Gene Rearrangement Analysis Research Articles

Minimal residual disease in acute lymphoblastic leukemia: technical aspects and implications for clinical interpretation.

Minimal residual disease (MRD) monitoring has proven to be one of the fundamental independent prognostic factors for patients with acute lymphoblastic leukemia (ALL). Sequential monitoring of MRD using sensitive and specific methods, such as real-time quantitative polymerase chain reaction (qPCR) or flow cytometry (FCM), has improved the assessment of treatment response and is currently used for therapeutic stratification and early detection. Although both FCM and qPCR yield highly consistent results with sensitivities of 10‒4, each method has several limitations. For example, qPCR is time-consuming and laborious: designing primers that correspond to the immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements at diagnosis can take 3‒4 weeks. In addition, the evolution of additional clones beyond the first or index clone during therapy cannot be detected, which might lead to false-negative results. FCM requires experienced technicians and sometimes does not achieve a sensitivity of 10‒4. Accordingly, a next generation sequencing (NGS)-based method has been developed in an attempt to overcome these limitations. With the advent of high-throughput NGS technologies, a more in-depth analysis of IG and/or TCR gene rearrangements is now within reach, which impacts all applications of IG/TR analysis. However, standardization, quality control, and validation of this new technology are warranted prior to its incorporation into routine practice.

Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.

Deep transcriptome profiling sheds light on key players in nucleus implantation induced immune response in the pearl oyster Pinctada martensii

Complete mitochondrial genome (mitogenome) provides important information for better understanding of gene rearrangement, molecular evolution and phylogenetic analysis. Here we determined the complete mitogenome sequence of Chiromantes eulimene (Brachyura: Sesarmidae) for the first time. The total length is 15,894 bp and includes 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs, as well as a putative control region. The genome composition is highly A + T biased (75.5%), and exhibits a negative AT-skew (−0.017) and GC-skew (−0.206). All of the 13 PCGs are initiated by the start codon ATN, with an exception (GTG) in ND1. The typical stop codon (TAA or TAG) is detected in ten PCGs, whereas the remaining three PCGs (COI, COII and Cyt b) terminate by an incomplete T. The gene order in C. eulimene mitogenome was rearranged compared with that of the ancestor of Decapoda. The gene order of F-ND5-H changed to H-F-ND5. Like other sesarmid crabs, the I-Q-M gene cluster in the pancrustacean ground pattern became Q-I-M order in C. eulimene genome. Tandem duplication-random loss model and slipped-strand mispairing mechanism are determined as most likely to explain the observed gene rearrangements. Phylogenetic analysis places all Sesarmidae species into one group. Almost all families except Xanthidae, Gecarcinidae and Homolidae form a monophyletic clade and the polyphyly of Eriphioidea, Ocypodoidea and Grapsoidea is well supported. These results will help to better understand the gene rearrangements and evolutionary position of C. eulimene and lay a foundation for further phylogenetic studies of Brachyura.

PB2009 PERIPHERAL BLOOD INVOLVEMENT BY FLOW CYTOMETRY IS NOT AN UNUSUAL FEATURE IN IGM MGUS/WALDENSTRÖM MACROGLOBULINEMIA WITH MYD88 L265P MUTATION.

