

The Microscopic Complexities of C3 in Breast Cytology

Background: Fine-needle aspiration (FNA) of difficult breast lesions often results in an atypical (C3) report. The assortment of outcomes generated by C3 reports varies widely, and this has given rise to different clinical management pathways. Objective: To identify and objectively assess microscopic features associated with atypical/C3 breast FNA cases. Materials and Methods: A total of 230 atypical breast FNAs were subjected to a blind microscopic rescreen using a range of robust qualitative and quantitative cytological criteria including cellularity, architectural qualities, cytomorphology and background features. A logistic regression with a receiver operating characteristic (ROC) curve and the resultant forward stepwise analysis were conducted to assess the results. This statistical testing was measured against malignant, benign proliferative and benign non-proliferative outcomes. Results: The malignant and benign proliferative outcomes showed a mixture of opposing protective and predictive individual cytological criteria. The stepwise analysis produced models demonstrating the best combination of individual cytological criteria for malignancy, proliferative and benign non-proliferative entities. In the malignancy model, discohesion, nuclear crowding within sheets, diminished numbers of bare bipolar nuclei and myoepithelial cells, the presence of tubules or necrosis and the absence of a cystic background were important features. The benign proliferative model suggested the same criteria but with the opposite implication and with the addition of several others, such as the presence of apocrine metaplasia, retained polarity and a speckled or coarse chromatin pattern. Age was a significant factor in malignant and proliferative outcomes. The benign non-proliferative stepwise analysis produced a model with fewer criteria (complex sheets, bare bipolar nuclei and a cystic background) limiting clinical application. Conclusion: Atypical/C3 breast cytology remains a legitimate reporting category. However, it is associated with a number of different histological outcomes. The incidence of the C3 category can be significantly reduced by controlling extrinsic factors and understanding the associated microscopic features.

Sequence-Level Mechanisms of Human Epigenome Evolution

DNA methylation and chromatin states play key roles in development and disease. However, the extent of recent evolutionary divergence in the human epigenome and the influential factors that have shaped it are poorly understood. To determine the links between genome sequence and human epigenome evolution, we examined the divergence of DNA methylation and chromatin states following segmental duplication events in the human lineage. Chromatin and DNA methylation states were found to have been generally well conserved following a duplication event, with the evolution of the epigenome largely uncoupled from the total number of genetic changes in the surrounding DNA sequence. However, the epigenome at tissue-specific, distal regulatory regions was observed to be unusually prone to diverge following duplication, with particular sequence differences, altering known sequence motifs, found to be associated with divergence in patterns of DNA methylation and chromatin. Alu elements were found to have played a particularly prominent role in shaping human epigenome evolution, and we show that human-specific AluY insertion events are strongly linked to the evolution of the DNA methylation landscape and gene expression levels, including at key neurological genes in the human brain. Studying paralogous regions within the same sample enables the study of the links between genome and epigenome evolution while controlling for biological and technical variation. We show DNA methylation and chromatin divergence between duplicated regions are linked to the divergence of particular genetic motifs, with Alu elements having played a disproportionate role in the evolution of the epigenome in the human lineage.

Cyto-histopathological and outcome features of the prepuce squamous cell carcinoma of a mixed breed dog.

BackgroundSquamous cell carcinomas (SCCs) are uncommon, high-grade tumors, predominantly composed of round cells in the prepuce. The aim of this study is to better define the clinicopathologic features of this neoplasm.Case reportWe conducted cyto-histopathologic analysis on the manifestations of the prepuce SCC by H & E staining in a terrier mix dog. Grossly, tumor was large, multiple erythematous patch, and ulcerated masses frequently affecting the prepuce and deeply invading to distal prepuce out from the ventro-lateral of penis and the tumor covered by a necrotic discharge. Cytological evaluation of fine-needle aspirates from the cutaneous mass from the prepuce comprised of round nuclei, coarse chromatin pattern, distinct nucleoli and nuclear pleomorphism. Furthermore, the neoplastic cells were pleomorphic, round to caudate in shape, exhibiting prominent anisokaryosis and anisocytosis with rare mitotic features. Microscopically, the lesions were predominantly composed of atypical round cells disposed in interlacing fascicles. Frequent findings include keratin formation, horn pearls, mitoses and cellular atypia. The cells showed distinct borders, ranged from polygonal to round or elongate and had moderate amounts of eosinophilic cytoplasm.ConclusionThe histopathologic features coupled with the cytopathology findings led to a diagnosis of squamous cell carcinoma. To the authors’ knowledge, this is the first time that multiple erythematous plaques have undergone malignant transformation in a terrier mix dog.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5748771971272873

