ctDNA improves prognostic prediction for patients with relapsed/refractory MM receiving ixazomib, lenalidomide, and dexamethasone.

ABSTRACT: It remains elusive how driver mutations, including those detected in circulating tumor DNA (ctDNA), affect prognosis in relapsed/refractory multiple myeloma (RRMM). Here, we performed targeted-capture sequencing using bone marrow plasma cells (BMPCs) and ctDNA of 261 RRMM cases uniformly treated with ixazomib, lenalidomide, and dexamethasone in a multicenter, prospective, observational study. We detected 24 and 47 recurrently mutated genes in BMPC and ctDNA, respectively. In addition to clonal hematopoiesis-associated mutations, varying proportion of driver mutations, particularly TP53 mutations (59.2% of mutated cases), were present in only ctDNA, suggesting their subclonal origin. In univariable analyses, ctDNA mutations of KRAS, TP53, DIS3, BRAF, NRAS, and ATM were associated with worse progression-free survival (PFS). BMPC mutations of TP53 and KRAS were associated with inferior PFS, whereas KRAS mutations were prognostically relevant only when detected in both BMPC and ctDNA. A total number of ctDNA mutations in the 6 relevant genes was a strong prognostic predictor (2-year PFS rates: 57.3%, 22.7%, and 0% for 0, 1, and ≥2 mutations, respectively) and independent of clinical factors and plasma DNA concentration. Using the number of ctDNA mutations, plasma DNA concentration, and clinical factors, we developed a prognostic index, classifying patients into 3 categories with 2-year PFS rates of 57.9%, 28.6%, and 0%. Serial analysis of ctDNA mutations in 94 cases revealed that TP53 and KRAS mutations frequently emerge after therapy. Thus, we clarify the genetic characteristics and clonal architecture of ctDNA mutations and demonstrate their superiority over BMPC mutations for prognostic prediction in RRMM. This study is a part of the C16042 study, which is registered at www.clinicaltrials.gov as #NCT03433001.

Anti-HTLV-1 immunity combined with proviral load as predictive biomarkers for adult T-cell leukemia-lymphoma.

Human T-cell leukemia virus type 1 (HTLV-1) establishes chronic infection in humans and induces a T-cell malignancy called adult T-cell leukemia-lymphoma (ATL) and several inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Persistent HTLV-1 infection is established under the pressure of host immunity, and therefore the immune response against HTLV-1 is thought to reflect the status of the disease it causes. Indeed, it is known that cellular immunity against viral antigens is suppressed in ATL patients compared to HAM/TSP patients. In this study, we show that profiling the humoral immunity to several HTLV-1 antigens, such as Gag, Env, and Tax, and measuring proviral load are useful tools for classifying disease status and predicting disease development. Using targeted sequencing, we found that several carriers whom this profiling method predicted to be at high risk for developing ATL indeed harbored driver mutations of ATL. The clonality of HTLV-1-infected cells in those carriers was still polyclonal; it is consistent with an early stage of leukemogenesis. Furthermore, this study revealed significance of anti-Gag proteins to predict high risk group in HTLV-1 carriers. Consistent with this finding, anti-Gag cytotoxic T lymphocytes (CTLs) were increased in patients who received hematopoietic stem cell transplantation and achieved remission state, indicating the significance of anti-Gag CTLs for disease control. Our findings suggest that our strategy that combines anti-HTLV-1 antibodies and proviral load may be useful for prediction of the development of HTLV-1-associated diseases.

Whole-genome landscape of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform-specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3'-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell-like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

Synthesis of fluorescent 5-heteroarylpyrimidine-containing oligonucleotides via post-synthetic trifluoromethyl conversion.

Post-synthetic conversion of the trifluoromethyl group to a heteroaryl group at the C5 position of the pyrimidine base in DNA oligonucleotides was achieved. Specifically, the oligonucleotides containing 5-trifluoromethylpyrimidine bases were treated with o-phenylenediamines and o-aminothiophenols as nucleophiles to afford the corresponding 5-(benzimidazol-2-yl)- and 5-(benzothiazol-2-yl)-pyrimidine-modified bases. Furthermore, evaluation of the fluorescence properties of the obtained oligonucleotides revealed that among them the oligonucleotide containing 5-(5-methylbenzimidazol-2-yl)cytosine exhibited the highest fluorescence intensity. These results indicated that post-synthetic trifluoromethyl conversion, which is practical and operationally simple, is a powerful tool for exploring functional oligonucleotides.