Background:The IgM monoclonal gammopathy of undetermined significance (MGUS) is defined as a serum IgM paraprotein concentration <30 g/L, bone marrow (BM) lymphoplasmacytic infiltration by clonal population <10% and no symptoms or end‐organ damage. Waldenström Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma (LPL) with BM involvement and an IgM monoclonal gammopathy of any concentration. Patients fullfilling criteria for WM and not requiring inmediate treatment are classified as smoldering WM. More than 90% of LPL/WM cases and 60% of IgM MGUS display the MYD88 L265P mutation (MYD88mut), suggesting that IgM MGUS is closer to mature B‐cell neoplasms than to plasma cell myeloma according to 2016 WHO update. From the immunophenotypic point of view, clonal B cells are reported in 75% of IgM MGUS in BM on multiparameter flow cytometry (FC). However, data about peripheral blood (PB) involvement in IgM MGUS/WM are scarce.Aims:To analyze the incidence of PB involvement detected by FC in a series of IgM MGUS/WM with MYD88mut in BM consecutively diagnosed in a single institution.Methods:69 patients (47 male/22 female; median age 77 years, range 38‐92) diagnosed with IgM MGUS/WM MYD88mut between 2000 and 2019 with PB FC availability were selected. The diagnosis was made in BM in all cases. For FC analysis, 100,000 events were acquired in a FACSCanto II (BD) cytometer and were analyzed with the Infinicyt software (Cytognos). The study of the MYD88 L265P mutation was performed in PB by allele‐specific qPCR and the analysis of the IGH gene rearrangement by PCR.Results:Monoclonal gammopathy was detected in routine blood tests in asymptomatic patients in 52/69 (75%) cases. Median lymphocyte count was 2.04x109/L (0.77‐8.81). Only 3/69 (4.3%) patients presented with ≥4x109/L lymphocytes. Leukemic involvement by FC was detected in 36/69 (52%) patients (see Table 1). The phenotype of clonal lymphocytes was the usually described in WM (CD19dimCD22dimCD25dim) in most cases. In those patients, the median percentage of B‐cells from the total lymphocytes was 8% (0.6‐40%) with only 5 (7%) patients presenting ≥20% B‐lymphocytes. The median percentage of clonal B‐cells in respect of the total number of B‐cells was 60% (3‐100%). However, in 9 patients the clonal B‐lymphocytes could not be quantified because of the minor representation of this abnormal population with non‐specific phenotype. In 5 (7%) patients an additional abnormal B‐cell population was detected, 4/5 being phenotypically consistent with chronic lymphocytic leukemia. PB involvement was most frequently observed in patients displaying ≥30% lymphocytes in BM (15/20, 75% vs. 21/48, 44% in patients with <30% lymphocytes, P = 0.032). No differences were observed with regard to the amount of serum monoclonal component (PB involvement: 12/24 cases with ≥15 g/L vs. 24/45 patients with <15 g/L, P = NS). MYD88mut was detected in 31/34 (91%) cases with leukemic expression. In 13/31 (42%) patients without PB involvement by FC, molecular data were consistent with circulating disease (only MYD88mut 2/31, only clonal IGH rearrangement 5/29, both 6/29).Summary/Conclusion:In our series, PB involvement by FC was not unusual in patients with IgM MGUS/MW MYD88mut. However, nearly all patients had a normal lymphocyte count and a percentage of B‐cells within the normal ranges. Leukemic expression by FC correlated with the degree of BM infiltration. Finally, PB involvement could be detected by molecular techniques (MYD88 L265P mutation and/or clonal IGH rearrangement) in almost half of the patients with negative FC.image

Molecular analysis of glycosylphosphatidylinositol anchor deficient aerolysin resistant isolates in gulf war i veterans exposed to depleted uranium.

During the First Gulf War (1991) over 100 servicemen sustained depleted uranium (DU) exposure through wound contamination, inhalation, and shrapnel. The Department of Veterans Affairs has a surveillance program for these Veterans which has included genotoxicity assays. The frequencies of glycosylphosphatidylinositol anchor (GPIa) negative (aerolysin resistant) cells determined by cloning assays for these Veterans are reported in Albertini RJ et al. (2019: Environ Mol Mutagen). Molecular analyses of the GPIa biosynthesis class A (PIGA) gene was performed on 862 aerolysin-resistant T-lymphocyte recovered isolates. The frequencies of different types of PIGA mutations were compared between high and low DU exposure groups. Additional molecular studies were performed on mutants that produced no PIGA mRNA or with deletions of all or part of the PIGA gene to determine deletion size and breakpoint sequence. One mutant appeared to be the result of a chromothriptic event. A significant percentage (>30%) of the aerolysin resistant isolates, which varied by sample year and Veteran, had wild-type PIGA cDNA (no mutation). As described in Albertini RJ et al. (2019: Environ Mol Mutagen), TCR gene rearrangement analysis of these isolates indicated most arose from multiple T-cell progenitors (hence the inability to find a mutation). It is likely that these isolates were the result of failure of complete selection against nonmutant cells in the cloning assays. Real-time studies of GPIa resistant isolates with no PIGA mutation but with a single TCR gene rearrangement found one clone with a PIGV deletion and several others with decreased levels of GPIa pathway gene mRNAs implying mutation in other GPIa pathway genes. Environ. Mol. Mutagen. 60:470-493, 2019. © 2019 Wiley Periodicals, Inc.

Outcomes of patients with childhood B-cell precursor acute lymphoblastic leukaemia with late bone marrow relapses: long-term follow-up of the ALLR3 open-label randomised trial

