Ying S. Zou

Mucinous tubular and spindle cell carcinoma of the kidney with prominent papillary component, a non-classic morphologic variant. A histologic, immunohistochemical, electron microscopic and fluorescence in situ hybridization study

Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare type of kidney tumor with relatively indolent behavior. Non-classic morphological variants have not been well studied and rarely been reported. We report a challenging case MTSCC with a peculiar morphology in a 42-year-old man, arising in a background of end-stage renal disease (ESRD). Predominant areas with extensive papillary architecture, psammoma bodies and stromal macrophageal aggregates, reminiscent of a papillary renal cell carcinoma (papillary RCC), were intermixed with foci that transitioned into a MTSCC-like morphology exhibiting elongated tubules and a low grade spindle cell component in a background of mucinous stroma. Immunohistochemistry demonstrated diffuse positivity for P504s/AMACR and vimentin in tumor cells. Focal positivity for RCC, CD10 and CK7 was also noted. Kidney-specific cadherin, cytokeratin 34betaE12 and TFE3 stains were negative in the tumor. The major differential diagnostic considerations were papillary RCC, clear cell papillary RCC, and Xp11.2 translocation carcinoma. Negative FISH studies for trisomy 7 and 17 in both papillary and spindled components supported the diagnosis of MTSCC. The ultrastructural profile was not entirely indicative of the cellular origin of the tumor. Cytogenetic analysis should be performed in atypical cases of MTSCC for precise diagnosis.

Jumping translocations of 3q21 in an acute monocytic leukemia (M5) patient reveal mechanisms of multistage telomere shortening in pathogenesis of AML.

Jumping translocations (JTs) are rare chromosome rearrangeents characterized by the relocalization of the same part of a onor chromosome to more than one recipient chromosome. To ate, fewer than 70 JTs have been reported in hematological maligancies and solid tumors [1]. Only 18 JTs have been described in yeloid-lineage hematological disorders. Of these, 5 involve the ong arm of a chromosome 3 (3q) JTs with gain of a chromosome (trisomy 8) [2]. Among these, 2 had acute myeloid leukemia AML)-M5b, 1 had AML-M5a, 1 had an unspecified myeloprolifrative neoplasm (MPN(u)), and 1 had jCMML → unspecified AML 2]. Here, we describe the 6th 3q JT case with trisomy 8 in a patient ith AML-M5. But notably it is the first case with quantitative FISH nalyses (Q-FISH) of telomere lengths in normal and abnormal cells f a 3q JT patient, as well as with chromosome microarray analyes. The 3q JT donor breakpoint is centric to the RPN1 gene and the ajority of recipient breakpoints are within pure telomere repeat equences. Telomere length was significantly decreased in abnoral cells with trisomy 8 only and JT with trisomy 8 compared to ormal cells. This is supporting evidence that telomere shortening ontributes to the development and formation of 3q JTs, which is ost likely a multi-stage process.

A novel TTC40–MSI2 fusion in de novo acute myeloid leukemia with an unbalanced 10;17 translocation

