\(\textit{rbc}\)L DNA region revealed as the best DNA barcode for identification of \(\textit{Mahonia}\) and \(\textit{Berberis}\) species (Berberidaceae)

Berberidaceae contains 17 genera and nearly 650 species, in which the genus Berberis and the genus Mahonia are sisters with a very close relationship and share many similar characteristics. In the past, the two genera were merged into the genus Berberis. At present, the Berberidaceae is at risk and is prioritized for conservation, especially the species of Mahonia and Berberis. In the fact, identification for the Berberidaceae species based on the morphology of parts for sale (such as roots, stems, leaves) without reproductive organs (flowers and fruits) is impossible. In recent years, molecular biology techniques are being applied widely and effectively in research on the evolution, classification and genetic diversity of populations. Study based on DNA has highly accurate and particularly useful for closely related species which morphological observations are not sufficient to distinguish. Results from DNA analysis allow authentic species, populations or individuals from un-intact specimens accurately and especially, it is not affected by objective factors such as the environment or human. In this study, we assessed the taxonomy ability of three commonly DNA barcoding regions used for classification including rbcL, trnH-psbA and ITS2 on 12 samples of the Berberidaceae family, in which 7 samples are genus Mahonia, 4 samples are genus Berberis and one sample of Epimedium was used as the control sample. The results of the study will contribute to the selection of suitable DNA barcode for the identification of Mahonia and Berberis samples. The results demonstrated that the rbcL region showed the most obvious ability to distinguish the 12 Berberidaceae species. The ITS2 sequences of Berberis julianeae and Mahonia bealei of Vietnam were submitted to GenBank with accession numbers MT073031 and MT008067, respectively. The sequence of rbcL of Mahonia bealei was also submitted to GenBank with accession number MT457415.1.

Seedling Morphology of some selected members of Commelinaceae and its bearing in taxonomic studies

Seedling morphology of eight species from four genera of the family Commelinaceae viz. Commelina appendiculata C.B. Clarke, C. benghalensis L., C. caroliniana Walter, C. paludosa Blume, Cyanotis axillaris (L.) D. Don ex Sweet, C. cristata (L.) D. Don, Murdannia nudiflora (L.) Brenan and Tradescantia spathacea Sw. are investigated using both light and scanning electron microscopy. The seedling morphological features explored include germination pattern, seed shape, surface and hilum, root system, cotyledon type, cotyledonary hyperphyll (apocole), cotyledonary hypophyll (cotyledonary sheath), hypocotyl, first leaf and subsequent leaves. All taxa studied had hypogeal and remote tubular cotyledons. However, differences in cotyledon structure (apocole, cotyledonary sheath), seed, hypocotyl, internodes, first leaf and subsequent leaves were observed. Variations of those characters were used to prepare an identification key for the investigated taxa. Commelina spp. and Murdannia nudiflora of the tribe Commelineae were found to differ from Cyanotis spp. and Tradescantia spathacea of tribe Tradescantieae in the petiolate first leaf with papillate margins on upper surface with 6-celled stomata and the glabrous epicotyl. The presence of an elongated cotyledonary sheath, long apocole and extended periblast region appear to be synapomorphies for Commelina spp. and T. spathacea. The affinity of the investigated taxa as revealed through multivariate analysis supports some of the relationships inferred by pollen morphology, floral morphology and DNA (rbc-L, 5S NTS, trnL-trnF) data stated by previous authors.

