To identify the origin and track the migratory pathway of specific subpopulations of GABAergic interneurons, we studied tangential migration in a recently developed GAD65-GFP transgenic mouse strain. First, we used immunohistochemical methods to characterize the expression of specific neurochemical markers in the GAD65-GFP neurons. Then, organotypic cultures were used in combination with birth-dating studies to determine the time of generation, place of origin and migratory route of these cells. From E14 to E15, the highest density of GAD65-GFP cells was seen in the lower intermediate zone; however, at later stages more GAD65-GFP cells were observed in the subventricular zone. Migratory GAD65-GFP cells express GAD65, but not calretinin or reelin. Surprisingly, only 4% were calbindin immunopositive. At P21, GAD65-GFP cells were found predominantly in layers II-III and expressed calretinin and neuropeptide Y. Remarkably, almost all cholecystokinin-positive but very few parvalbumin-positive neurons expressed GFP. In vitro studies demonstrated that the caudal ganglionic eminence gives rise to a large proportion of GAD65-GFP interneurons and in vivo birth-dating experiments showed that GAD65-GFP interneurons in supragranular layers are born at late embryonic development. Taken together these results support the idea that the destination layer of GABAergic interneurons is closely linked to their place of origin and time of generation.

Objective: The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum,” are commonly associated with immune‐mediated haemolytic anaemia (IMHA) in UK dogs. Methods: Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non‐anaemic dogs (n=113). DNA was extracted from 100 μl of blood and subjected to real‐time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co‐amplification of canine glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an endogenous internal control. Results: Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or “Candidatus M. haematoparvum” DNA. Clinical Significance: Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.

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Background: In the treatment of advanced Hodgkin lymphoma, it is increasingly common UK practice to modify escalated BEACOPP (eBPP) by removing oral procarbazine and replacing it with intravenous dacarbazine (250mg/m2 D2-3) to reduce haematopoietic stem cell and gonadal toxicity. However, published data of the "escalated BEACOPDac (eBPDac)" regimen are very limited. Methods: This is a retrospective study of 225 patients from 20 centres in the UK, Ireland and France who were treated with eBPDac first line for advanced stage Hodgkin Lymphoma. Toxicity outcomes were compared with 58 matched patients treated with eBPP at 4 UK centres and survival outcomes were compared with 2073 eBPP patients in the HD18 trial 1 and with 1088 patients aged 18-59y in the RATHL trial 2,3. Most eBPDac patients were treated as per HD18 protocol. The 34 patients treated in Paris followed the AHL2011 protocol with two courses of eBPDac given upfront and if iPET2 negative were deescalated to 4 cycles of ABVD. Toxicity outcomes: Toxicity was compared between eBPDac patients (n=225; median follow-up 22.1 months) and matched real-world UK eBPP patients (n=58; median follow-up 52.7 months) over the first 4 cycles. eBPP and eBPDac patients were well matched with no significant differences in age (median 23 y vs 26 y), sex, stage (stage 3/4 82% vs 83%) and international prognostic score (IPS 3+ 74% vs 65%). 55% of eBPDac patients received only 4 cycles (vs 12% of eBPP patients; p<0.001) reflecting publication of HD18 trial data. Mean day 8 (D8) ALT was similar between the two regimens. Mean D8 neutrophil count tended to be lower in eBPDac than eBPP patients (2.04 vs 2.45; p=0.072; G-CSF given D9), however it increased to 6.48 in eBPDac patients given GCSF from D4. There were fewer non-elective days of inpatient care for eBPDac compared with eBPP (mean 3.35 vs 5.84; p=0.022), and eBPDac patients received fewer red cell transfusions compared with eBPP patients (mean 1.79 units vs 4.16 units; p<0.001). Women aged <35, who completed ≥4 cycles of eBPDac/eBPP had a similar rate of return of menstrual cycles (eBPP 22/25; eBPDac 41/41), although eBPDac patients appeared to restart menstruation earlier post chemotherapy (mean 4.64 months vs 9.12 months, p=0.0026). However, this could also reflect the higher mean chemotherapy cycle number completed by the eBPP women (5.86 vs 4.60; p<0.001). The use of Goserelin to suppress ovulation varied between centres. Disease outcomes: The eBPDac patients (n=225) were younger than the HD18 patients (median age 26 y vs 35 y, p<0.001) and the RATHL patients (median age 26 y vs 31 y). However, they had higher risk disease than HD18 (IPS 4+ 36% vs 16%, p<0.001) and RATHL patients (IPS 3+ 65% vs 33%, p<0.001) and more