The Musashi-2 (MSI2) gene, at chromosome band 17q22, is recurrently associated with regulating normal hematopoiesis and promoting leukemia progression [1-2]. MSI2 is overexpressed in human myeloid leukemia cell lines and its knockdown leads to decreased proliferation and increased apoptosis [2]. High expression of MSI2 protein may predict unfavorable outcome in acute myeloid leukemia (AML) and adult B-cell acute lymphoblastic leukemia [2-4]. MSI2 rearrangement was first reported in two patients who presented with chronic myelogenous leukemia and cryptic balanced translocations involving chromosomes 7 and 17 were discovered during disease progression [5]. The translocation involved identical breakpoints in chromosome 17q22 within the MSI2 gene in both cases [5]. One of these translocations resulted in a MSI2-HOXA9 chimeric fusion gene [5]. MSI2 rearrangement has also been reported in patients with myeloid leukemia and a 3;17 translocation leading to EVI1 gene overexpression [6]. Here, we present a case of de novo AML exhibiting an unbalanced 10;17 translocation leading to a TTC40-MSI2 fusion gene. To our knowledge this is the first report of this specific translocation. A previously healthy 66-year-old woman presented with fatigue and was found to have abnormal blood counts: hemoglobin 11.1 g/dL, white blood cells 58 × 10k/ul with 80% blasts, and platelets 87 × 10k/ul. Her peripheral smear showed marked leukocytosis with increased blast forms that were intermediate in size and had round nuclei, dispersed chromatin, indistinct nucleoli, and scant cytoplasm with rare azurophilic granules. No Auer rods were identified. Red blood cells and platelets were unremarkable. The bone marrow was hypercellular (95% cellularity), with a predominance (90-95% of cellularity) of intermediate-sized blasts, similar to those seen in the blood. Maturing myeloid and erythroid cells and megakaryocytes were rare. Immunophenotyping by flow cytometry identified a population of abnormal myeloid blasts (96.9% of total cells) expressing CD117 (partial), CD13 (increased), CD15 (partial), CD33 (increased), CD38 (decreased), CD45, CD56 (partial), CD64 (dim/equivocal), and CD71, but not CD34, HLA-DR, or CD14. Fluorescence in situ hybridization (FISH) using probe sets for the LSI PML-RARA and RARA break-apart (Abbott Molecular) did not show a 15;17 translocation or RARA gene rearrangement. A diagnosis of AML without maturation (or with minimal differentiation) was made. The patient underwent three cycles of induction chemotherapy with two chemo drugs and achieved complete remission. Then, she had one cycle of consolidation chemotherapy with high dose cytarabine (1 g/m2, 6 days). 9 months after complete remission, she had relapsed. She was treated with 5 days of intravenous mitoxantrone (8 mg/m2), etoposide (100 mg/m2) and cytarabine (1 g/m2) along with 4 doses of MDX-1338 (1000 mg). She expired shortly, 14 months after the initial diagnosis. Cytogenetic analysis was performed on bone marrow cells at the time of diagnosis. All 20 metaphase cells analyzed had an abnormal chromosome 10 with additional material of unknown origin on the distal long arm of chromosome 10, 46,XX,add(10)(q26)[20] (Figure 1A). Whole genome CNV/SNP chromosomal microarray assay (CMA) with a total of 2.6 million copy number variant and SNP markers (Affymetrix CytoscanHD array) was performed on the DNA extracted from bone marrow cells and revealed a 25.6 Mb gain of the distal long arm of chromosome 17 (17q22-qter) and a 783 Kb loss of the distal long arm of chromosome 10 (10q26.3), arr[hg19]10q26.3(134644013-135427143)×1,17q22q25.3(55401945-81041938)×3 (Figure 1B). Based on the CMA findings the breakpoint of the translocation on chromosome 10 was mapped within the tetratricopeptide repeat domain 40 (TTC40) gene at band 10q26.3 and the breakpoint on chromosome 17 was within the MSI2 gene at band 17q22. The TTC40 gene contains 58 exons spanning 134 Kb, and is a novel gene with high GC content. Figure 1 Cytogenetic and molecular analyses of unbalanced 10;17 translocation with TTC40-MSI2 fusion gene. (A) Representative metaphase showing a derivative chromosome 10 with additional material of unknown origin on 10q. (B) Chromosomal microarray assay revealing ... FISH analysis was performed using BAC clones covering the MSI2 gene on 17q22 and the TTC40 gene on 10q26.3, as well as chromosome 10 centromere probe and the 17q subtelomere probe D17S928 mapping to 17q25 (Abbott Molecular). Analysis of abnormal metaphase cells confirmed the presence of chromosome 17 material on the derivative chromosome 10 (Figure 1C) and revealed the presence of the TTC40-MSI2 gene fusion on interphase and metaphase cells (Figure 1D).Sequencing analysis following long-range polymerase chain reaction using the primer pair TTC40-1F:5′-GACGGGACTGGACGCTGCAC-3′ and MSI2-1R: 5′-GGTGCATGTGGCCCAGTCAGT-3′ revealed that the MSI2 gene was fused to the TTC40 gene with breaks in introns 4 and 50, respectively (Figure 1E). These findings confirm that the derivative chromosome 10 is the product of an unbalanced translocation between chromosome bands 10q26.3 and 17q22, der(10)t(10;17)(q26.3;q22), leading to a TTC40-MSI2 fusion gene. The unbalanced 10;17 translocation also resulted in a small terminal deletion of the long arm of chromosome 10 distal to 10q26.3 and a large duplication of the long arm of chromosome 17 including the 17q22-q25.3 region. The deleted 10q26.3 region contains 29 genes (10 OMIM genes) (http://genome.ucsc.edu). This 10q26.3 deletion has also been reported in normal individuals without phenotype, suggesting that the region might be a population variant /polymorphic and does not contain haploinsufficient genes (http://dgv.tcag.ca). However, the duplicated 17q22-q25.3 region contains ∼ 480 genes (235 OMIM genes and known disease genes) (http://genome.ucsc.edu). Among them, at least 20 genes are involved in solid tumors, including ASPSCR1, AXIN2, BCAS3, BRIP1, CLTC, DDX5, ENPP7, MIR21, PPMID,PRKAR1A, RAC3, SLC9A3R1, SLC16A3, SOCS3, SPSF1, ST6GALNAC1, TBX2, TIMP2, USP32, VMP1 (http://atlasgeneticsoncology.org). A number of other genes are thought to play a role in the pathogenesis of hematologic malignancies. The RNF213 and CLTC genes are engaged in anaplastic large cell lymphoma [7], the SEPT9 gene (a fusion partner gene of MLL) is involved in de novo and treatment related leukemia [8], and the GRB2 gene is implicated in the pathogenesis of Philadelphia chromosome positive leukemia [9]. This case illustrates the importance of comprehensive morphologic, cytogenetic, chromosomal microarray and sequence analysis in characterizing a de novo AML with unbalanced 10;17 translocation, leading to discovery of a novel TTC40-MSI2 fusion gene. This unbalanced translocation der(10)t(10;17)(q26.3;q22) in our case is the first to be reported, and further studies are warranted to elucidate the roles of the involved genes in leukemogenesis. Collecting and reporting data on rare chromosomal abnormalities in AML will add important information regarding disease pathogenesis and prognosis, and may eventually translate to targeted therapies.

