Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): a single-blind, randomised, controlled, phase 2/3 trial.

BACKGROUND: Older adults (aged ≥70 years) are at increased risk of severe disease and death if they develop COVID-19 and are therefore a priority for immunisation should an efficacious vaccine be developed. Immunogenicity of vaccines is often worse in older adults as a result of immunosenescence. We have reported the immunogenicity of a novel chimpanzee adenovirus-vectored vaccine, ChAdOx1 nCoV-19 (AZD1222), in young adults, and now describe the safety and immunogenicity of this vaccine in a wider range of participants, including adults aged 70 years and older. METHODS: In this report of the phase 2 component of a single-blind, randomised, controlled, phase 2/3 trial (COV002), healthy adults aged 18 years and older were enrolled at two UK clinical research facilities, in an age-escalation manner, into 18-55 years, 56-69 years, and 70 years and older immunogenicity subgroups. Participants were eligible if they did not have severe or uncontrolled medical comorbidities or a high frailty score (if aged ≥65 years). First, participants were recruited to a low-dose cohort, and within each age group, participants were randomly assigned to receive either intramuscular ChAdOx1 nCoV-19 (2·2 × 1010 virus particles) or a control vaccine, MenACWY, using block randomisation and stratified by age and dose group and study site, using the following ratios: in the 18-55 years group, 1:1 to either two doses of ChAdOx1 nCoV-19 or two doses of MenACWY; in the 56-69 years group, 3:1:3:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY; and in the 70 years and older, 5:1:5:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY. Prime-booster regimens were given 28 days apart. Participants were then recruited to the standard-dose cohort (3·5-6·5 × 1010 virus particles of ChAdOx1 nCoV-19) and the same randomisation procedures were followed, except the 18-55 years group was assigned in a 5:1 ratio to two doses of ChAdOx1 nCoV-19 or two doses of MenACWY. Participants and investigators, but not staff administering the vaccine, were masked to vaccine allocation. The specific objectives of this report were to assess the safety and humoral and cellular immunogenicity of a single-dose and two-dose schedule in adults older than 55 years. Humoral responses at baseline and after each vaccination until 1 year after the booster were assessed using an in-house standardised ELISA, a multiplex immunoassay, and a live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) microneutralisation assay (MNA80). Cellular responses were assessed using an ex-vivo IFN-γ enzyme-linked immunospot assay. The coprimary outcomes of the trial were efficacy, as measured by the number of cases of symptomatic, virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were by group allocation in participants who received the vaccine. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. This study is ongoing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Between May 30 and Aug 8, 2020, 560 participants were enrolled: 160 aged 18-55 years (100 assigned to ChAdOx1 nCoV-19, 60 assigned to MenACWY), 160 aged 56-69 years (120 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY), and 240 aged 70 years and older (200 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY). Seven participants did not receive the boost dose of their assigned two-dose regimen, one participant received the incorrect vaccine, and three were excluded from immunogenicity analyses due to incorrectly labelled samples. 280 (50%) of 552 analysable participants were female. Local and systemic reactions were more common in participants given ChAdOx1 nCoV-19 than in those given the control vaccine, and similar in nature to those previously reported (injection-site pain, feeling feverish, muscle ache, headache), but were less common in older adults (aged ≥56 years) than younger adults. In those receiving two standard doses of ChAdOx1 nCoV-19, after the prime vaccination local reactions were reported in 43 (88%) of 49 participants in the 18-55 years group, 22 (73%) of 30 in the 56-69 years group, and 30 (61%) of 49 in the 70 years and older group, and systemic reactions in 42 (86%) participants in the 18-55 years group, 23 (77%) in the 56-69 years group, and 32 (65%) in the 70 years and older group. As of Oct 26, 2020, 13 serious adverse events occurred during the study period, none of which were considered to be related to either study vaccine. In participants who received two doses of vaccine, median anti-spike SARS-CoV-2 IgG responses 28 days after the boost dose were similar across the three age cohorts (standard-dose groups: 18-55 years, 20 713 arbitrary units [AU]/mL [IQR 13 898-33 550], n=39; 56-69 years, 16 170 AU/mL [10 233-40 353], n=26; and ≥70 years 17 561 AU/mL [9705-37 796], n=47; p=0·68). Neutralising antibody titres after a boost dose were similar across all age groups (median MNA80 at day 42 in the standard-dose groups: 18-55 years, 193 [IQR 113-238], n=39; 56-69 years, 144 [119-347], n=20; and ≥70 years, 161 [73-323], n=47; p=0·40). By 14 days after the boost dose, 208 (>99%) of 209 boosted participants had neutralising antibody responses. T-cell responses peaked at day 14 after a single standard dose of ChAdOx1 nCoV-19 (18-55 years: median 1187 spot-forming cells [SFCs] per million peripheral blood mononuclear cells [IQR 841-2428], n=24; 56-69 years: 797 SFCs [383-1817], n=29; and ≥70 years: 977 SFCs [458-1914], n=48). INTERPRETATION: ChAdOx1 nCoV-19 appears to be better tolerated in older adults than in younger adults and has similar immunogenicity across all age groups after a boost dose. Further assessment of the efficacy of this vaccine is warranted in all age groups and individuals with comorbidities. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.