Crystallization kinetics of atomic crystals revealed by a single-shot and single-particle X-ray diffraction experiment.

Crystallization is a fundamental natural phenomenon and the ubiquitous physical process in materials science for the design of new materials. So far, experimental observations of the structural dynamics in crystallization have been mostly restricted to slow dynamics. We present here an exclusive way to explore the dynamics of crystallization in highly controlled conditions (i.e., in the absence of impurities acting as seeds of the crystallites) as it occurs in vacuum. We have measured the early formation stage of solid Xe nanoparticles nucleated in an expanding supercooled Xe jet by means of an X-ray diffraction experiment with 10-fs X-ray free-electron laser (XFEL) pulses. We found that the structure of Xe nanoparticles is not pure face-centered cubic (fcc), the expected stable phase, but a mixture of fcc and randomly stacked hexagonal close-packed (rhcp) structures. Furthermore, we identified the instantaneous coexistence of the comparably sized fcc and rhcp domains in single Xe nanoparticles. The observations are explained by the scenario of structural aging, in which the nanoparticles initially crystallize in the highly stacking-disordered rhcp phase and the structure later forms the stable fcc phase. The results are reminiscent of analogous observations in hard-sphere systems, indicating the universal role of the stacking-disordered phase in nucleation.

Expansion of Phosphoramidite Chemistry in Solid-Phase Oligonucleotide Synthesis: Rapid 3'-Dephosphorylation and Strand Cleavage.

In solid-phase oligonucleotide synthesis, a solid support modified with a universal linker is frequently used to prepare oligonucleotides bearing non-natural- or non-nucleosides at the 3'-end. Generally, harsh basic conditions such as hot aqueous ammonia or methylamine are required to release oligonucleotides by 3'-dephosphorylation via the formation of cyclic phosphate with the universal linker. To achieve 3'-dephosphorylation under milder conditions, we used O-alkyl phosphoramidites instead of the commonly used O-cyanoethyl phosphoramidites at the 3'-end of oligonucleotides. Alkylated phosphotriesters are more alkali-tolerant than their cyanoethyl counterparts because the latter generates phosphodiesters via E2 elimination under basic conditions. Among the designed phosphoramidites, alkyl-extended analogs exhibited rapid and efficient 3'-dephosphorylation compared to conventional cyanoethyl and methyl analogs under mild basic conditions such as aqueous ammonia at room temperature for 2 h. Moreover, nucleoside phosphoramidites bearing 1,2-diols were synthesized and incorporated into oligonucleotides. 1,2,3,4-Tetrahydro-1,4-epoxynaphthalene-2,3-diol-bearing phosphoramidite behaved like a universal linker at the 3'-terminus, allowing dephosphorylation and strand cleavage of the oligonucleotide chain to occur efficiently. Our strategy using this new phosphoramidite chemistry is promising for the tandem solid-phase synthesis of diverse oligonucleotides.

Synthesis of purine derivatives of Me-TaNA and properties of Me-TaNA-modified oligonucleotides.

We previously reported that pyrimidine derivatives of methylated 2'-O,4'-C-methyleneoxy-bridged nucleic acid (Me-TaNA), a unique consecutive three-acetal-containing nucleic acid, are promising building blocks for chemically modified oligonucleotides. Herein, purine derivatives of Me-TaNA (Me-TaNA-A and -G) were synthesized and introduced into oligonucleotides. During the synthesis, we found stereoselective introduction of a substituent on the 4' carbons by using 2',3'-carbonate compounds as substrates. When forming duplexes with single-stranded RNA, the modified oligonucleotides, including purine derivatives of Me-TaNA, showed higher duplex stability than the natural oligonucleotide. This study enabled the use of Me-TaNA for the chemical modification of various oligonucleotide sequences because synthesis of Me-TaNAs with all four nucleobases was achieved.

Synthesis of Nucleobase-Modified Oligonucleotides by Post-Synthetic Modification in Solution.