Features of atypical granular cell tumor on fine needle aspiration in an 8-month-old child: a case report

Granular cell tumors (GCT) are uncommon, generally benign soft tissue neoplasms that can affect virtually any site of the body, the most common locations being tongue, breast, upper respiratory tract, skin and soft tissues of upper extremities. Most patients are middle-aged, with a peak incidence in the fourth to sixth decade of life. GCT commonly presents as an asymptomatic slowly growing, painless nodule. Atypical and malignant counterparts are rare. Herein, we report on the case of an atypical GCT in an 8-months-old baby who presented at our Department with well-defined papulomatous lesion of one month duration on his back. A solitary, firm, painless nodule, about 1.5 cm in size was found on clinical examination. Fine needle aspiration cytology (FNAC) of the lesion was performed using a 22-gauge needle. Smears were stained with May-Grunwald-Giemsa and Papanicolaou stain. Cytomorphology revealed highly cellular tumor composed of loosely dispersed group of cells and stripped bare nuclei. The cells were large, polygonal to spindle-shaped with an abundant eosinophilic granular and fragile cytoplasm and round to oval nucleus with fine chromatin pattern. Focally cells were larger with prominent nucleoli. Rare mitotic figures were present. Diagnosis of malignant mesenchymal tumor was suggested and excision was advised. Histological examination revealed tumor tissue composed of large, polygonal cells with granular eosinophilic cytoplasm, vesicular nuclei, moderate nuclear pleomorphism and focally prominent nucleoli. There were 2 mitosis per 10 HPF. The tumor cells were positive for S-100 and vimentin. A diagnosis of atypical GCT was established. In conclusion, benign GCT has a distinctive cytomorphologic appearance and can be diagnosed on the basis of the cytological examination alone. Nuclear pleomorphism, spindling of the cells, prominent nucleoli and mitotic figures are worrisome features that suggest malignancy and necessity of histological examination. Considering the patient's age and atypical morphology, to the best of our knowledge, there have been up to 15 GCT cases described in medical literature by now in the children less than one year.

Slight variations in the SC35 ESE sequence motif among human chromosomes: a computational approach.

Gene expression is initiated by the binding of transcription factors to cis-regulatory modules such as enhancer elements binding to the Serine/Arginine proteins. Recently, we noticed an increased ability to identify the location as well as the motifs of enhancers using genome-wide information on spliceosomal factor occupancy, cofactor recruitment and chromatin modifications. In this study, we have undertaken a large-scale genomic analysis in an attempt to uncover if the exonic splicing enhancer motif binding to the SC35 and the SRp40 SR proteins is conserved among several groups of human genes. For the SRp40, the results showed that the ESE consensus is conserved among human genes. Concerning the SC35 SR protein, results showed an ESE motif conserved among human tissues and between different levels of muscular cell differentiation and within the same chromosome. However, this motif displays subtle discrepancies between genes localized in different chromosomes. These results emphasize the presence of different translational isoforms of the SFRS2 gene encoding for the SC35, or different post-translational protein maturations in different chromosomes, confirming that chromatin structure is another layer of gene regulation. These links between chromatin pattern and splicing give further mechanistic support to functional interconnections between splicing, transcription and chromatin structure, and raise the intriguing possibility of the existence of a memory for splicing patterns to be inherited through epigenetic modifications.

Mitochondrial Patterns in Bovine Oocytes with Different Meiotic Competence Related to Their in vitro Maturation

This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.