Safety and immunogenicity of seven COVID-19 vaccines as a third dose (booster) following two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK (COV-BOOST): a blinded, multicentre, randomised, controlled, phase 2 trial.

BACKGROUND: Few data exist on the comparative safety and immunogenicity of different COVID-19 vaccines given as a third (booster) dose. To generate data to optimise selection of booster vaccines, we investigated the reactogenicity and immunogenicity of seven different COVID-19 vaccines as a third dose after two doses of ChAdOx1 nCov-19 (Oxford-AstraZeneca; hereafter referred to as ChAd) or BNT162b2 (Pfizer-BioNtech, hearafter referred to as BNT). METHODS: COV-BOOST is a multicentre, randomised, controlled, phase 2 trial of third dose booster vaccination against COVID-19. Participants were aged older than 30 years, and were at least 70 days post two doses of ChAd or at least 84 days post two doses of BNT primary COVID-19 immunisation course, with no history of laboratory-confirmed SARS-CoV-2 infection. 18 sites were split into three groups (A, B, and C). Within each site group (A, B, or C), participants were randomly assigned to an experimental vaccine or control. Group A received NVX-CoV2373 (Novavax; hereafter referred to as NVX), a half dose of NVX, ChAd, or quadrivalent meningococcal conjugate vaccine (MenACWY)control (1:1:1:1). Group B received BNT, VLA2001 (Valneva; hereafter referred to as VLA), a half dose of VLA, Ad26.COV2.S (Janssen; hereafter referred to as Ad26) or MenACWY (1:1:1:1:1). Group C received mRNA1273 (Moderna; hereafter referred to as m1273), CVnCov (CureVac; hereafter referred to as CVn), a half dose of BNT, or MenACWY (1:1:1:1). Participants and all investigatory staff were blinded to treatment allocation. Coprimary outcomes were safety and reactogenicity and immunogenicity of anti-spike IgG measured by ELISA. The primary analysis for immunogenicity was on a modified intention-to-treat basis; safety and reactogenicity were assessed in the intention-to-treat population. Secondary outcomes included assessment of viral neutralisation and cellular responses. This trial is registered with ISRCTN, number 73765130. FINDINGS: Between June 1 and June 30, 2021, 3498 people were screened. 2878 participants met eligibility criteria and received COVID-19 vaccine or control. The median ages of ChAd/ChAd-primed participants were 53 years (IQR 44-61) in the younger age group and 76 years (73-78) in the older age group. In the BNT/BNT-primed participants, the median ages were 51 years (41-59) in the younger age group and 78 years (75-82) in the older age group. In the ChAd/ChAD-primed group, 676 (46·7%) participants were female and 1380 (95·4%) were White, and in the BNT/BNT-primed group 770 (53·6%) participants were female and 1321 (91·9%) were White. Three vaccines showed overall increased reactogenicity: m1273 after ChAd/ChAd or BNT/BNT; and ChAd and Ad26 after BNT/BNT. For ChAd/ChAd-primed individuals, spike IgG geometric mean ratios (GMRs) between study vaccines and controls ranged from 1·8 (99% CI 1·5-2·3) in the half VLA group to 32·3 (24·8-42·0) in the m1273 group. GMRs for wild-type cellular responses compared with controls ranged from 1·1 (95% CI 0·7-1·6) for ChAd to 3·6 (2·4-5·5) for m1273. For BNT/BNT-primed individuals, spike IgG GMRs ranged from 1·3 (99% CI 1·0-1·5) in the half VLA group to 11·5 (9·4-14·1) in the m1273 group. GMRs for wild-type cellular responses compared with controls ranged from 1·0 (95% CI 0·7-1·6) for half VLA to 4·7 (3·1-7·1) for m1273. The results were similar between those aged 30-69 years and those aged 70 years and older. Fatigue and pain were the most common solicited local and systemic adverse events, experienced more in people aged 30-69 years than those aged 70 years or older. Serious adverse events were uncommon, similar in active vaccine and control groups. In total, there were 24 serious adverse events: five in the control group (two in control group A, three in control group B, and zero in control group C), two in Ad26, five in VLA, one in VLA-half, one in BNT, two in BNT-half, two in ChAd, one in CVn, two in NVX, two in NVX-half, and one in m1273. INTERPRETATION: All study vaccines boosted antibody and neutralising responses after ChAd/ChAd initial course and all except one after BNT/BNT, with no safety concerns. Substantial differences in humoral and cellular responses, and vaccine availability will influence policy choices for booster vaccination. FUNDING: UK Vaccine Taskforce and National Institute for Health Research.