Oligonucleotides containing modified nucleobases have applications in various technologies. In general, to synthesize oligonucleotides with different nucleobase structures, each modified phosphoramidite monomer needs to be prepared over multiple steps and then introduced onto the oligonucleotides, which is time-consuming and inefficient. Post-synthetic modification is a powerful strategy for preparing many types of modified oligonucleotides, especially nucleobase-modified ones. Depending on the stage of modification, post-synthetic modification can be divided into two stages: "solid-phase modification," wherein an oligonucleotide attaches to the resin, and "solution-phase modification," wherein an oligonucleotide detaches itself from the resin. In this review, we focus on post-synthetic modification in solution for the synthesis of nucleobase-modified oligonucleotides, except the modifications to linkers for conjugation. Moreover, the reactions are summarized for each modified position of the nucleobases.

Synthesis and Properties of Oligonucleotides Containing 2'-O,4'-C-Methyleneoxy-Bridged Pyrimidine Derivatives.

In this study, 2'-O,4'-C-methyleneoxy-bridged nucleic acid, a unique consecutive three-acetal-containing nucleic acid (TaNA), was designed. Pyrimidine derivatives of methylated TaNA (Me-TaNA) were also synthesized and introduced into oligonucleotides via solid-phase synthesis. The Me-TaNA-modified oligonucleotides exhibited higher stabilities when forming duplexes with single-stranded RNA or triplexes with double-stranded DNA, relative to the natural oligonucleotides and modified oligonucleotides containing another 2',4'-bridged 5-methyluridine, such as 2',4'-BNA/LNA and 2',4'-ENA. Furthermore, Me-TaNA within oligonucleotides significantly enhanced nuclease resistance.

Sarcoidosis and sarcoidal foreign body reaction after permanent eye makeup application: Analysis by immunohistochemistry with commercially available antibodies specific to Cutibacterium acnes and Mycobacteria.

We report two cases of eyebrow granulomas in patients who underwent a permanent eye makeup procedure. A rash was observed 16 months after the procedure in Case 1, and 10 years after the procedure in Case 2. Histopathologically, both patients exhibited noncaseating epithelioid cell granulomas. In Case 1, most of the black-brown granules of the permanent makeup were not present in the granulomas but were localized in the upper dermis. In contrast, in Case 2, some of the black-brown granules were phagocytized in the granulomas, preferentially within the giant cells. Based on systemic examinations, the patients from Cases 1 and 2 were diagnosed with sarcoidosis and sarcoidal foreign body reaction, respectively. To clarify the pathogenesis of our cases, we performed immunohistochemistry using commercially available monoclonal antibodies specific to Cutibacterium acnes, previously Propionibacterium acnes (PAB), and Mycobacteria (LAM antibody). PAB antibody results were positive in granulomas only in Case 1, and the LAM antibody results were negative in both cases. Immunohistochemical detection of C. acnes in granulomas could provide useful information for differentiating between cutaneous sarcoidosis and sarcoidal foreign body reactions.

Clinicopathological factors and tumor microenvironment markers predicting watch-and-wait discontinuation in 82 patients with follicular lymphoma.

OBJECTIVES: In this study, we aimed to determine the clinicopathological factors influencing the treatment-free period in patients with follicular lymphoma (FL) using a watch-and-wait (WW) strategy. METHODS: We retrospectively assessed histopathological parameters of 82 patients with FL. RESULTS: The median time from diagnosis to WW discontinuation was 62 months (range, 3-138), and median follow-up was 86 months (range, 3-183). Intermediate or high-risk Follicular Lymphoma International Prognostic Index score (P = .012), non-duodenal-type (P = .011), higher numbers of interfollicular CD4+ (P = .038) and intrafollicular FOXP3+ cells (P = .024) in the tumor microenvironment, and Ki-67 index ≥10% (P = .031) were significant adverse factors for WW discontinuation in univariate analyses. CONCLUSION: Patients with adverse factors for WW discontinuation should be carefully observed during follow-up.

Synthesis and properties of oligonucleotides bearing thymidine derivatives with 1,6-dioxaspiro[4.5]decane skeleton.

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.

Use of Core-Needle Biopsy for the Diagnosis of Malignant Lymphomas in Clinical Practice.