SummaryBackgroundThe ALLR3 trial investigated outcomes of children with B-cell precursor acute lymphoblastic leukaemia who had late bone marrow relapses. We analysed long-term follow-up outcomes of these patients.MethodsALLR3 was an open-label randomised clinical trial that recruited children aged 1–18 years with B-cell precursor acute lymphoblastic leukaemia who had late bone marrow relapses. Eligible patients were recruited from centres in Australia, Ireland, the Netherlands, New Zealand, and the UK. Patients were randomly assigned from Jan 31, 2003, to Dec 31, 2007, and the trial closed to recruitment on Oct 31, 2013. Randomly assigned patients were allocated to receive either idarubicin or mitoxantrone in induction by stratified concealed randomisation; after randomisation stopped in Dec 31, 2007, all patients were allocated to receive mitoxantrone. After three blocks of therapy, patients with high minimal residual disease (≥10−4 cells) at the end of induction were allocated to undergo allogeneic stem-cell transplantation and those with low minimal residual disease (<10−4 cells) at the end of induction were allocated to receive chemotherapy. Minimal residual disease level was measured by real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements. The primary endpoint of the original ALLR3 clinical trial was progression-free survival of randomly assigned patients. The primary endpoint of this long-term follow-up analysis was progression-free survival of patients with late bone marrow relapses stratified by minimal residual disease level. Outcomes were correlated with age, site, time to recurrence, and genetic subtypes, and analysed by both intention to treat and actual treatment received. This trial is registered on the ISRCTN registry, number ISRCTN45724312, and on ClinicalTrials.gov, number NCT00967057.FindingsBetween Feb 2, 2003, and Oct 28, 2013, 228 patients with B-cell precursor acute lymphoblastic leukaemia and late bone marrow relapses were treated. After a median follow-up of 84 months (IQR 48–109), progression-free survival of all randomly assigned patients was 60% (95% CI 54–70). 220 patients achieved second complete remission, and minimal residual disease was evaluable in 192 (87%). 110 patients with late bone marrow relapses and high minimal residual disease at the end of induction were allocated to undergo stem-cell transplantation, and 82 patients with low minimal residual disease at the end of induction were allocated to receive chemotherapy. In the patients allocated to undergo stem-cell transplantation, four relapses and three deaths were reported before the procedure, and 11 patients were not transplanted. Of the 92 patients transplanted, 58 (63%) remained in second complete remission, 13 (14%) died of complications, and 21 (23%) relapsed after stem-cell transplantation. In patients allocated to receive chemotherapy, one early treatment-related death was reported and 11 patients were transplanted. Of the 70 patients who continued on chemotherapy, 49 (70%) remained in second complete remission, two (3%) died of complications, and 19 (27%) relapsed. Progression-free survival at 5 years was 56% (95% CI 46–65) in those with high minimal residual disease and 72% (60–81) in patients with low minimal residual disease (p=0·0078). Treatment-related serious adverse events were not analysed in the long-term follow-up.InterpretationPatients with B-cell precursor acute lymphoblastic leukaemia with late bone marrow relapses and low minimal residual disease at end of induction had favourable outcomes with chemotherapy without undergoing stem-cell transplantation. Patients with high minimal residual disease benefited from stem-cell transplantation, and targeted therapies might offer further improvements in outcomes for these patients.FundingBloodwise (Formerly Leukaemia and Lymphoma Research) UK, Cancer Research UK, Sporting Chance Cancer Foundation, National Health and Medical Research Council Australia, KindreneKankervrij Netherlands, European Union Seventh Framework Programme, India Alliance Wellcome DBT Margdarshi Fellowship.

Large-scale mitochondrial gene rearrangements in the hermit crab Pagurus nigrofascia and phylogenetic analysis of the Anomura

Complete mitochondrial genome (mitogenome) provides important information for better understanding of gene rearrangement, molecular evolution and phylogenetic analysis. Currently, only a few Paguridae mitogenomes have been reported. Herein, we described the complete mitogenome of hermit crab Pagurus nigrofascia. The total length was 15,423 bp, containing 13 protein-coding genes (PCGs), two ribosomal RNA, 22 transfer RNA genes, as well as an AT-rich region. The genome composition was highly A + T biased (71.4%), and exhibited a negative AT-skew (−0.006) and GC-skew (−0.138). Eight tRNA genes, two PCGs and an AT-rich region found to be rearranged with respect to the pancrustacean ground pattern gene order. Duplication-random loss and recombination model were adopted to explain the large-scale gene rearrangement events. Two phylogenetic trees of Anomura involving 12 families were constructed. The results showed that all Paguridae species were clustered into one clade except Pagurus longicarpus, which for the first time imposed raises doubt about the morphological taxonomy of this species. Furthermore, the present study found that higher- level phylogenetic relationships within Anomura were controversial, compared with the previous studies. Our results help to better understand gene rearrangements and the evolutionary status of P. nigrofascia and lay foundation for further phylogenetic study of Anomura.