Personalized and Optimized Low-Dose and Intensive Chemotherapy Treatments for Patients with Acute Myeloid Leukemia (AML)

Patients with AML have heterogeneous features, including those specific to the patient as well as those specific to the disease, such as leukemic burden and dynamic sub-clonal populations. Outside of clinical trials, few of these components are used to determine treatment. In order to move towards precision medicine, we have developed πiChemo, a computational application based on a dynamic mathematical modelling framework, using patient-, leukemia- and treatment-specific data to predict outcomes and optimize chemotherapy regimens for patients with AML. The model consists of a pharmacokinetic and pharmacodynamic (PK/PD) module that calculates the concentration and effect of Cytarabine Arabinoside (Ara-C) and Daunorubicin (DNR) in bone marrow (BM); and a population balance models (PBMs) module that describes normal populations (stem cells, progenitors, precursors) and abnormal populations (leukemic sensitive blasts (LSB) and leukemic resistant blasts (LRB)) in BM. The PBMs module also determines mature cell numbers in three lineages found in BM and peripheral blood (PB): (1) red blood cells (RBC), (2) white blood cells (WBC) and (3) lymphocytes (L). Model structure was analysed by global sensitivity analysis, which identified the most significant parameters on outcome predictions, re-estimated for each patient. The final integrated PK/PD & PBMs model has 1,295 differential equations, 8,044 algebraic equations, 9,335 variables, 25 fixed parameters and 4 degrees of freedom or variables to be optimized (Ara-C dose, Ara-C injection duration, DNR dose and DNR injection duration). Model validation, predictions and optimizations were performed using anonymised retrospective data from 28 patients with AML. The model required: (i) patient features: height, weight, age and gender, (ii) patient status: initial BM differential and PB cell counts, (iii) leukemia data: cellularity, presence of dysplasia and initial blast percentage and, (iv) treatment data: type (low-dose (LD) or intensive (DA)), dose, administration route (SC vs IV), administration mode (bolus injection vs infusion), time between injections and between cycles. The model predicted the absolute numbers of stem cells, progenitors, precursors, WBC, RBC, L, LSB and LRB in BM, and WBC, RBC, L and neutrophil count in PB during treatment for all patients. Model simulations predicted outcomes for 18 patients who achieved complete remission (7 LD & 11 DA), 4 patients who entered partial remission (2 LD & 2 DA) and 6 patients who relapsed (2 LD & 4 DA). The most remarkable results are those of prediction for BM blast percentage after each chemotherapy cycle and the PB neutrophil count for all patients. The notable fit between model predictions and daily patient data demonstrate model robustness and accuracy in the capacity to track patient-specific restaging BM and daily PB count evolution before, during and after treatment. The same patient datasets were used to apply an optimization algorithm that could maximize normal cell number and reduce leukemia burden, to personalize chemotherapy dose and administration for best outcomes. The results show that doses and administration methods vary between patients and between chemotherapy cycles for the same patient, depending on the evolution of normal and abnormal populations in BM. Low-dose continuous Ara-C infusions were more effective than rapid bolus injections, due to reduced chemotherapy effects on normal cells and subsequent quicker recovery in the normal BM compartments. RBC progenitors and precursors recovered faster than WBC and L lineages, and the recovery of normal BM cells was faster than that of normal mature cells in PB. The πiChemo tool requires only patient- and leukemia-specific initial conditions at diagnosis, easily obtained in standard clinical practice, for outcome predictions and treatment optimizations. Real-time model-fit testing and comparison of model results against daily PB cell counts would enable the re-estimation of significant parameters, increasing model accuracy and treatment effectiveness whilst therapy is ongoing. The πiChemo precision therapy tool has the potential to personalize optimal standard and novel treatments for AML in real-time. Disclosures No relevant conflicts of interest to declare.

