Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.

High diversity of coralline algae in New Zealand revealed: Knowledge gaps and implications for future research.

Coralline algae (Corallinophycideae) are calcifying red algae that are foundation species in euphotic marine habitats globally. In recent years, corallines have received increasing attention due to their vulnerability to global climate change, in particular ocean acidification and warming, and because of the range of ecological functions that coralline algae provide, including provisioning habitat, influencing settlement of invertebrate and other algal species, and stabilising reef structures. Many of the ecological roles corallines perform, as well as their responses to stressors, have been demonstrated to be species-specific. In order to understand the roles and responses of coralline algae, it is essential to be able to reliably distinguish individual species, which are frequently morphologically cryptic. The aim of this study was to document the diversity and distribution of coralline algae in the New Zealand region using DNA based phylogenetic methods, and examine this diversity in a broader global context, discussing the implications and direction for future coralline algal research. Using three independent species delimitation methods, a total of 122 species of coralline algae were identified across the New Zealand region with high diversity found both regionally and also when sampling at small local spatial scales. While high diversity identified using molecular methods mirrors recent global discoveries, what distinguishes the results reported here is the large number of taxa (115) that do not resolve with type material from any genus and/or species. The ability to consistently and accurately distinguish species, and the application of authoritative names, are essential to ensure reproducible science in all areas of research into ecologically important yet vulnerable coralline algae taxa.

Confirmation of Caprella scauroides Mayer, 1903 (Crustacea: Amphipoda) from New Zealand, using integrative techniques.

The presence of caprellid amphipod, Caprella scauroides is confirmed for the first time from New Zealand waters through two separate sample submissions received by the Ministry for Primary Industries (MPI) Marine Invasives Taxonomic Service (MITS) during May 2017, both from Okahu Bay, Waitemata Harbour, from swing mooring ropes. A subsequent report (November 2017) recorded the species from the Whangarei Harbour Marine High Risk Site Surveillance (MHRSS) survey. Caprella scauroides, non-indigenous to New Zealand, is morphologically similar and closely related to the known invasive, Caprella californica, with which it is often confused. Caprella scauroides from New Zealand is described in detail using both morphological and molecular methods.

A new species of sponge-dwelling amphipod, Polycheria spongoteras sp. nov., from Spirits Bay, Northland, New Zealand.

A new species of the often-cryptic genus Polycheria (Crustacea; Amphipoda) was discovered living in a small specimen of the sponge, Homaxinella erecta (Brøndsted, 1924) (Demospongiae, Suberitida, Suberitidae), in Spirits Bay, on the northern tip of the North Island of New Zealand. Polycheria spongoteras sp. nov. is described using integrative techniques (morphologically, molecularly and ecologically), with discussions on the New Zealand records of the genus and related taxa. The host sponge is redescribed and placed in the Spirits Bay context. [Zooban URL: urn:lsid:zoobank.org:act:FB60B77B-6B98-4102-A41F-D980B03204EB].

Oral fludarabine and rituximab as initial therapy for chronic lymphocytic leukemia or small lymphocytic lymphoma: population-based experience matches clinical trials.

Clinical trials report that fludarabine and rituximab (FR) as initial therapy for chronic lymphocytic leukemia (CLL) improves progression-free and overall survival (OS) when compared historically to fludarabine alone. To determine whether similar results are achievable with oral FR in a community-based setting, we conducted a population-based analysis of patients treated for CLL or small lymphocytic lymphoma (SLL) in British Columbia, where FR is standard initial therapy. Ninety-eight patients received FR for CLL/SLL from 2004 to 2009. Two- and 4-year OS was 90% and 73%, respectively (median not reached); 2- and 4-year treatment-free survival (TFS) was 69% and 54% (median 4.0 years). Age ≥ 60 years or ≥ 70 years had no effect on OS or TFS. Toxicity led to treatment discontinuation in 13%. FR with oral fludarabine was safely, conveniently and successfully given to community-based patients, irrespective of age, for first-line therapy for CLL/SLL, achieving OS and TFS similar to those in clinical trials.

Alemtuzumab in clinical practice: a British Columbia experience.

Limited information is available on alemtuzumab in the nonclinical trial setting. We evaluated its efficacy and safety in 42 consecutive unselected patients who received alemtuzumab monotherapy in British Columbia between October 2002 and August 2006. Information on patient demographics, baseline clinical characteristics, dose and schedule, clinical response, survival, and toxicities associated with alemtuzumab was collected retrospectively. Thirty-nine of 42 patients had chronic lymphocytic leukemia, two had mycosis fungoides, and one had T-cell post-transplant lymphoproliferative disorder. In contrast to previous reports, 42% were treated by community practitioners and 83% received alemtuzumab subcutaneously. The median time from diagnosis to alemtuzumab was 58 months. One of 42 patients (2%) achieved a complete response, 20 (48%) achieved a partial response, and 13 (31%) had stable disease. The post-alemtuzumab median overall survival was 15.1 months. Response to alemtuzumab correlated with an increased progression-free survival (11 vs. 3.6 months, p = 0.001) compared to that seen in non-responders. Significant adverse events included grade 3/4 neutropenia (76%), thrombocytopenia (45%), infections (60%) and death (12%). With careful monitoring, alemtuzumab can be safely administered in a wide variety of clinical settings, including community practice, and is associated with a high level of activity in situations with few available alternative treatment options.