Cytologic features of the normal pineal gland on squash preparations

As primary pineal lesions are extremely rare, many surgical pathologists are unfamiliar with normal pineal cytologic features. We describe cytologic features of the normal pineal gland in patients of varying ages and identify common diagnostic pitfalls. We performed a retrospective review of pineal gland biopsies performed at our institution, where approximately 30,000 surgical specimens are accessioned yearly, for the last 23 years. Only two pineal gland biopsies were found. Although both cases were initially diagnosed as low-grade gliomas on frozen section, the final diagnosis was benign pineal tissue based on light microscopy and immunohistochemistry results. Additionally, we performed squash preparations of five normal pineal gland autopsy specimens with Papanicolaou and Diff-Quik® (Dade Behring, Newark, DE) stains. Infant preparations were highly cellular smears composed of numerous, uniform, single cells with indistinct cytoplasm, small round-to-oval nuclei, fine chromatin, and absent nucleoli and calcifications. The vague microfollicular pattern mimicked a pineocytoma and the fine fibrillary background mimicked a glial neoplasm. Young adult smears were similar; however, microcalcifications were present with fewer background single cells. Older patients had much less cellular smears composed of small clusters of cells with fusiform-to-spindle nuclei, a fine chromatin pattern, and indistinct cytoplasmic borders. There were fewer background single cells and more microcalcifications. The cytologic features of the native pineal gland vary with age. Normal pineal tissue can be confused with a pineocytoma or low-grade glioma. Familiarity with normal pineal gland cytological features will help to avoid a potential misdiagnosis.

Homozygous Pelger-Huet anomaly in three different crossbred rabbits: a case report

In this case report, three different crossbreedings of pet rabbits were performed producing affected as well as healthy bunnies. All affected rabbits were smaller and had local alopecia, exophthalmus, and limb deviations compared to their healthy littermates; thus, a homozygous form of Pelger-Huet anomaly was suspected. This anomaly was confirmed by blood examination in which granulocytes with oval nuclei and a very coarse chromatin pattern, as well as lymphocytes with micronuclei were noticed. Karyotype analyses of the lymphocytes revealed many chromosomal aberrations in affected rabbits. Moreover, severe arterial abnormalities in the pelvic cavity and proximal part of the pelvic limbs were also found in these rabbits. Our findings suggest a multigenic origin of Pelger-Huet anomaly in rabbits, because only male and female offspring with the otter colour of fur were severely affected by this congenital disorder.  

Gastrointestinal: Pancreatic tuberculosis masquerading as malignancy

A 73 year old man presented with a 2 months history of abdominal pain, occasional night sweats, and 4 Kg weight loss. Laboratory evaluation showed a mild normocytic anemia and erythrocyte sedimentation rate (ESR) of 78 mm. Serum levels of CA 19-9, amylase, and liver enzymes were within normal limits. An abdominal CT scan revealed a heterogeneous mass lesion in the pancreatic head (Figure 1; black arrow), and lymphadenopathies in the portahepatis and para-aortic regions. Endoscopic ultrasound (EUS) demonstrated a 20 mm solidcystic mass lesion in the pancreatic head (Figure 2). EUS-guided fine needle aspiration (FNA) from the pancreatic mass was performed. Cytology showed aggregation of epithelioid histiocytes with regular oval nuclei with smooth nuclear borders and fine chromatin pattern on HE 600X magnification). The smears stained by Ziehl Neelsen method showed beaded, rod shaped acid fast bacilli (Figure 3b; black arrow; 1500X magnification). Polymerase chain reaction of the FNA sample became positive for Mycobacterium tuberculosis. The patient received a 6-month course of anti-tubeculosis therapy. His symptoms completely resolved on treatment and ESR returned back to normal at the end of treatment. The patient is doing well one year after completion of therapy. Pancreatic tuberculosis (TB) is a very rare disorder. It is speculated that the antibacterial effects of the pancreatic enzymes may prevent the development of this disorder. Pancreatic TB presents as a mass lesion in the pancreas and mimics pancreatic malignancy. As it is evident in Figure 2, the mass lesion was solid-cystic on EUS. The main differential diagnoses of pancreatic solid-cystic masses include cystic neuroendocrine tumor, mucinous cystadenocarcinoma, serous cystadenoma, solid pseudopapillary neoplasm and cystic degeneration of pancreatic adenocarcinoma. Pancreatic TB should also be considered in the differential diagnosis of pancreatic solid-cystic mass lesions. The diagnosis of pancreatic TB has traditionally been made on biopsy samples taken during surgery. However, EUS-guided FNA is increasingly being used to make the diagnosis. Pancreatic TB should be suspected in patients with pancreatic solid-cystic mass lesions who have conditions such as living in or emigrating from endemic areas, presence of predominant constitutional symptoms, normal serum CA 19-9 level, and associated lymphadenopathies in the portahepatis and paraaortic regions.