stage 4 disease than HD18 (66% vs 36%, p<0.001) and RATHL (66% vs 28%, p<0.001). Of the 225 patients who started eBPDac, 77% achieved iPET2 Deauville score (DS) ≤3, similar to RATHL (DS ≤3: 83.7%) and HD18 (DS ≤3: 76%). Of the eBPDac patients, one patient had primary refractory disease, and ten have relapsed at 6 to 36 months. One 56-year-old eBPDac patient with high IPS died with bowel perforation during cycle 1 and one 34-year-old with alcoholic liver disease died 8 months after treatment while still in remission. There have been no lymphoma-related deaths to date. Figure 1 shows Kaplan-Meier plots for progression-free survival (PFS) and overall survival (OS). The PFS at 22 months (median follow-up) of eBPDac patients was 94.9% (91.7-98.3%) which is similar to HD18 3 year PFS of 92.3% (91.1-93.5%) and appears superior to RATHL 5 year PFS 81.4% (78.9-83.7%). The difference in PFS between eBPDac and RATHL is most marked in IPS3+ patients. The OS rates with all 3 regimens are excellent, with 22 month eBPDac OS estimate of 98.9% (97.4-100%). Summary/Conclusion: Accepting the limitations of a retrospective study, we suggest that substituting dacarbazine for procarbazine is unlikely to compromise the efficacy of eBPP and may have some toxicity benefits. Despite the clear preference of clinicians to offer this regimen to high-risk advanced stage patients, with nearly 2 years median follow-up we have observed similar PFS and OS compared to HD18 but superior survival estimates compared with 18-59y RATHL patients, suggesting that eBPDac is highly efficacious for the treatment of Hodgkin lymphoma. References: 1Borchmann P et al. Lancet 2017; 390:2790-2802 2Johnson P et al. NEJM. 2016; 374:2419-2429 3Russell J et al. Ann Hematol 2021; 100:1049-1058 Figure 1 Figure 1. Disclosures Brice: MSD: Research Funding; Amgen: Other: Travel/accommodations/expenses; Roche: Other: Travel/accommodations/expenses; Takeda: Research Funding. Menne: Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Astra Zeneca: Research Funding; Jazz: Other: Travel grants; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Bayer: Other: Travel grants; Kyowa Kirin: Other: Travel grants; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Atara: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Honoraria for Lectures; Roche: Other: Honoraria for Lectures. Osborne: Roche: Other; Takeda: Other; Pfizer: Other; Servier: Other; Gilead: Other; Novartis: Other; MSD: Other. Ardeshna: Gilead, Beigene, Celegene, Novartis and Roche: Membership on an entity's Board of Directors or advisory committees; Gilead, Beigene, Celegene, Novartis and Roche: Honoraria; Norvartis, BMS, Autolus, ADCT, Pharmocyclics and Jansen: Research Funding. Collins: Pfizer: Honoraria; Amgen: Research Funding; AstraZeneca: Honoraria, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Speakers Bureau; Celgene: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celleron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau. Cwynarski: Adienne, Takeda, Roche, Autolus, KITE, Gilead, Celgene, Atara, Janssenen: Other. Davies: Janssen: Honoraria, Research Funding; Karyopharm Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma/AstraZeneca: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; BioInvent: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel to scientific conferences, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel to scientific conferences, Research Funding. Furtado: Abbvie: Other: Conference support. Gallop-Evans: Takeda: Membership on an entity's Board of Directors or advisory committees; Kyowa-Kirin: Membership on an entity's Board of Directors or advisory committees. Iyengar: Gilead: Membership on an entity's Board of Directors or advisory committees, Other: conference support, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: conference support, Speakers Bureau; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: conference support; Janssen: Other: conference support, Speakers Bureau. Linton: Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Aptitude Health: Honoraria; Hartley Taylor: Honoraria; University of Manchester: Current Employment; Celgene: Research Funding; BeiGene: Research Funding; Genmab: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Calle: Abbvie: Other. McKay: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Other: Travel Support; KITE: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Janssen: Honoraria, Other: Travel Support; Beigene: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Nagumantry: Takeda, Alexion, Abbvie: Other. Shah: Abbvie, Janssen and Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Uttenthal: Jazz: Other; Takeda: Other; Roche: Other. McMillan: Pfizer: Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Abvie: Membership on an entity's Board of Directors or advisory committees. Follows: Janssen, Abvie, Roche, AZ: Other.