CHOP-R therapy overcomes the adverse prognostic influence of BCL-2 expression in diffuse large B-cell lymphoma.

BCL-2 protein expression correlates with shorter survival in patients with diffuse large B cell lymphoma (DLBCL) who are treated with CHOP chemotherapy. We report a retrospective analysis of the prognostic significance of BCL-2 status in patients who received CHOP with the addition of rituximab (CHOP-R) for DLBCL. Patients over 15 years of age with de novo, HIV negative DLBCL, without CNS involvement, and known BCL-2 protein status were identified from the BCCA Lymphoid Cancer Database. BCL-2 tumour positivity was defined as over 50% of tumour cells with BCL-2 protein expression. 140 patients who received CHOP-R were analysed. The majority (59%) of patients were over 60 years of age. Disease stage distribution was limited (22%) and advanced (78%). BCL-2 protein expression was observed in 90 (64%) cases. IPI score was similar in both BCL-2 positive and negative cases. Median follow-up time for living patients is 40 months. BCL-2 status did not predict for either progression-free or overall survival. IPI score was predictive for progression-free survival but not overall survival. The addition of rituximab to CHOP chemotherapy negates the adverse prognostic influence of BCL-2 protein expression on progression free and overall survival in DLBCL.

Rapid infusion rituximab in combination with corticosteroid-containing chemotherapy or as maintenance therapy is well tolerated and can safely be delivered in the community setting.

The increasing usage of rituximab in the management of non-Hodgkin lymphoma (NHL) has created huge logistical challenges with respect to the delivery of this time- and labor-intensive drug. To address these challenges, we developed and tested the feasibility of a 90-minute infusion schedule for rituximab (20% of the dose administered in the first 30 minutes, remaining 80% administered over 60 minutes). A safety analysis performed in 150 patients receiving rituximab with corticosteroid-containing chemotherapy and 56 patients receiving rituximab as maintenance therapy demonstrated that this schedule was well tolerated, with no grade 3 or 4 infusion reactions observed. In addition, no increase in minor reactions was noted. More than 1200 patients have been treated with this rapid rituximab infusion schedule in the province of British Columbia (BC), demonstrating its safety in the community setting. The adoption of this 90-minute schedule as standard practice has had a positive impact on resource utilization.

The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity, with patients exhibiting a wide range of outcomes. The addition of rituximab to CHOP chemotherapy (R-CHOP)has led to a marked improvement in survival and has called into question the significance of previously recognized prognostic markers. Since randomized controlled trials of R-CHOP in DLBCL have included select subgroups of patients, the utility of the International Prognostic Index (IPI) has not been reassessed. We performed a retrospective analysis of patients with DLBCL treated with R-CHOP in the province of British Columbia to assess the value of the IPI in the era of immunochemotherapy. The IPI remains predictive, but it identifies only 2 risk groups. Redistribution of the IPI factors into a revised IPI (R-IPI) provides a more clinically useful prediction of outcome. The R-IPI identifies 3 distinct prognostic groups with a very good (4-year progression-free survival [PFS] 94%, overall survival [OS] 94%), good (4-year PFS 80%, OS 79%), and poor (4-year PFS 53%, OS 55%) outcome, respectively (P < .001). The IPI (or R-IPI) no longer identifies a risk group with less than a 50% chance of survival. In the era of R-CHOP treatment, the R-IPI is a clinically useful prognostic index that may help guide treatment planning and interpretation of clinical trials.

Introduction of combined CHOP plus rituximab therapy dramatically improved outcome of diffuse large B-cell lymphoma in British Columbia.

PURPOSE: For more than two decades, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) has been the standard therapy for diffuse large B-cell lymphoma (DLBCL). The addition of rituximab to CHOP has been shown to improve outcome in elderly patients with DLBCL. We conducted a population-based analysis to assess the impact of this combination therapy on adult patients with DLBCL in the province of British Columbia (BC). METHODS: We compared outcomes during a 3-year period; 18 months before (prerituximab) and 18 months after (postrituximab) institution of a policy recommending the combination of CHOP and rituximab for all patients with newly diagnosed advanced-stage (stage III or IV or stage I or II with "B" symptoms or bulky [> 10 cm] disease) DLBCL. RESULTS: A total of 292 patients were evaluated; 140 in the prerituximab group (median follow-up, 42 months) and 152 in the postrituximab group (median follow-up, 24 months). Both progression-free survival (risk ratio, 0.56; 95% CI, 0.39 to 0.81; P = .002) and overall survival (risk ratio, 0.40; 95% CI, 0.27 to 0.61, P < .0001) were significantly improved in the postrituximab group. After controlling for age and International Prognostic Index score, era of treatment remained a strong independent predictor of progression-free survival (risk ratio, 0.59; 95% CI, 0.41 to 0.85; P = .005) and overall survival (risk ratio, 0.43; 95% CI, 0.29 to 0.66; P < .001). The benefit of treatment in the postrituximab era was present regardless of age. CONCLUSION: The addition of rituximab to CHOP chemotherapy has resulted in a dramatic improvement in outcome for DLBCL patients of all ages in the province of BC.