Cytodiagnosis of scar endometriosis

Sir, Many non-neoplastic and neoplastic lesions that occur on anterior abdominal wall can be confused with scar endometriosis on clinical grounds. Non-neoplastic lesions include hematoma, suture granuloma, abscess, incisional hernia, focal fibrosis, desmoid tumor, keloid and melanocytic nevus. Neoplasms to be differentiated may be primary (benign or malignant) or metastatic (sister Mary Josephs nodule). Scar endometriosis can be differentiated from most of these only on the basis of detailed history about the swelling, careful clinical examination and characteristic cytological features.[1] We present a case that initially posed a problem in diagnosis as patient could not be seen by the reporting cytopathologist and important clinical indicators were missed. A 35-year-old female patient presented with the complaints of gradually increasing swelling and pain in the infra-umbilical region since last 3 years. Patient did not have swellings at any other site or any other complaints. She gave history of two full term deliveries by cesarean section; her last childbirth was 5 years back. A 3 cm × 3 cm, firm non-mobile swelling was present on anterior abdominal wall below umbilicus. Skin over the swelling was adherent and discolored [Figure 1]. Clinical diagnosis of soft-tissue tumor was offered and cytology study was advised. Abdominal sonography of patient revealed a poorly defined lesion in the subcutaneous plane with minimal vascularity and without peritoneal communication. The impression given was abdominal wall desmoid. Figure 1 Anterior abdominal wall swelling 3×3 cm with discoloured overlying skin near a scar mark Fine needle aspiration cytology smears from the tender infraumbilical swelling showed high cellularity of glandular and stromal cells [Figure 2]. Glandular cells were arranged in tight clusters and sheets, had round nuclei with uniform chromatin pattern [Figure 3]. Altered blood, debris and foamy macrophages were seen in the background [Figure 4]. Some macrophages showed presence of hemosiderin pigment. Figure 2 Bimodal population of endometrial glandular and stromal cells seen as compact fragments in haemorrhagic background (PAP ×100) Figure 3 Honeycomb sheets of glandular cells without nuclear atypia (PAP ×400) Figure 4 A group of macrophages in the background of blood and debris (PAP ×400) Inset-Hemosiderin laden macrophage High cellularity in a dirty background observed in cytology smears initially puzzled us. After carefully examining the swelling and eliciting, the history of increase in the size of the swelling and pain during the menstrual cycle: Noticing the location of the swelling near a scar mark and Appreciating the bland nuclear pattern in glandular and stromal cells in all the smears diagnosis of scar endometriosis could be given. Endometriosis is defined as the presence of functioning endometrium outside the uterus. Occurrence of endometriosis is described usually in the pelvic region and in many unusual and uncommon extra pelvic sites.[2] Only cutaneous and subcutaneous endometriosis are easily accessible to cytological study. Scar endometriosis-a form of cutaneous endometriosis is generally seen in women of reproductive age group.[3] History of catamenial exacerbation, i.e., increase in pain and tenderness in a lump during menstruation and location of a firm, tender swelling near a scar either of C section, laparotomy, hysterectomy or episiotomy point toward the diagnosis of scar endometriosis.[4] On radiological examination, the swelling is in the subcutaneous plane with no intraperitoneal communication. Characteristic cytological features include good cellularity of glandular and stromal cells with uniform, bland nuclei in the background of altered blood, debris and foamy macrophages, some of them containing hemosiderin. The cytological features of endometriosis are related to cyclic hormonal changes. In proliferative phase, glandular cells are seen as honeycomb sheets and tight clusters, have round or oval nuclei with evenly distributed chromatin. Stromal cells form syncytial clusters and have plump spindle nuclei with fine chromatin. In the secretory phase, glandular cells show an increase in cell size and microvacuolation while stromal cells reveal abundant cytoplasm.[4] Diagnosis of endometriosis is not always straight forward. Difficulties arise when it arises in unusual clinical setting and without characteristic bimodal pattern in the smears. When atypical cytological features are seen in smears histological examination is advisable in order to differentiate endometriosis from malignancy.[5] Fine needle aspiration cytology remains a reliable and relatively atraumatic investigation for primary pre-operative diagnosis in most cases if the cytopathologist is aware of the entity and interprets the cytological features in the light of clinical findings of: A reproductive age group female with discolored swelling near a scar mark on anterior abdominal wall History of catamenial exacerbation Location of swelling in the subcutaneous plane on radiological examination. Cytology is a method of choice to monitor treatment and possible malignant transformation in a case of endometriosis.