The study of haematopoiesis has been revolutionized in recent years by the application of single cell RNA sequencing technologies. The technique coupled with rapidly developing bioinformatic analysis has provided great insight into the cell type compositions of many populations previously defined by their cell surface phenotype. Moreover, transcriptomic information enables the identification of individual molecules and pathways which define novel cell populations and their transitions including cell lineage decisions. Combining single cell transcriptional profiling with molecular perturbations allows functional analysis of individual factors in gene regulatory networks and better understanding of the earliest stages of malignant transformation. In this chapter we describe a comprehensive protocol for scRNA-Seq analysis of the mouse bone marrow, using both plate-based (low throughput) and droplet-based (high throughput) methods. The protocol includes instructions for sample preparation, an antibody panel for flow cytometric purification of haematopoietic progenitors with index sorting for plate-based analysis or in bulk for droplet-based methods. The plate-based protocol described in this chapter is a combination of the Smart-Seq2 and mcSCRB-Seq protocols, optimized in our laboratory. It utilizes off-the-shelf reagents for cDNA preparation, is amenable to automation using a liquid handler, and takes 4 days from preparation of the cells for sorting to producing a sequencing-ready library. The droplet-based method (using for instance the 10x Genomics platform) relies on the manufacturer's user guide and commercial reagents, and takes 3 days from isolation of the cells to the production of a library ready for sequencing.

Introduction Since interim results from the EuroNet-PHL-C1 study1were published in 2013 showing that the gonadotoxic drug procarbazine in COPP can be safely replaced with dacarbazine (COPDac) it is increasingly common practice to modify escalated BEACOPP (eBPP) by removing oral procarbazine and replacing it with intravenous dacarbazine (250mg/m2 D2-3), hoping to reduce haematopoietic stem cell and gonadal toxicity. However, published data of 'escalated BEACOPDac (eBPDac)' regimen are very limited. Methods We collected retrospective data from 15 centres in the UK, Ireland and France, that offer eBPDac therapy for first line advanced stage Hodgkin Lymphoma, and compared outcomes with matched patients treated with eBPP at 4 UK centres. Most patients were treated as per HD15 or HD18 protocol. The 24 patients treated in Paris followed the AHL2011 protocol with two courses of eBPDac given upfront and if iPET2 negative were deescalated to 4 cycles of ABVD. Results From 2009, 141 patients were managed first line with either eBPP (n=52) or eBPDac (n=89) with median follow-up 40 months for eBPP and 12.7 months for eBPDac patients. Patients were well matched with no significant differences in age (median: 28), sex, stage (stage 3/4: 82%) and international prognostic score (IPS3+:65%). More patients treated with eBPDac received only 4 cycles of treatment (43% vs 12%; p<0.001) reflecting recent publication of HD18 trial data2. In total, 74% patients achieved iPET2 Deauville score 2 or 3 and 96% patients achieved PET negative remission by end of treatment. Of eBPDac patients, 77% achieved iPET Deauville 2 or 3 which was statistically similar to the eBPP cohort (69%; p=0.391) and matched the 76% iPET D2/3 reported in HD181. Of 141 patients, 139 are alive and 136 continue in first remission. Two eBPP patients have relapsed at 13 and 41 months