INTRODUCTION: Excisional biopsy (EB) is considered the gold standard for lymphoma diagnosis. Although recent advances in interventional radiology enable sampling with core-needle biopsy (CNB), only few studies evaluated the utility of CNB compared to that of EB. METHODS: We analyzed patients with lymphoma who had a diagnostic biopsy at the National Cancer Center Hospital during 2002-2017. We investigated the clinical and pathological characteristics of CNB in 2017. RESULTS: The proportion of CNB utility in total biopsy procedures had increased from 11 to 48% during the 15 years. In 2017, CNB was opted more frequently than EB for a biopsy of superficial, abdominal, or anterior mediastinal lesions. Only one out of 72 patients who had CNB required re-biopsy with EB because of insufficiency. The incidence of complications was comparable between CNB and EB: 2 (4%) cases of minor bleeding with CNB and 1 (8%) case of minor bleeding with EB. The median time from the first visit to biopsy was significantly shorter with CNB (5.5 days) than with EB (15 days). CONCLUSION: There is an increasing trend in the utility of CNB. CNB is a less invasive method with shorter time to biopsy and can be considered an alternative to EB.

Rhinoceros beetle horn development reveals deep parallels with dung beetles.

Beetle horns are attractive models for studying the evolution of novel traits, as they display diverse shapes, sizes, and numbers among closely related species within the family Scarabaeidae. Horns radiated prolifically and independently in two distant subfamilies of scarabs, the dung beetles (Scarabaeinae), and the rhinoceros beetles (Dynastinae). However, current knowledge of the mechanisms underlying horn diversification remains limited to a single genus of dung beetles, Onthophagus. Here we unveil 11 horn formation genes in a rhinoceros beetle, Trypoxylus dichotomus. These 11 genes are mostly categorized as larval head- and appendage-patterning genes that also are involved in Onthophagus horn formation, suggesting the same suite of genes was recruited in each lineage during horn evolution. Although our RNAi analyses reveal interesting differences in the functions of a few of these genes, the overwhelming conclusion is that both head and thoracic horns develop similarly in Trypoxylus and Onthophagus, originating in the same developmental regions and deploying similar portions of appendage patterning networks during their growth. Our findings highlight deep parallels in the development of rhinoceros and dung beetle horns, suggesting either that both horn types arose in the common ancestor of all scarabs, a surprising reconstruction of horn evolution that would mean the majority of scarab species (~35,000) actively repress horn growth, or that parallel origins of these extravagant structures resulted from repeated co-option of the same underlying developmental processes.

[Utility of core-needle biopsy as a primary diagnostic method for detecting aggressive B-cell lymphoma in the intensive care unit].

A 68-year-old male presented with appetite loss and abdominal distention. The whole-body computed tomography scan revealed an ileocecal mass with a large amount of ascites, which was consistent with malignant lymphoma. Due to the worsening of his general condition following admission, he was intubated and admitted to the intensive care unit (ICU). In the ICU, we performed a core-needle biopsy (CNB) on the left peritoneal mass, the findings of which showed a pathological diffuse infiltration of CD20+ middle-sized lymphoid cells. After chemotherapy was initiated, the patient showed complete response, suggesting that CNB can be performed immediately and safely even on a critically ill patient.

Chemical Characterization of a Volatile Dubnium Compound, DbOCl3.

The formation and the chemical characterization of single atoms of dubnium (Db, element 105), in the form of its volatile oxychloride, was investigated using the on-line gas phase chromatography technique, in the temperature range 350-600 °C. Under the exactly same chemical conditions, comparative studies with the lighter homologues of Group 5 in the Periodic Table clearly indicate the volatility sequence being NbOCl3 > TaOCl3 ≥ DbOCl3 . From the obtained experimental results, thermochemical data for DbOCl3 were derived. The present study delivers reliable experimental information for theoretical calculations on chemical properties of transactinides.

Immunohistochemical CD20-negative change in B-cell non-Hodgkin lymphomas after rituximab-containing therapy.