Endoscopic ultrasound-guided fine-needle aspiration cytology findings from a case of ectopic pancreas of the stomach – cytological differential diagnosis with acinar cell carcinoma

Ectopic pancreas is a relatively common lesion as gastric submucosal tumor (SMT), but it makes little mention of cytological findings in endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). Herein, we present a case of ectopic pancreas diagnosed by EUS-FNA, and discuss the cytological differential diagnosis from ACC. A 41-year-old woman was admitted to our hospital with gastric SMT, measuring 19 mm in diameter. EUS-FNA was performed. Cytologic specimens showed clustered or scattered small cells. Nuclei were relatively small and slightly irregular shaped. Their chromatin pattern was fine granular, and nucleoli were small but conspicuous. Most cells had pink granular substance on Pap staining. Histologic specimens were prepared from tiny pieces simultaneously obtained at EUS-FNA, which revealed benign pancreatic tissue with small nucleoli and eosinophilic granules. Immunohistology showed positivity for chymotrypsin. The lesion was diagnosed as ectopic pancreas. Ectopic pancreas has been reported to show slightly nuclear atypia and small nucleoli in EUS-FNA. In this case, benign acinar cells also showed slightly nuclear atypia. The findings indicate some similarities with ACC. However, ACC usually shows high cellularity compared with ectopic pancreas. It also lacks pink granular substance on Pap staining. These findings are useful to differentiate from ACC.

Histopathologic Findings of Small Renal Tumor Biopsies Performed Immediately After Cryoablation Therapy

To evaluate the histopathology of small renal tumor biopsies following cryoablation. We retrospectively evaluated small renal tumor biopsy specimens after cryoablation treatment for renal cell carcinoma and determined the ability to differentiate tumor types, effect on nuclear grading, immunohistochemical staining, and if the number of freeze cycles affected interpretation. Of the biopsy specimens, 66% were diagnostic of tumor and 34% showed normal renal parenchyma. Tumor subtype was determined in 91% of diagnostic cases. Nuclear grading was affected due to freeze effect, complicating the assessment of chromatin pattern and nucleolar details at low magnification. In particular, the distinction between Fuhrman nuclear grades I and II was compromised; these were designated as low nuclear grade. Immunohistochemical staining was retained similar to untreated tumors. Tumor subtyping was not affected after one or two freeze cycles. Biopsies performed immediately after cryoablation can be used to render an optimal histologic diagnosis.

Chromatin states reveal functional associations for globally defined transcription start sites in four human cell lines.

BackgroundDeciphering the most common modes by which chromatin regulates transcription, and how this is related to cellular status and processes is an important task for improving our understanding of human cellular biology. The FANTOM5 and ENCODE projects represent two independent large scale efforts to map regulatory and transcriptional features to the human genome. Here we investigate chromatin features around a comprehensive set of transcription start sites in four cell lines by integrating data from these two projects.ResultsTranscription start sites can be distinguished by chromatin states defined by specific combinations of both chromatin mark enrichment and the profile shapes of these chromatin marks. The observed patterns can be associated with cellular functions and processes, and they also show association with expression level, location relative to nearby genes, and CpG content. In particular we find a substantial number of repressed inter- and intra-genic transcription start sites enriched for active chromatin marks and Pol II, and these sites are strongly associated with immediate-early response processes and cell signaling. Associations between start sites with similar chromatin patterns are validated by significant correlations in their global expression profiles.ConclusionsThe results confirm the link between chromatin state and cellular function for expressed transcripts, and also indicate that active chromatin states at repressed transcripts may poise transcripts for rapid activation during immune response.