Clear cell papillary renal cell carcinoma: A chromosomal microarray analysis of two cases using a novel Molecular Inversion Probe (MIP) technology

Chromosomal microarray analysis using novel Molecular Inversion Probe (MIP) technology demonstrated 2,570 kb copy neutral LOH of 10q11.22 in two clear cell papillary renal cell carcinomas. In addition, one of the tumors had a big 29,784 kb deletion of 13q11-q14.2. There were two variants of unknown significance, a 2,509 kb gain of Xp22.33 and a 257 kb homozygous deletion of 8p11.22. The somatic mutation panel containing 74 mutations in nine genes did not reveal any mutations. Besides identification of submicroscopic duplications or deletions, SNP microarrays can reveal abnormal allelic imbalances including LOH and copy neutral LOH, which cannot be recognized by chromosome, FISH, and non-SNP microarray arrays. To the best of our knowledge, this is the first study demonstrating copy neutral LOH of 10q11.22 in clear cell papillary renal cell carcinomas using the new MIP SNP OncoScan FFPE Assay Kit on formalin-fixed paraffin-embedded tumor samples.

Concomitant amplification of the MLL gene on a ring chromosome and a homogeneously staining region (hsr) in acute myeloid leukemia: mechanistic implications.

Gene amplification is one mechanism by which oncogenes are overexpressed. Although MLL (KMT2A) gene rearrangements (or 11q23 rearrangements) are well known in acute myeloid leukemia (AML), MLL gene...

Cryptic ETV6–PDGFRB fusion in a highly complex rearrangement of chromosomes 1, 5, and 12 due to a chromothripsis-like event in a myelodysplastic syndrome/myeloproliferative neoplasm

Myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) are hematological malignancies characterized by simultaneous presence of both myelodysplastic and myeloproliferative features [1]. C...

Novel t(5;11)(q32;q13.4) with NUMA1-PDGFRB fusion in a myeloid neoplasm with eosinophilia with response to imatinib mesylate

We report a NUMA1-PDGFRB fusion in a myeloproliferative neoplasm with eosinophilia in a 61-year old man, with response to imatinib mesylate therapy. A t(5;11) chromosome translocation involving bands 5q32 and 11q13.4 was identified by metaphase chromosome analysis, and rearrangement of the platelet-derived growth factor receptor beta (PDGFRB) gene on 5q32 was demonstrated by FISH using a PDGFRB break-apart probe set. Bacterial artificial chromosome (BAC) FISH mapping of the PDGFRB fusion partner gene narrowed the breakpoint at 11q13.4 to a 150 kb genomic region containing three genes, including NUMA1. Mate pair sequencing analysis demonstrated NUMA1-PDGFRB fusion. The fusion protein includes coiled-coil domains of nuclear mitotic apparatus protein 1 (NuMA1, involved in protein homodimerization and heteroassociation) and tyrosine kinase domains of PDGFRB. Diverse rearrangements involving the PDGFRB gene have been identified in myeloid and lymphoid neoplasms with eosinophilia, but rearrangement of the nuclear mitotic apparatus protein 1 (NUMA1) gene has previously been reported in a human malignancy in only one instance, a NUMA1-RARA fusion caused by a t(11;17) translocation in a patient with acute promyelocytic leukemia. The NUMA1-PDGFRB fusion is the second instance of rearrangement of NUMA1, encoding an element of the mitotic apparatus, in human cancer.