and the latter died of refractory disease. One eBPDac patient had primary refractory disease, another relapsed seven months after treatment, and one 56-year-old eBPDac patient with high IPS died with bowel perforation during cycle 1. Toxicity was compared over the first 4 cycles. There was no difference in day 8 ALT between the two regimens although the mean day 8 neutrophil count was lower in eBPDac than eBPP patients (1.84 vs 2.35; p=0.043; G-CSF given day 9). There was a trend to fewer non-elective days of in-patient care for eBPDac compared with eBPP (mean: 2.78 vs 6.00; p=0.0846), and eBPDac patients received fewer red cell transfusions during cycles 1 to 4 compared with eBPP patients (Mean 1.87 units vs 4.33 units; p<0.001). Women aged < 35, who completed ≥4 cycles of eBPDac/eBPP with > 6 months post chemotherapy follow-up had a similar rate of return of menstrual cycles (eBPP: 20/21; eBPDac: 13/16), although eBPDac patients appeared to restart menstruation earlier post chemotherapy completion (mean: 3.85 months vs 8.65 months, p=0.0018). However, this could also reflect the higher mean chemotherapy cycle number completed by the eBPP women (5.86 vs 4.67; p<0.001). The use of monthly Goserelin to suppress ovulation varied between centres. Conclusions Accepting the limitations of a retrospective study, we suggest that substituting dacarbazine for procarbazine is unlikely to compromise the efficacy of eBPP in frontline therapy of patients with advanced Hodgkin Lymphoma and may have some toxicity benefits. As it is highly unlikely that this single drug substitution will ever be tested in a prospective trial, publishing real-world data from eBPDac patients is important. References 1. EuroNet-PHL-C1 study interim report published 14/08/2013 http://www.anzchog.org/docs/public-resources/euronet-recommendation.pdf?sfvrsn=0 2. Borchmann P et al. 2018;390:2790-2802 Disclosures Brice: BMS: Honoraria; Takeda France: Consultancy, Honoraria; Millennium Takeda: Research Funding. Menne:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Kyowa Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Osborne:Novartis: Other: Travel; Pfizer: Honoraria, Speakers Bureau; Servier: Consultancy; MSD: Consultancy; Takeda: Consultancy, Honoraria, Other: Travel, Speakers Bureau; Gilead: Consultancy; Roche: Consultancy, Honoraria, Other: Travel, Speakers Bureau. Ardeshna:Gilead: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Collins:Gilead: Consultancy, Honoraria. Cwynarski:Adienne: Consultancy; Takeda: Consultancy, Other: conference and travel support , Speakers Bureau; Gilead: Consultancy, Other: conference and travel support, Speakers Bureau; Celgene: Consultancy; Roche,: Consultancy, Other: conference and travel support, Speakers Bureau; Autolus: Consultancy; KITE: Consultancy; Atara: Consultancy; Janssen: Other: conference and travel support, Speakers Bureau. Iyengar:Takeda: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Abbvie: Honoraria; Roche: Honoraria. Martinez-Calle:ABBVIE: Other: Travel support. McKay:Epizyme: Consultancy, Honoraria. Nagumantry:Takeda: Honoraria, Speakers Bureau; Alexion: Speakers Bureau; Abbvie: Honoraria. Shah:Abbvie: Consultancy. Uttenthal:Roche: Honoraria; Takeda: Honoraria; Jazz: Honoraria. McMillan:BMS: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Sandoz: Honoraria; Celgene: Honoraria, Speakers Bureau; Novartis: Honoraria; Gilead: Honoraria; MSD: Honoraria; Pfizer: Honoraria, Research Funding. Follows:Roche: Consultancy, Honoraria, Speakers Bureau; AZ: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Speakers Bureau.