L-Asparaginase Allergic Patients Treated with L-Asparaginase Loaded into Red Blood Cells in an Expanded Access Program. Report of Four Cases

L-asparaginase (L-ASP) is an important drug in the treatment of acute lymphoblastic leukemia (ALL) demonstrating efficacy in a broad range of patients. However the toxicity profile, including allergy, has been a major drawback. There is an unmet medical need for patients who cannot receive L-ASP current formulations, especially due to allergy. E-Coli L-Asparaginase encapsulated into red blood cells (RBC/L-ASP) is a new product under development with the aim of improving the tolerability of this enzyme. Asparagine is actively transported through the membrane of red blood cells (RBC) where it is hydrolyzed by the encapsulated L-ASP, the erythrocytes acting as “bioreactors”. In addition, the RBC membrane shields against the anti-L-ASP antibody then avoiding binding to encapsulated L-ASP. Clinical trials have demonstrated a reduction in allergy with encapsulated L-ASP, and the enzyme activity is sustained even in presence of anti-L-ASP antibodies. Four patients, who were not able to receive any current L-ASP due to allergy, were enrolled in an Expanded Access Program (# NCT02197650) allowing to receive RBC/L-ASP: Patient 1: 48 year old, female, with T-cell ALL Ph-, normal karyotype, without neuro-meningeal infiltration, without extra-medullar localization. Treated according to GRAALL-2005 protocol. In complete remission (CR) after induction therapy, with negative MRD. During consolidation therapy, a grade 3 allergy (anaphylaxis) was observed after 6 injections of native E-Coli L-ASP. For late intensification, the patient was switched to Erwinia and after 3 injections a grade 3 allergy occurred. This patient was switched to RBC/L-ASP (150IU/Kg) receiving 2 injections to complete the late intensification phase with no occurrence of allergy. To date this patient is treated by radiotherapy which will be followed by the maintenance therapy. Patient 2: 30 year old, male, with T-cell ALL Ph-, normal karyotype, without neuro-meningeal infiltration, with extra-medullar localization (cutaneous and renal involvement). Treated according to GRAALL-2005 protocol, corticosensitive and chemosensitive. In CR after induction therapy, with negative MRD after 35 and 70 days. During consolidation phase, a grade 3 allergy (anaphylaxis) was observed after 8 injections of native E.Coli L-ASP. The patient was switched to Erwinia and after 2 injections a grade 2 allergy was observed. For late intensification this patient received RBC/L-ASP (150IU/Kg) at day 2 and at day 15. No allergy occurred. The patient remains in CR and has initiated maintenance therapy. Patient 3: 9 year old, male, with relapsing B-cell ALL Ph-, with hyperdipliody, without neuro-meningeal infiltration, with no extra-medullar localization. Treated according to INTREALL 2010 protocol UKALL R3 arm. In CR after re-induction therapy, with negative MRD on day 35. Grade 1 L-ASP related allergy was observed after 17 injections of native E-coli L-ASP. Erwinia was initiated and a grade 1 allergy observed after 5 injections. Peg-ASP then resulted in a grade 1 allergy after 1 injection. This patient received RBC/L-ASP (150IU/Kg) at day 6 and at day 41 of the consolidation phase. A grade 1 allergy was observed in hours following the first administration. One dose of hydrocortisone and an anti histamine treatment for 2 days resulted in full resolution. 35 days later, the patient received a second injection with prophylactic anti allergic treatment. No allergy occurred. 21 days later the patient died due to bacterial infection with ARDS. Patient 4: 3 year old, male, with T-cell ALL Ph-, with no extramedullary localization, treated in the VHR group of EORTC 58081 protocol (poor prednisone response to the prephase). In CR at the end of induction (Ia), with negative MRD after induction and consolidation (<10-4). Grade 3 allergy to native E Coli L-ASP occurred during the first injection of the consolidation phase and the patient received 6 injections of Erwinase . During the subsequent phase (VANDA) a grade 3 allergy occurred at the first Erwinase injection. The patient was switched to RBC/L-ASP (150IU/Kg) for the re-induction phase. No allergy occurred. The second re-induction phase is scheduled in 3 months. Circulating L-ASP activity was sustained over 100 IU/L for at least 14 days. In conclusion, for patients allergic to the native E-Coli and/or Erwinia L-ASP, RBC/L-ASP seems to strongly reduce the risk of allergy in maintaining L-ASP activity. Disclosures Godfrin: ERYTECH: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.