CD20- change after rituximab-containing therapy is considered one of the main reasons of rituximab resistance of B-cell non-Hodgkin lymphomas (B-NHLs). However, the clinicopathological characteristics of B-NHL with CD20- change are not entirely understood. In this study, 252 B-NHL patients who were CD20+ at initial diagnosis, whose diseases relapsed or were refractory after rituximab-containing therapy, and who were re-biopsied between 2000 and 2018, were included. The median number of rituximab administration was 11 (range, 1-48). Completely negative (cCD20-) and partially negative (pCD20-) change of CD20 was observed in 49 (20%) and 16 (6%) cases, respectively. Among cCD20- and pCD20- cases, 74% and 62% of the cases changed to CD20- at the second relapse or later, respectively. Overall survival was significantly shorter in cCD20- follicular lymphoma (FL) cases than in CD20+ FL cases. Seven histopathological patterns, such as CD20- change without histological change, histological transformation (HT) to CD20- diffuse large B-cell lymphoma, and proliferation of plasmablastic/plasmacytoid tumor cells, were associated with CD20- change. HT occurred more frequently in FLs with CD20- change than in FLs continuously expressing CD20 (P < 0.0001), regardless of the timing of HT. Nine out of 25 cases (36%) showed regain or heterogeneous regain of CD20 expression. In conclusion, 20% and 6% of the 252 B-NHL cases show cCD20- and pCD20- changes with 7 histological patterns after rituximab-containing therapy. Because changes in morphology and CD20 expression after rituximab-containing therapy vary, and recovery of CD20 expression is not rare, careful follow-up and re-biopsy in B-NHL patients are recommended.

Synthesis of Oligonucleotides Containing 2'-N-alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) Moieties Using Post-Synthetic Modification Strategy.

The post-synthetic modification of an oligonucleotide is a powerful strategy for the synthesis of various analogs of the oligonucleotide, aiming to achieve the desired functions. In this study, we synthesized the thymidine phosphoramidite of 2'-N-pentafluorophenoxycarbonyl-2'-amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2'-N-pentafluorophenoxycarbonyl-2'-amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM K2CO3 in MeOH at room temperature for 4 h). Moreover, by treatment with various amines as a post-synthetic modification, the oligonucleotides were successfully converted into the corresponding 2'-N-alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) derivatives. The duplex- and triplex-forming abilities of the synthesized oligonucleotides were evaluated by UV-melting experiments, which showed that 2'-urea-LNAs could stabilize the nucleic acid complexes, similar to the proto-type, 2'-amino-LNA. Thus, 2'-urea-LNAs could be promising units for the modification of oligonucleotides; the design of a substituent on urea may aid the formation of useful oligonucleotides. In addition, pentafluorophenoxycarbonyl, an amino moiety, acted as a precursor of the substituted urea, which may be applicable to the synthesis of oligonucleotide conjugates.

Ultrafast Structural Dynamics of Nanoparticles in Intense Laser Fields.

Femtosecond laser pulses have opened new frontiers for the study of ultrafast phase transitions and nonequilibrium states of matter. In this Letter, we report on structural dynamics in atomic clusters pumped with intense near-infrared (NIR) pulses into a nanoplasma state. Employing wide-angle scattering with intense femtosecond x-ray pulses from a free-electron laser source, we find that highly excited xenon nanoparticles retain their crystalline bulk structure and density in the inner core long after the driving NIR pulse. The observed emergence of structural disorder in the nanoplasma is consistent with a propagation from the surface to the inner core of the clusters.

Synthesis of the Methyl Analog of 2'-O,4'-C-Ethylene-Bridged 5-Methyluridine via Intramolecular Radical Cyclization and Properties of Modified Oligonucleotides.

The synthesis of 6'S-Me-2'-O,4'-C-ethylene-bridged 5-methyluridine (6'S-Me-ENA-T) was achieved using visible light-mediated stereoselective radical cyclization as a key step. This is the first example of a method for constructing a 2',4'-bridged structure from a 4'-carbon radical intermediate. The 6'S-Me-ENA-T monomer was successfully incorporated into oligonucleotides, and their properties were examined. The oligonucleotides containing 6'S-Me-ENA-T exhibited a highly selective hybridization affinity toward single-stranded RNA and an excellent enzymatic stability, compared to the corresponding LNA- and ENA-modified oligonucleotides.