Progressive multifocal leukoencephalopathy – report of a case with touch cytology

Progressive multifocal leukoencephalopathy (PML) is an opportunistic demyelinating disease caused by the ubiquitous, usually polyomavirus (DNA virus) belonging to Papovaviridae group. Up to 85% of all cases are associated with HIV. PML is the only known clinical manifestation of JC virus infection. There is lysis of myelin producing oligodendrocytes. Perivascular inflammation is usually absent. Case report We report a case on the basis of clinical features, neuroradiology and brain biopsy/cytology smears. A 34-year-old woman was admitted with a new-onset seizure disorder. On computed tomography, there were right frontoparietal cortex, right external capsule and right basal ganglia lucencies. Core needle biopsy of the radiolucent area revealed foci of white matter demyelination and a few eosinophilic inclusions in oligodendrocytes plus abnormal giant astrocytes, ’Creutzfeldt astrocytes’. Smears showed moderate cellularity, consisting of moderately to markedly atypical cells with enlarged hyperchromatic nuclei. The chromatin pattern showed smudging, with or without clumping, similar in pattern to the human polyomavirus-infected ’decoy’ cells seen in urine cytology. Nuclei were predominantly round to oval, smoothly contoured and often stripped of cytoplasm. Occasional bizarre lobulated or multinucleated forms were seen. Some atypical cells had abundant cytoplasm exhibiting stellate projections. Histologic sections of the biopsy material confirmed the PML diagnosis. The present findings suggest that PML should be considered in the differential diagnosis of marked cytologic atypia in brain aspirates from immunocompromised patients. Positive immunohistochemical staining of oligodendrocytes for JC virus proteins. Conclusion Diagnostic cranial CT-guided FNAB, with cytologic and histologic studies, is extremely valuable in evaluating the nature of central nervous system demyelinated and space-occupying lesions.

New Translocation (7:17) In Variant Acute Promyelocytic Leukemia With ATRA Resistance

BackgroundAcute promyelocytic leukemia (APL) is characterized by pathologic promyelocytes, chromosomal translocations t(15:17) (q22; q21) and clinical and cytological response to all-transretinoic acid (ATRA). A significant minority of patients with APL have variant translocations involving RARα gene with a different partner, not involving PML. These APL variant translocations may or not respond to ATRA treatment. Case reportA routine laboratory evaluation, before minor surgery, revealed leukocytosis, thrombocytopenia and blasts presence in peripheral blood (PB), in an otherwise asymptomatic 70-year-old man. He had a previous history of hypertension, dyslipidemia, ischemic heart disease, double coronary bypass and prostatectomy for non-malignant prostatic hypertrophy.Peripheral blood (PB) count: hemoglobin concentration 12g/dL, platelet count: 21x109/L and white blood cell count (WBC): 35x109/L. Peripheral blood smear: myelemia exhibiting 74% blasts (including myeloid blast and promyelocytes) with fine chromatin pattern, heterogeneous and frequently convoluted nuclei and one to three prominent nucleoli. The cytoplasm of these cells was basophilic with scanty purple granules. No Auer rods were found. The basic coagulation tests were normal(INR, APTT and fibrinogen) but D Dimer was 33.87 µg/ml. Normal biochemistry was shown, except for GGT 85 U/L and lactate dehydrogenase 2244 U/L.Bone marrow (BM) aspirate was unsuccessful (”dry tap”) but the bone trephine revealed an infiltration of 80% myeloid blasts. Cytochemistry: myeloperoxidase stain was deeply positive and chloroacetate and non-specific esterase stains were negative. Flow cytometry immunophenotype on PB was consistent with AML, likely M3 leukemia, with an atypical phenotype (MPO+, CD117+low, CD34+, CD13++, CD45+, CD15-,CD133-, CD33+, HLA-DR+low het , CD5-, CD7-, CD11b- , CD56+het, CD10-, CD8-,CD4-, CD45RA+, CD19-, CD2-, Glya-, CD71+low, CD38-, CD1a-, CD41-, CD14-).The analysis of the PML/RARα fusion gene, according to standard protocols, was negative. FISH studies showed 2 PML signals and 3 RARα copies, suggesting a possible variant rearrangement of this gene. FISH and molecular biology did not detect transcript for t(15:17), (5:17) or (11:17); so RARα was translocated with a strange partner.Suspecting of a variant APL, the patient was treated with ATRA plus Idarrubicin. After 4 weeks treatment, deep neutropenia was observed but no granulocytic maturation. That suggested us resistance to ATRA. Thus, we changed the treatment to a classical induction scheme for AML. The patient attained cytologic remission, which he mantains so far with conventional chemotherapy.Karyotype study was subsequently performed, identified an unbalanced rearrangement between 7q and 17q. Currently, we are identifying and sequencing the fusion partner of RARα. ConclusionWe report a novel case of variant acute promyelocytic leukemia with the karyotype der (7) t(7;17) (q11;q21). The morphology of PB and BM was critical for initial diagnosis and treatment decisions. Disclosures:No relevant conflicts of interest to declare.

Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer

Malignancies are characterized by extensive global reprogramming of epigenetic patterns, including gains or losses in DNA methylation and changes to histone marks. Furthermore, high-resolution genome-sequencing efforts have discovered a wealth of mutations in genes encoding epigenetic regulators that have roles as 'writers', 'readers' or 'editors' of DNA methylation and/or chromatin states. In this Review, we discuss how these mutations have the potential to deregulate hundreds of targeted genes genome wide. Elucidating these networks of epigenetic factors will provide mechanistic understanding of the interplay between genetic and epigenetic alterations, and will inform novel therapeutic strategies.

Adult Immunohistochemical Markers Fail to Detect Intratubular Germ Cell Neoplasia in Prepubertal Boys with Cryptorchidism

Intratubular germ cell neoplasia is a precursor to testicular germ cell cancer. The condition is characterized by large germ cells with large nuclei with a hyperchromatic, coarse chromatin pattern, large prominent nucleoli and abundant pale cytoplasm. In prepubertal boys these cells are located centrally and peripherally mixed with normal cells in the seminiferous tubules. We evaluated the impact of adult intratubular germ cell neoplasia marking immunohistochemistry in screening for intratubular germ cell neoplasia in boys with cryptorchidism. Histology sections of 236 testicular biopsies were retrieved from 170 boys 1 month to 15 years old operated on for cryptorchidism (excluding disorders of sex development). Specimens were incubated with primary antibodies, including anti-placental-like alkaline phosphatase, anti-Oct3/4, anti-C-kit and anti-D2-40 receptor. A 1-year, 1-month-old boy had intratubular germ cell neoplasia and all positive markers. The prevalence of placental-like alkaline phosphatase positive staining of germ cells in testicular biopsies was 98% in boys younger than 1 year, 82% in those 1 to less than 2 years old, 74% in those 2 to less than 3 years old and 60% in those 3 to 15 years. Similarly the prevalence of C-kit positive staining was 71% in boys younger than 1 year, 49% in those 1 to less than 2 years, 16% in those 2 to less than 3 years and 34% in those 3 to 15 years. Placental-like alkaline phosphatase negative germ cells did not express any of the other described antigens. In none of the 116 testes from boys older than 1 year and 7 months were any Oct3/4 or D2-40 positive germ cells identified. Up to that age 33% and 8% of biopsies were Oct3/4 and D2-40 positive, respectively. Adult intratubular germ cell neoplasia/cancer immunohistochemical markers cannot be used alone for intratubular germ cell neoplasia screening in male infants with cryptorchidism because positive immunohistochemistry is commonly seen within this age group, when most orchiopexies are performed. It is generally not plausible that intratubular germ cell neoplasia originates during fetal development in patients with cryptorchidism.