Blinatumomab As a Successful Bridge to Hematopoietic Stem Cell Transplant in a Down Syndrome Patient with Relapsed B-Precursor Acute Lymphoblastic Leukemia

Introduction Children with Down syndrome (DS) carry a 20-fold higher risk of developing acute lymphoblastic leukemia (ALL) than other children and have increased rates of treatment-related mortality (TRM) and relapse. The prognosis for relapsed DS-ALL patients is grim with long-term survival rates of 20%. Blinatumomab, a CD19/CD3 bispecific antibody, is FDA approved for relapsed pediatric pre-B ALL, and recently approved for patients with minimal residual disease (MRD). However there are few reports of the use of blinatumomab in DS-ALL patients, and no reports of blinatumomab as a bridge to stem cell transplant (SCT) in this population. Case Description A 19 year old male with DS presented with thrombocytopenia and was diagnosed with pre-B ALL. Upfront therapy included fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, pegaspargase, and rituximab for 8 cycles, and he achieved complete remission (CR). Maintenance therapy included 26 months of 6-mercaptopurine, vincristine, methotrexate, and prednisone. He relapsed 5 months after completion of therapy and was found to have a P2RY8-CRLF2 fusion. IgH gene rearrangement analysis by next generation sequencing (LymphoTrack assay) demonstrated clonal rearrangement. Re-induction therapy included vincristine, pegaspargase, and prednisone. He completed 2 additional courses of chemotherapy but continued to have MRD both by flow cytometry and LymphoTrack. Given persistent MRD with CD19+ lymphoblasts, he was treated with blinatumomab at the FDA recommended dosing; he had no signs of toxicity. He achieved negative MRD by flow cytometry and LymphoTrack after 1 cycle. He then proceeded to a T-cell depleted peripheral blood SCT from a 10/10 matched sibling after conditioning with total body irradiation, thiotepa, and cyclophosphamide. Post-SCT course was uncomplicated and he is now 1 year post-SCT with no evidence of leukemia and 100% donor chimerism. Conclusion Blinatumomab is clinically effective and safe for patients with relapsed/refractory pre-B ALL, yet there is limited information on its use in DS-ALL patients. These patients have been largely excluded from blinatumomab trials given their altered immune system and unknown level of innate T cell dysfunction, potentially altering the efficacy of blinatumomab. We present here the case of a 22 year old male with relapsed DS-ALL who remained MRD positive after several cycles of re-induction chemotherapy, but achieved a CR following 1 cycle of blinatumomab with no toxicities, and safely proceeded to SCT. Although this is a single case, it demonstrates clinical efficacy of blinatumomab in relapsed DS-ALL, suggesting that these patients' T cells can be engaged to recognized CD19-positive leukemic blasts. Furthermore, blinatumomab represents a particularly attractive bridging therapy to SCT for DS-ALL patients who experience high rates of TRM with traditional agents.

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B‐cell neoplasms

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.

Dysregulated TRAF3 and BCL2 Expression Promotes Multiple Classes of Mature Non-hodgkin B Cell Lymphoma in Mice.

TNF-Receptor Associated Factor (TRAF)-3 is a master regulator of B cell homeostasis and function. TRAF3 has been shown to bind and regulate various proteins involved in the control of innate and adaptive immune responses. Previous studies showed that TRAF3 overexpression renders B cells hyper-reactive to antigens and Toll-like receptor (TLR) agonists, while TRAF3 deficiency has been implicated in the development of a variety of B cell neoplasms. In this report, we show that transgenic mice overexpressing TRAF3 and BCL2 in B cells develop with high incidence severe lymphadenopathy, splenomegaly and lymphoid infiltrations into tissues and organs, which is the result of the growth of monoclonal and oligoclonal B cell neoplasms, as demonstrated by analysis of VHDJH gene rearrangement. FACS and immunohistochemical analyses show that different types of mature B cell neoplasms arise in TRAF3/BCL2 double-transgenic (tg) mice, all of which are characterized by the loss of surface IgM and IgD expression. However, two types of lymphomas are predominant: (1) mature B cell neoplasms consistent with diffuse large B cell lymphoma and (2) plasma cell neoplasms. The Ig isotypes expressed by the expanded B-cell clones included IgA, IgG, and IgM, with most having undergone somatic hypermutation. In contrast, mouse littermates representing all the other genotypes (TRAF3-/BCL2-; TRAF3+/BCL2-, and TRAF3-/BCL2+) did not develop significant lymphadenopathy or clonal B cell expansions within the observation period of 20 months. Interestingly, a large representation of the HCDR3 sequences expressed in the TRAF3-tg and TRAF3/BCL2-double-tg B cells are highly similar to those recognizing pathogen-associated molecular patterns and damage-associated molecular patterns, strongly suggesting a role for TRAF3 in promoting B cell differentiation in response to these antigens. Finally, allotransplantation of either splenocytes or cell-containing ascites from lymphoma-bearing TRAF3/BCL2 mice into SCID/NOD immunodeficient mice showed efficient transfer of the parental expanded B-cell clones. Altogether, these results indicate that TRAF3, perhaps by promoting exacerbated B cell responses to certain antigens, and BCL2, presumably by supporting survival of these clones, cooperate to induce mature B cell neoplasms in transgenic mice.