Utilization of magnetic-activated cell sorting and high-density single nucleotide polymorphism microarrays improves diagnostic yield and prognostic value in clinical testing for patients with multiple myeloma and normal routine chromosome study.

Multiple myeloma (MM) is a clonal plasma cell disorder. Chromosome and FISH studies are used to provide prognostic information that is useful for refining risk stratification and therapeutic response [1]. Frequently, chromosome study is limited by the inability of plasma cells to proliferate in vitro, and FISH is restricted to small probe panels. To alleviate this problem, we have implemented a protocol of magnetic activated cell sorting (MACs) to isolate abnormal plasma cells followed by high-density single nucleotide polymorphism (SNP) microarray testing to improve our ability to clearly resolve chromosomal copy number abnormalities and to identify loss of heterozygosity (LOH) and copy number-neutral LOH (CNN-LOH). LOH/CNN-LOH have been shown to be important in cancer biology where they can lead to tumor suppressor gene inactivation [2]. By applying this methodology we have been able to increase our diagnostic yield and provide prognostic information on these clonal populations that previously would have been masked by the normal cells in the patient samples. Three consecutive MM patients with enough bone marrow volume were used for this study. They had plasma cell ratios ranging from 16% to 77% based on morphology and from 4% to 20% based on flow cytometry (Table 1). All patients had normal karyotype results by chromosome studies. Interphase MM FISH panel including probe sets of D13S319/13q34, IGH/FGFR3, P53/CEP17, and MLL break-apart (Abbott Molecular) revealed only 1-2 anomalies per sample (Table 1). After MACs enrichment treatments (Stem Cell Technologies), SNP microarray was performed on both the plasma-enriched and the plasma-depleted fractions with a total of 2.6 million probes (Affymetrix CytoscanHD array). The microarray result of the plasma-depleted fraction was normal with no pathogenic deletions or duplications; however, microarray results of the plasma-enriched fraction revealed 8 - 12 chromosomal abnormalities. Follow-up FISH studies confirmed microarray findings using CBFB break-apart, LSI 1p36/1q25, BCR, LSI 21, and centromere probes (CEP) for X, 2, 3, 5, 7, 9, 11, 15, 17, 19 (Abbott Molecular) (Table 1) . Table 1 Summary of patients’ clinical, karyotype (chromosome), FISH and SNP microarray data Microarrays were also performed on the total cell fraction of specimens from Patients 1 and 3 because of their high clonal plasma cell population (77% and 27% by morphology, respectively). By comparing microarray data between the total cell and plasma-enriched fractions of patients 1 and 3 (T and P+ in Table 1, respectively), it is evident that a more refined and accurate profile of chromosome copy number abnormalities is revealed from the plasma-enriched fraction than from the total fraction. For example, for chromosomes 5, 9, and 15 in patient 1, microarray revealed the presence of both trisomies and tetrasomies in the plasma-enriched fraction compared to only trisomies observed in the total cell fraction. Chromosomes 3, 7, 11, 17, 19, and 21 in patient 1 that were clearly trisomies in the plasma-enriched fraction dissolved into partial gain signals in the total cell fraction that were much more difficult to interpret with accuracy. For patient 3, microarray data from the total cell fraction only found the presence of mosaic loss of chromosome 13 and missed other eight additional changes, which were revealed by microarray data from the plasma-enriched fraction (Table 1). In all cases, the prognostic information from the microarray of plasma-enriched factions exceeded that of chromosome and FISH combined. According to microarray data, Patients 1 and 2 represent hyperdiploidy while Patient 3 is an example of hypodiploidy with 1q triplication that is also associated with a poor prognosis [3]. Besides identification of submicroscopic duplications or deletions, SNP microarrays can reveal abnormal allelic imbalances including LOH and CNN-LOH, which cannot be recognized by chromosome and FISH. CNN-LOH is the occurrence of LOH in the absence of allelic loss (copy number ≥ 2) and mosaic CNN-LOH is the mixture of normal and abnormal cells with CNN-LOH (Fig. 1a). Mosaic CNN-LOH of 16q was identified in Patient 2 (Fig. 1b), which has been associated with adverse prognosis [4]. Patient 3 was found to have complex abnormalities at chromosome 11q including mosaic deletion at 11q14.1-q22.1, a deletion at 11q22.1-q22.3, and mosaic CNN-LOH at 11q22.3-qter (Fig. 1c). Thus, plasma enrichment and SNP microarray provides a clear improvement in identification unbalanced chromosome abnormalities, LOH and CNN-LOH. Figure 1 Regions of CNN-LOH by SNP microarray. a Examples of different allele peak patterns determined by the ChAS software (Affymetrix). b Mosaic CNN-LOH at 16q in patient 2. c Complex 11q abnormalities in patient 3. del. = Deletion In summary, SNP microarray testing of MACs isolated abnormal plasma cells in our MM patients provided a much more comprehensive overview of the genome-wide chromosome copy number abnormalities compared to chromosome and FISH studies. SNP Microarray testing is useful in clinical practice to refine our diagnostic and prognostic indicators. This technique can be easily incorporated into every cytogenetic laboratory. Although SNP microarray testing cannot reveal any balanced structural abnormalities such as IGH-FGFR3 fusions, FISH on MACs isolated abnormal plasma cells will increase detection rate of clinically relevant genomic abnormalities to overcome the overall low percentage of plasma cells present in primary bone marrow aspirates. Therefore, combination of SNP microarray results of MACs enriched plasma cells and FISH/chromosome results will present a more complete picture of chromosome abnormalities and provide insights into understanding mechanisms of MM formation and development.

