Optimal sequencing of ibrutinib, idelalisib, and venetoclax in chronic lymphocytic leukemia: results from a multicenter study of 683 patients.

Background: Ibrutinib, idelalisib, and venetoclax are approved for treating CLL patients in the United States. However, there is no guidance as to their optimal sequence. Patients and methods: We conducted a multicenter, retrospective analysis of CLL patients treated with kinase inhibitors (KIs) or venetoclax. We examined demographics, discontinuation reasons, overall response rates (ORR), survival, and post-KI salvage strategies. Primary endpoint was progression-free survival (PFS). Results: A total of 683 patients were identified. Baseline characteristics were similar in the ibrutinib and idelalisib groups. ORR to ibrutinib and idelalisib as first KI was 69% and 81%, respectively. With a median follow-up of 17 months (range 1-60), median PFS and OS for the entire cohort were 35 months and not reached. Patients treated with ibrutinib (versus idelalisib) as first KI had a significantly better PFS in all settings; front-line [hazard ratios (HR) 2.8, CI 1.3-6.3, P = 0.01], relapsed-refractory (HR 2.8, CI 1.9-4.1, P < 0.001), del17p (HR 2.0, CI 1.2-3.4, P = 0.008), and complex karyotype (HR 2.5, CI 1.2-5.2, P = 0.02). At the time of initial KI failure, use of an alternate KI or venetoclax had a superior PFS when compared with chemoimmunotherapy. Furthermore, patients who discontinued ibrutinib due to progression or toxicity had marginally improved outcomes if they received venetoclax (ORR 79%) versus idelalisib (ORR 46%) (PFS HR .6, CI.3-1.0, P = 0.06). Conclusions: In the largest real-world experience of novel agents in CLL, ibrutinib appears superior to idelalisib as first KI. Furthermore, in the setting of KI failure, alternate KI or venetoclax therapy appear superior to chemoimmunotherapy combinations. The use of venetoclax upon ibrutinib failure might be superior to idelalisib. These data support the need for trials testing sequencing strategies to optimize treatment algorithms.

First Report of Powdery Mildew (Pseudoidium neolycopersici) on Croton (Codiaeum variegatum var. pictum) in China.

Croton (Codiaeum variegatum (Linn.) var. pictum (Lodd.)) is an ornamental plant commonly grown in southern China. In March 2014, severe powdery mildew infections were observed on crotons in gardens of Hainan University (20.1°N and 110.3°E), Haikou, Hainan province. Disease incidence was estimated in a random batch of 100 plants in three replicates, with the average value approaching 80%. Symptoms first appeared as white circular patches on the adaxial surface and expanded to the abaxial surface, petioles, and stems. The top leaves were the most affected. Upper surfaces of the infected leaves were covered by white, dense mycelia. As the disease progressed, infected leaves turned purple on the lower surfaces and curly before becoming necrotic and abscising from the plant. Powdery mildew was more severe in shaded environments, especially during rainy or foggy weather in early spring. Two hundred conidiophores and conidia were observed microscopically. The conidiophores were straight or occasionally flexuous, 62.3 to 127.6 × 6.2 to 10.2 µm, consisting of two to three straight cells. Conidia were born in solitary on the top of conidiophores. Conidia were hyaline, ellipsoidal, 26.4 to 42.2 × 11.7 to 23.4 µm (average 32.5 × 16.5 µm), contained no distinct fibrosin bodies, and produced a subterminal germ tube. The wrinkling pattern of the outer walls of older conidia was angular or reticulated. Appressoria were single and multilobed. Cleistothecia were not observed. Based on morphological characteristics, the fungus was identified as Oidium neolycopersici (2), which was recently renamed Pseudoidium neolycopersici (L. Kiss) (3). The identity was confirmed by sequence analysis. Genomic DNA was extracted from the foliar powdery mildew colonies using Chelex-100 (Bio-Rad, Shanghai, China). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 (5). The ITS sequence of the representative isolates C01 (GenBank Accession No. KJ890378.1) and four other powdery mildew samples collected from crotons in Hainan University was 100% identical to that of P. neolycopersici isolates from tomato plants such as JQ972700 and AB163927. Inoculations were made by gently pressing diseased leaves onto leaves of five healthy plants of croton and tomato ('Money maker'). Five non-inoculated croton and tomato plants served as controls. Inoculated and non-inoculated plants were maintained in an incubator at 25°C with a 12-h photoperiod. After eight days, typical powdery mildew symptoms developed on 93% of the inoculated plants, while no symptom developed on the non-inoculated plants. The pathogenicity tests were repeated three times. The same fungus was always re-isolated from the diseased tissue according to Koch's postulates. The pathogenicity tests further confirmed that the pathogen from crotons is P. neolycopersici (Basionym. Oidium neolycopersici (KJ890378.1)), which is commonly known as the tomato powdery mildew. P. neolycopersici is also a pathogen of Normania triphylla (1) and papaya (4). To our knowledge, this is the first report of P. neolycopersici infecting croton. The avenue of this pathogen entering gardens of Hainan University remains unknown. The gardens are located far away from tomato farms. Also no symptom of powdery mildew on croton was observed during surveys in other locations in Haikou. The origin of the pathogen warrants additional research. References: (1) D. Delmail et al. Mycotaxon 113:269, 2010. (2) L. Kiss et al. Mycol. Res. 105:684, 2001. (3) L. Kiss et al. Mycol. Res. 115:612, 2011. (4) J. G. Tsay et al. Plant Dis. 95:1188, 2011. (5) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Target Spot of Cotton Caused by Corynespora cassiicola in China.