Flow cytometric, gene rearrangement, and karyotypic analyses of 110 cases of primary thyroid lymphoma: a single-institutional experience in Japan.

Ancillary studies for primary nodal lymphomas have been well documented; however, studies of primary thyroid lymphoma (PTL) are limited. Here, we aimed to clarify the clinicopathological, flow cytometric, gene rearrangement, and karyotypic characteristics of PTL by investigation of a large series at a single institute. We performed flow cytometric, IgH rearrangement, and karyotypic analyses of 110 PTL tissues surgically resected at Kuma Hospital between January 2012 and April 2017. All PTLs were of B-cell origin, including mucosa-associated lymphoid tissue lymphoma (MALTL; 89 patients, 80.9%), diffuse large B-cell lymphoma (DLBCL; 18 patients, 16.4%), and follicular lymphoma (FL; three patients, 2.7%). In 96 (87.3%) patients, anti-thyroid antibodies were positive. For flow cytometry using aspirated and resected materials, light chain restriction was observed in 73.7% and 69.2% of examined cases, respectively. Heavy chain JH DNA rearrangement was observed in 65.4% of PTLs (58.1% of MALTL cases, 100% of DLBCL cases, and 100% of FL cases). Chromosomal abnormalities were detected in 49.0% of PTLs, and translocation was most frequently detected (24.0%), followed by addition (20.8%) and trisomy (18.8%). The most frequent (9.4%) karyotype was t(3;14)(q27;q32). Both FLs harbored t(14;18)(q32;q21), and the karyotype was not detected in patients with MALTL and DLBCL. The negative rate for all three examinations was 3.8%. We concluded that thyroid MALTL was cytogenetically different from that in other organs. Our results suggested that pre-operative flow cytometry analysis using aspirated materials was as reliable as that using resected materials.

Minimal Residual Disease (MRD) Status after Induction Therapy Is a Strong Prognostic Factor in the Treatment of Adult Ph (-) Acute Lymphoblastic Leukemia (ALL): Results of a Prospective Study (ALL MRD2008 Study)

IntroductionA clinical indication for allogeneic hematopoietic stem cell transplantation (HSCT) in adult Philadelphia-chromosome negative [Ph (-)] acute lymphoblastic leukemia (ALL) patients in complete remission 1 (CR1) remains to be clarified. Minimal residual disease (MRD) status has been reported to be a strong prognostic factor in adult ALL patients (Blood. 2006 107:1116, 2009 113:4153)( J Hematol Oncol. 2013 6:14). We prospectively monitored MRD status during induction and consolidation therapy in adult Ph (-) ALL patients to determine a clinical indication for HSCT.Materials & MethodsFrom December 2008 to November 2013, 103 adult ALL patients were enrolled in this study. Eligibility criteria included non-L3 ALL, 16-65 years of age, and adequate organ function. Of these patients, 95 were eligible for this study and 59 were Ph (-). The treatment protocol used in this study was modified CALGB 19802. Treatment consisted of 6 courses given in the order of A-B-C-A-B-C, followed by a maintenance phase. Induction chemotherapy (course A) consisted of cyclophosphamide (1,200 mg/m2), daunorubicin (60 mg/m2 x 3), vincristine (VCR) (1.3 mg/m2 (max 2mg) x 4), L-asparaginase (3,000 U/m2 x 6) and prednisolone. Granulocyte colony-stimulating factor was started on day 4 and continued until neutrophil recovery. Consolidation B consisted of mitoxantrone (10 mg/m2 x 2), cytarabine (2,000 mg/m2/day x 4) and intrathecal (IT) administration of methotrexate (MTX). Consolidation C consisted of VCR (1.3 mg/m2 (max 2mg) x 3) and MTX (1,500 mg/m2 x 3) with leucovorin rescue and IT MTX. Patients received maintenance chemotherapy on a monthly basis: prednisolone 60 mg/m2 on days 1-5, VCR (1.3mg/m2 (max 2mg) x 3) on day 1, oral MTX 20 mg/m2 weekly, and oral 6-mercaptopurine 60 mg/m2 daily. MRD status was evaluated after induction therapy (first course A) and after second consolidation therapy (first course C). When MRD statuses after the consolidation were positive, patients were supposed to proceed to HSCT whenever possible.ResultsAmong the 59 Ph-negative ALL patients, 51 patients (86%) could be monitored for MRD status, and the remaining 8 patients were not because of no clonal TCR/Ig targets or chimeric mRNA. The MRD status was determined by PCR analysis of major gene rearrangements and/or chimeric mRNAs (18 were positive for TCRγ, 16 for TCRδ, 13 for IgH, 1 for TCRγ and MLL-AF4, 1 for TCRγ and ETV6-RUNX1, 1 for IgH and MLL-AF4, and 1 for IgH and MLL-ENL). MRD quantifications were performed using ASO-primers with a sensitivity of ≤1 × 10−4, and MRD positivity was defined as a lower limit of detection of ≥1 × 10−3. The median follow-up time was 1597 days (range, 16-2870 days). A total of 51 patients included 29 males and 22 females, and their median age was 29 years ranging from 16 to 65. The median white blood cell count at presentation was 8.5 × 103/L (range 1.2-650.4). CR was achieved in 45 patients (88%). One patient died during induction due to intestinal bleeding. There were 29 survivors after the median follow-up period. The probability of 3-year OS and DFS in these patients with Ph-negative ALL was 69% (95%CI 54-80) and 50% (95%CI 36-63, respectively. In terms of CR1 status, MRD-negative patients after induction chemotherapy A in the first course (n = 17) showed a better 3-year DFS (65%) compared with MRD-positive patients (n = 31; 43%), as shown in Figure 1. The difference was not statistically significant (p = 0.07). There was no patient who proceeded to allogeneic HSCT among 17 MRD-negative patients after induction therapy in CR. In contrast, patients who were MRD-positive after induction but became MRD-negative after consolidation chemotherapy C in the first course (n = 11) showed a worse 3-year DFS compared with patients who were MRD-negative after induction chemotherapy A in the first course (45% vs. 65%, p = 0.08). Fourteen patients were MRD-positive after consolidation chemotherapy C in the first course. Among them, 5 patients proceeded to allogeneic HSCT in 1CR, and 9 did not. Three-year DFS with or without allogeneic HSCT were 60% (95%CI 13-88) vs 44% (95%CI 14-72, p=0.52), respectively.DiscussionThe present study indicates that MRD status after induction and consolidation therapies is a strong prognostic factor. Post-induction MRD-negative patients have been identified to have good-prognosis with chemotherapies, not suitable for up-front allogeneic HSCT. [Display omitted] DisclosuresTakamatsu:Taisho Toyama Pharmaceutical: Research Funding; TAIHO Pharmaceutical: Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Research Funding; Chugai Pharma: Research Funding; Takeda Pharmaceutical: Research Funding; Celgene: Honoraria. Akashi:Celgene: Research Funding, Speakers Bureau; Novartis pharma: Research Funding; Ono Pharmaceutical: Research Funding; Eisai: Research Funding; sanofi: Research Funding; Pfizer: Research Funding; Chugai Pharma: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Asahi-kasei: Research Funding; MSD: Research Funding; Otsuka Pharmaceutical: Research Funding; Taiho Pharmaceutical: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Eli Lilly Japan: Research Funding.