Familial 25.3 Mb inverted duplication of bands q32.1 to q35.1 on chromosome 4 with psychomotor impairments

Partial 4q trisomy has been described in more than 80 patients. In most patients, the 4q trisomic segment was derived from a parent carrying a reciprocal balanced translocation or an inversion on chromosome 4 and was accompanied by a concurrent deletion of another chromosomeor a short armof chromosome4, respectively. Clinical heterogeneity of partial 4q trisomy has been reported, possibly due to the variability of the 4q duplicated segment and its accompanied anomalies. Pure duplications of 4q without a concurrent deletion are infrequent and have been described in fewer than 25 patients [Celle et al., 2000; Rinaldi et al., 2003; Lin et al., 2004; Otsuka et al., 2005; Egritas et al., 2010]. Here we present a three-generation family with pure 4q32.1-q35.1 duplication, never described before (Fig. 1). This 25.3Mb 4q duplication was detected by conventional chromosome study and was further characterized by SNP microarray analyses and FISH studies. The patient was a 4-month-old male with failure to progress, who was born to a 33-year-old mother at 41 weeks gestational age by cesarean. At birth, he was in the neonatal intensive care unit for 6 days because of respiratory distress. Subsequently, at 2months of age, he had an episode of apnea andwas hospitalized. At this time, he was diagnosed with a fenestrated atrial shunt, mild supravalvar pulmonic stenosis, and gastroesophageal reflux disease. At the age of 4 months (Fig. 1, III-2), his height was at the 50th centile, his weight was at the 90th centile, and his OFC was at the 75th centile. He had developmental delay, large anterior fontanelle, broad cheeks, bilateral epicanthal folds, and long philtrum. He had bilateral single transverse palmar creases, deep creases in the soles of his feet, hypoplastic nails, and right-side cryptorchidism. Otherwise, his general physical examination was unremarkable. His 34-year-oldmother, his 56-year-oldmaternal grandmother, his 35-year-old maternal aunt (Fig. 1, II-1), and his 14-year-old maternal cousin (Fig. 1, III-1) had psychomotor impairments and needed special education. His 59-year-old maternal grandfather (Fig. 1, I-2) is phenotypically normal and his father is not available. The patient’s blood karyotype is 46,XY,dup(4)(q35q32) resulting in an interstitial duplication of the 4q32-q35 segment (Fig. 2a), which was further delineated by chromosomemicroarray analyses. Affymetrix Genome-wide 6.0 SNP-microarray genotyping (using Chromosome Analysis Suite software for the analysis) showed a 25.3Mb duplication of a chromosome 4 [arr 4q32.1q35.1 (158,425,313–183,681,864)X3] using the assembly hg19/GRCh 37 of the human genome (Fig. 2b). FISH was performed using home-brewed BAC probes including RP11-719M18 at 4q32 and RP11-295A7 at 4q35. FISH analysis confirmed this 4q duplication since these probes showed three copies in each of 200 interphase