Cytological criteria of high‐grade epithelial atypia in the cyst fluid of pancreatic intraductal papillary mucinous neoplasms

The recognition of epithelial cells with high-grade atypia (HGA) in the cyst fluid of an intraductal papillary mucinous neoplasm (IPMN) identifies a cyst at high risk of invasive carcinoma. To the best of the authors' knowledge, the cytological features of HGA have not been systematically analyzed to define diagnostic criteria. Cell groups from patients with histologically confirmed branch-duct IPMNs were evaluated by 2 cytopathologists with expertise in pancreatic cytology. A consensus interpretation categorized the cell groups as having either low-grade (LG) or high-grade (HG) morphology. Characteristics regarding cell size and architecture, nuclear and cytoplasmic features, and background necrosis were analyzed. Performance characteristics were assessed using the Fisher exact test at 95% confidence intervals. Sixty cell groups yielded 27 LG and 25 HG morphological groups. No consensus was reached for 8 groups, which were excluded from statistical analysis. Five features that were found to be significantly different between the LG and HG groups included: 1) cell size < a 12-μm duodenal enterocyte for HG and size equal for LG; 2) an increased nuclear-to-cytoplasmic (N/C) ratio; 3) marked nuclear membrane abnormalities; 4) abnormal chromatin pattern; and 5) background necrosis. The 3 most accurate features for the identification of HGA were background necrosis (88%), abnormal chromatin pattern (84%), and an increased N/C ratio (82%). IPMN cyst fluid at high-risk of malignancy can be recognized most accurately by the presence of epithelial cells with HGA showing an increased N/C ratio, an abnormal chromatin pattern, and background necrosis.

Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

Abstract B14: A biochemical basis for toggling enhancers from the active to poised state in B-cells

Abstract The BCL6 transcriptional repressor is required for development of germinal center (GC) B-cells and diffuse large B-cell lymphomas (DLBCL). The BCL6 BTB domain corepressor interaction is an important mediator of BCL6 actions and a potential therapeutic target. BTB point mutations that disrupt corepressor recruitment inactivate BTB domain repressor function. A similar effect can be achieved using specific BCL6 BTB groove binding peptides or small molecules. Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B-cells is unknown. In this study we addressed the question of how BCL6 mediates transcriptional repression in normal and malignant B-cells through its association with SMRT or NCOR corepressor complexes. We combined extensive genomic binding (ChIP-seq) analysis of BCL6, its corepressors SMRT and NCOR and epigenetic chromatin patterns both in DLBCL and in primary human GC B-cells, with functional and biochemical experiments performed in vitro, in cell-based assays and in vivo to reveal the biological and transcriptional mechanism of BCL6. We find that in B-cells, virtually all SMRT/NCOR binding sites overlap with BCL6 binding sites indicating that in these cells BCL6 uniquely sequesters SMRT/NCOR complexes from other factors to mediate cell specific functions. Interestingly, these BCL6-SMRT/NCOR complexes preferentially localize at B cell enhancers. Moreover, BCL6-SMRT/NCOR complexes can inactivate these enhancers by deacetylating H3K27. The SMRT/NCOR-dependent H3K27 de-acetylation is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Furthermore, we show that BCL6 BTB targeted inhibitors may reverse the enhancer silencing effect by disrupting the association of BCL6 with SMRT/NCOR complexes. Taken together these results point to an enhancer “toggling” mechanism that might provide the basis for B-cells to undergo rapid transcriptional changes in response to environmental cues. This mechanism might be particularly relevant to the shuttling of B-cells from the dark to the light zone during affinity maturation facilitating the rapid on/off gene expression changes necessary for this transition. For example genes associated with BCL6-SMRT/NCOR enhancers were highly downregulated in the dark zone compared to the light zone and they were also significantly enriched in genes upregulated by CD40 signaling which occurs in the light zone during B-cell activation by Tfh cells and disrupts the association of BCL6 and SMRT/NCOR. In summary we describe a novel mechanism of enhancer regulation. Reversible enhancer toggling may be critical for dynamic modulation of the BCL6 transcriptional program during the GC reaction as well for the therapeutic effects of BCL6 inhibitors. This mechanism is likely a general biological phenomenon that applies to enhancer regulation in a variety cell types and tumors. Citation Format: Katerina Chatzi, Yanwen Jiang, Chuanxin Huang, Paul Zumbo, Srividya Bhaskara, Alexander D. MacKerell, Jr., Fengtian Xue, Christopher E. Mason, Scott W. Hiebert, Leandro Cerchietti, Vivian J. Bardwell, Olivier Elemento, Melnick Ari. A biochemical basis for toggling enhancers from the active to poised state in B-cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B14.