Analysis of misdiagnosis of histiocytic necrotizing lymphadenitis

The histiocytic necrotizing lymphadenitis (HNL) is a common benign lesion in the clinic. Because of its specificity, it is easy to be misdiagnosed as lymphoma or other benign lesions. The author analyzed the reasons of misdiagnosis as follows: (1) pathologists ignored the background of reactive hyperplasia and the specificity of clinical pathological changes; (2) isolate one-sided understanding of nuclear debris in necrotic foci; (3) a false negative result could be presented in HNL gene rearrangement analysis. The pathologists must fully grasp the clinical and histopathological features and immunophenotypic characteristics of HNL to prevent the misdiagnosis. Key words: Histiocytic necrotizing lymphadenitis; Pathologic diagnosis; Misdiagnosis

PCR-based clonality analysis of antigen receptor gene rearrangements in canine cutaneous plasmacytoma

Plasmacytomas are discrete, B cell-derived, round cell tumours that sometimes are difficult to distinguish from canine cutaneous histiocytomas or T cell lymphosarcomas (lymphomas). Diagnosis of plasmacytomas relies on morphological observations and immunohistochemistry for multiple myeloma oncogene-1 (MUM-1) and cluster of differentiation 3 (CD3). Clonality testing often is used as an adjunct diagnostic tool to examine lymphoproliferative diseases. In this study, the sensitivity of PCR-based clonality analysis of antigen receptor gene rearrangements in canine cutaneous plasmacytomas was determined. Formalin-fixed paraffin-embedded sections of 29 canine plasmacytomas, 23 diffuse large B cell lymphomas (DLBCLs) and 23 lymph nodes without lymphoma were used for clonality analysis. New oligonucleotide primers for the framework (FR)2 and FR3 regions of the immunoglobulin heavy chain (IGH) V gene subgroup 3 were designed and used with previously reported FR3 primers. Although plasma cells are of B cell lineage, the detected frequency of IGH clonality in plasmacytoma was 0–34.5% with the seven primers used, whereas in DLBCLs it was 8.7–78.3%. In 23 lymph nodes without lymphoma, IGH clonality was detected in only one case with two out of the seven primers used. Sequence analysis of PCR products from plasmacytomas revealed mismatches in the annealing region of the FR3 primers. The sensitivity of detecting IGH clonality in canine plasmacytomas was lower than in DLBCLs. The low detection rate of IGH clonality in canine plasmacytoma may be due to somatic hypermutation of the variable region.