Cotton (Gossypium hirsutum L.) is one of the most important economic crops in China and fungal diseases are the major limiting factors in its production. In September 2013, cotton plants infected with leaf spots were observed in Sanya, Hainan Province, China. Initial symptoms developed as brick-red dots that led to the formation of irregular to circular lesions with gray centers surrounded by brown borders. Individual leaf spots formed concentric rings of alternating light and dark brown bands. Leaf tissue segments collected from the border between symptomatic and healthy tissue were surface disinfested in 75% ethanol for 1 min, then rinsed three times in sterile water with streptomycin sulfate. Fungal isolates obtained from these segments were purified by the single spore technique on potato dextrose agar (PDA) at 28°C. The initial color of the colonies was olivaceous, turning dark brown after 5 days. Conidiophores were scattered or clustered, brown, straight to curved, unbranched, and glabrous. Conidia had 4 to 12 pseudosepta and were 56 to 230 µm long and 5 to 15 µm wide, brown, straight to slightly curved, obclavate to cylindrical, glabrous, and apex obtuse. These characteristics were consistent with the description of Corynespora cassiicola (Berk. & M.A. Curtis.) C.T. Wei (3). A pathogenicity test was conducted with the four isolates on detached young cotton leaves (two to four true leaf stage). For each isolate, three slightly wounded and three unwounded leaves were inoculated with 5.5-mm-diameter mycelial plugs. For the control treatment, wounded and unwounded leaves were mock inoculated with sterile PDA plugs of the same size. The inoculated leaves were placed in a moist chamber and incubated with a 12-h photoperiod at 28°C. Necrotic lesions appeared on wounded spots after 2 days of incubation and on unwounded leaves 3 days after incubation. All symptoms were similar to those observed in the field. Symptoms were not observed on control leaves. The same fungus was always re-isolated from the diseased tissue according to Koch's postulates. To confirm the identity of the pathogen, DNA was extracted from a 1-week-old culture grown on PDA and the internal transcribed spacer region (ITS) of one isolate (GenBank Accession No. KF924624) was amplified using primers ITS1 and ITS4 (4) and sequenced. BLAST search in GenBank revealed 100% homology with sequences of C. cassiicola (EU364535.1, EU364536.1, FJ852574.1, and FJ852575.1). Based on the symptoms, fungal morphology, ITS sequence comparison, and pathogenicity test, this fungus was identified as C. cassiicola. Target spot of cotton associated with C. cassiicola has been reported in Georgia (2) and Alabama (1). To our knowledge, this is the first report that C. cassiicola can infect cotton in China inducing target spot of cotton (2). This report will establish a foundation for further study of C. cassiicola to aid disease measurement and control. References: (1) K. N. Conner et al. Plant Dis. 97:1379, 2013. (2) A. M. Fulmer et al. Plant Dis. 96:1066, 2012. (3) J. Y. Lu. Page 407 in: Plant Pathogenic Mycology. China Agricultural Press, Beijing, 2000. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

Isolation and characterization of nucleotide-binding site and C-terminal leucine-rich repeat-resistance gene candidates in bananas.

Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.

Cloning and structural characterization of ECTACC, a new member of the transforming acidic coiled coil (TACC) gene family: cDNA sequence and expression analysis in human microvascular endothelial cells.

Erythropoietin (Epo) transduces mitogenic and chemoattractant signals to human endothelial cells. Identifications of Epo-responsive genes are important for understanding the molecular nature of Epo signaling in endothelial cells. The effects of Epo on differential expression of various genes were examined in human microvascular endothelial cells (HMVEC) by differential display reverse transcriptase polymerase chain reaction (RT-PCR). In the current study we obtained from Epo-treated HMVEC a cDNA fragment with characteristics of the 3' end of mRNA. Using the cDNA fragment, we then selectively isolated a full-length clone by screening an unamplified endothelial cell cDNA library followed by 5' rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR). The nucleotide sequence of the longest cDNA revealed an open reading frame of 3311 nucleotides that encodes a protein consisting of approximately 906 amino acids with a predicted MW of approximately 100 kDa. The nucleotide sequence of the cDNA is nearly identical to that of transforming acidic coiled coil-containing (TACC2) and anti-zuai-1 (AZU-1) cDNA clones except at the 5'- and 3'-ends. Northern blot analysis showed an increase in endothelial-TACC-related mRNA levels in Epo-treated cells in comparison to that of the control cells. Endothelial-TACC-related mRNA was highly expressed in heart and skeletal muscle tissue. Placenta and brain tissue exhibited low levels of expression of endothelial-TACC-related gene. Southern blot analysis of genomic DNA from somatic cell hybrids showed that endothelial-TACC-related cDNA maps to chromosome 10. Immunofluorescence microscopy and the occurrence of several putative phosphorylation and SH3 binding sites on the deduced protein suggest that endothelial-TACC-related protein may be involved in Epo signaling cascades in endothelial cells.