A study detection of the ROS1 gene fusion by FISH and ROS1 protein expression by IHC methods in patients with ovarian malignant or borderline serous tumors

ObjectiveROS1 is an orphan receptor protein tyrosine kinase which is supposed to undergo genetic rearrangement in carcinogenesis. In the current study, we aimed to investigate the frequency and clinicopathologic features associated with ROS1 gene fusion and ROS1 protein expression in patients with ovarian serous carcinoma or serous borderline tumors. Materials and methodsTissue samples of 102 patients with high or low grade serous carcinoma and borderline serous tumors were selected randomly from the archives of Department of Gyneco-pathology, and analyzed for ROS1 gene expression. (Fluorescence in situ hybridization (FISH) method was used to assess ROS1 gene rearrangement, while ROS1 protein expression was analyzed using immunohistochemistry. ResultsThe study consisted of 94 cases of high-grade serous carcinoma (92.1%), 2 cases of low-grade serous carcinoma (%2) and 6 cases of serous borderline tumor (5.9%). ROS1 gene rearrangement analysis revealed that 4 patients (3.9%) were FISH-positive; whereas the immunohistochemical analysis yielded only 1 patient (0.9%) exhibiting faint positive expression of ROS1 protein. Given the low incidences of ROS1 gene rearrangement and protein expression, their relationships with clinicopathologic parameters could not be statistically analyzed. ConclusionAlthough rare, patients with ovarian serous carcinoma or serous borderline tumor may exhibit ROS1 gene rearrangement and ROS1 protein expression. Further large-scale studies are necessary to explore the clinicopathologic significance of ROS1 gene expression in ovarian serous carcinoma.

Immunoglobulin Heavy Chain Gene Rearrangements in Patients with Gaucher Disease.

SummaryBackgroundSeveral studies support the evidence of increased incidence of hematological complications in Gaucher disease including monoclonal and polyclonal gammopathies and blood malignancies, especially multiple myeloma.MethodsSerum concentrations of immunoglobulins and PCR analysis of the IGH gene rearrangements were performed. The clonal PCR products were directly sequenced and analyzed with the appropriate database and tools. Serum monoclonal proteins were detected and identified by electrophoresis.ResultsAmong 27 Gaucher patients, clonal IGH rearrangement was discovered in eight, with 5/8 having also serum monoclonal protein. Elevated immunoglobulins were detected in 9/27 patients. Follow-up data for 17 patients showed that the clonal rearrangement remained the same in four of them, however, in one patient it disappeared after the follow-up period. The remaining 12/17 patients were without previous IGH clonal rearrangement and remained so after the follow-up.ConclusionsAlthough clonal expansion may occur relatively early in the disease course, at least judging by the IGH gene rearrangements in Gaucher patients, the detected clones may be transient. A careful clinical follow-up in these patients is mandatory, including monitoring for lymphoid neoplasms, especially multiple myeloma.

Hyalinizing clear cell carcinoma of the bronchial glands: presentation of three cases and pathological comparisons with salivary gland counterparts and bronchial mucoepidermoid carcinomas

AbstractHyalinizing clear cell carcinoma of the bronchial glands is a very rare tumor. Since only five reports describing six tumors have been published to date, only a little is known about specific histologic findings and clinical features. Because of its rarity, hyalinizing clear cell carcinoma has not been described in the latest WHO classification of pulmonary tumors yet. Here we present three cases of bronchial hyalinizing clear cell carcinomas, confirmed by both fluorescence in situ hybridization (FISH) and RT-PCR, focusing on histologic and immunohistochemical characteristics in a comparison with three cases of salivary gland origin. In addition, we compared immunohistochemical features with bronchial mucoepidermoid carcinoma, a lesion that needs to be taken into account in differential diagnosis of hyalinizing clear cell carcinoma. All our bronchial hyalinizing clear cell carcinoma cases were surgically resected. Histologically, tumor cells showed clear to eosinophilic cytoplasm with hyalinizing stroma in various proportions, resembling those of salivary gland origin. Immunohistochemically, tumor cells were positive for CK7, CK5/6, p40, p63, and ATF1, while they were negative for TTF1, Napsin A, HMB45, and SOX10. The CK5/6 staining pattern varied in mucoepidermoid carcinomas, while that of hyalinizing clear cell carcinoma was uniformly positive. FISH revealed EWSR1-ATF1 fusion, and RT-PCR with sequencing confirmed specificity of the chimeric gene for hyalinizing clear cell carcinoma. Clinically, bronchial hyalinizing clear cell carcinoma was characterized by occurrence in the fourth to sixth decades, no link with smoking history, and a predilection for the right lung, in line with previous reports. In summary, our study confirmed that the bronchial hyalinizing clear cell carcinoma is a histologically and genetically identical tumor to that of salivary gland origin, and that gene rearrangement analysis can play a critical role in distinction from mucoepidermoid carcinoma.

Primary adrenal diffuse large B cell lymphoma: a clinicopathological and molecular study from China.

Primary adrenal lymphoma is a rare entity that, in most cases, is derived from B cells. The most commonly seen primary adrenal lymphoma is diffuse large B cell lymphoma (DLBCL). To better understand the clinicopathological and molecular features of these tumors, we studied 14 Chinese patients with DLBCL who initially presented with an adrenal tumor. The clinicopathological features of the 14 primary adrenal DLBCL cases were retrospectively reviewed using immunohistochemistry, immunoglobulin gene rearrangement analysis, evaluation of Epstein-Barr virus status, and fluorescence in situ hybridization. Patient age ranged from 43 to 69years, with a mean age of 58years. The patients most commonly presented with abdominal pain and adrenal mass. Ten patients exhibited bilateral adrenal masses, and four had unilateral adrenal masses (three left, one right). Thirteen of 14 DLBCLs were DLBCL not otherwise specified, and one was an intravascular large B cell lymphoma. According to the algorithm of Hans et al. (Blood 103:275-282, 2004), 13 and 1 cases were classified as the non-germinal center B-cell-like subtype and the germinal center B-cell-like subtype, respectively. The Ki-67 index ranged from 35 to 80%. Epstein-Barr virus-encoded RNA was detected by in situ hybridization in 6 of the 12 available cases. Two patients showed BCL-6 rearrangements. The follow-up period ranged from 1 to 87months. During the follow-up period, four patients died of the disease, five were alive with the disease, four were alive without disease, and one was lost during the follow-up period. In summary, most primary adrenal lymphomas are non-germinal-center B-cell-like subtype DLBCLs, which have high proliferative activity and a poor prognosis.

Clinicopathologic features of primary hepatic marginal zone lymphoma of mucosa-associated lymphoid tissue and hepatic pseudolymphoma

Objective: To study the clinicopathological features of primary hepatic extranodal marginal zone lymphoma of mucosa associated lymphoid tissue (MALT lymphoma) and hepatic pseudolymphoma, and to discuss their differential diagnosis, treatment and prognosis. Methods: Three primary hepatic MALT lymphomas and two hepatic pseudolymphomas collected from January 2012 to March 2017 in the First Affiliated Hospital of Nanjing Medical University were evaluated by HE and immunohistochemistry(IHC), in-situ hybridization and immunoglobulin (Ig) gene rearrangement detection, and the relevant literature reviewed. Results: In the three MALT lymphomas, tumor cells infiltrated the portal areas with nodular pattern, and invaded the surrounding normal liver with serpiginous configuration and formation of confluent sheets. A number of bile ducts were entrapped within the lesions, and showed lymphoepithelial lesion. Reactive lymphoid follicles were present and surrounded by tumor cells, consisting of predominantly centrocyte-like cells and monocytoid B cells. There were clusters of epithelioid histiocytes in one case. The tumor cells were positive for CD20, PAX5 and negative for CD5, CD23, CD10, bcl-6, and cyclin D1. In the two hepatic pseudolymphomas, the lesions presented as solitary nodules well-demarcated from the surrounding liver tissue; one case was partially encapsulated with fibrous tissue. Entrapped bile ducts were only found at the edge of the lesions without lymphoepithelial lesion. The lesions comprised of massive lymphoid proliferation consisting predominantly of reactive lymphoid follicles, but not monocytoid B-cells or atypical cells. By IHC, a mixture of B- and T-cell population was identified. A monoclonal rearrangement of the Ig gene was detected in all three MALT lymphomas but not in two pseudolymphomas. Interphase fluorescence in situ hybridiazation test for MALT1 break-apart gene was positive in two cases of MALT lymphomas and EBER was negative in all studied cases. Conclusions: Primary heptic MALT lymphoma and pseudolymphoma are both rare lymphoid proliferative lesions of liver. These two lesions have overlapping histological and IHC features and are top differential diagnosis to each other. A combination analysis of morphology, immunophenotype and Ig gene rearrangement is helpful to distinguish between them.

An uncommon diagnosis for a recurrent erythematous patch in a paediatric patient

A 14-year-old girl presented with a circular erythematous patch over the left buttock for approximately 10 years, with ongoing ulceration and papules developing over the last 4 years. Punch biopsies...