High-affinity memory B cells induced by conjugate vaccines against weak tumor antigens are vulnerable to nonconjugated antigen.

Induction of antibody-mediated immunity against hematologic malignancies requires CD4(+) T-cell help, but weak tumor antigens generally fail to induce adequate T-cell responses, or to overcome tolerance. Conjugate vaccines can harness alternative help to activate responses, but memory B cells may then be exposed to leaking tumor-derived antigen without CD4(+) T-cell support. We showed previously using lymphoma-derived idiotypic antigen that exposure to "helpless" antigen silences the majority of memory IgG(+) B cells. Transfer experiments now indicate that silencing is permanent. In marked contrast to IgG, most coexisting IgM(+) memory B cells exposed to "helpless" antigen survive. Confirmation in a hapten (NP) model allowed measurement of affinity, revealing this, rather than isotype, as the determinant of survival. IgM(+) B cells had Ig variable region gene usage similar to IgG but with fewer somatic mutations. Survival of memory B cells appears variably controlled by affinity for antigen, allowing a minority of low affinity IgG(+), but most IgM(+), memory B cells to escape deletion in the absence of T-cell help. The latter remain, but the majority fail to undergo isotype switch. These findings could apply to other tumor antigens and are relevant for vaccination strategies aimed to induce long-term antibody.

Persistence of immune responses after heterologous and homologous third COVID-19 vaccine dose schedules in the UK: eight-month analyses of the COV-BOOST trial.

BACKGROUND: COV-BOOST is a multicentre, randomised, controlled, phase 2 trial of seven COVID-19 vaccines used as a third booster dose in June 2021. Monovalent messenger RNA (mRNA) COVID-19 vaccines were subsequently widely used for the third and fourth-dose vaccination campaigns in high-income countries. Real-world vaccine effectiveness against symptomatic infections following third doses declined during the Omicron wave. This report compares the immunogenicity and kinetics of responses to third doses of vaccines from day (D) 28 to D242 following third doses in seven study arms. METHODS: The trial initially included ten experimental vaccine arms (seven full-dose, three half-dose) delivered at three groups of six sites. Participants in each site group were randomised to three or four experimental vaccines, or MenACWY control. The trial was stratified such that half of participants had previously received two primary doses of ChAdOx1 nCov-19 (Oxford-AstraZeneca; hereafter referred to as ChAd) and half had received two doses of BNT162b2 (Pfizer-BioNtech, hereafter referred to as BNT). The D242 follow-up was done in seven arms (five full-dose, two half-dose). The BNT vaccine was used as the reference as it was the most commonly deployed third-dose vaccine in clinical practice in high-income countries. The primary analysis was conducted using all randomised and baseline seronegative participants who were SARS-CoV-2 naïve during the study and who had not received a further COVID-19 vaccine for any reason since third dose randomisation. RESULTS: Among the 817 participants included in this report, the median age was 72 years (IQR: 55-78) with 50.7% being female. The decay rates of anti-spike IgG between vaccines are different among both populations who received initial doses of ChAd/ChAd and BNT/BNT. In the population that previously received ChAd/ChAd, mRNA vaccines had the highest titre at D242 following their vaccine dose although Ad26. COV2. S (Janssen; hereafter referred to as Ad26) showed slower decay. For people who received BNT/BNT as their initial doses, a slower decay was also seen in the Ad26 and ChAd arms. The anti-spike IgG became significantly higher in the Ad26 arm compared to the BNT arm as early as 3 months following vaccination. Similar decay rates were seen between BNT and half-BNT; the geometric mean ratios ranged from 0.76 to 0.94 at different time points. The difference in decay rates between vaccines was similar for wild-type live virus-neutralising antibodies and that seen for anti-spike IgG. For cellular responses, the persistence was similar between study arms. CONCLUSIONS: Heterologous third doses with viral vector vaccines following two doses of mRNA achieve more durable humoral responses compared with three doses of mRNA vaccines. Lower doses of mRNA vaccines could be considered for future booster campaigns.

Safety, immunogenicity, and reactogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines given as fourth-dose boosters following two doses of ChAdOx1 nCoV-19 or BNT162b2 and a third dose of BNT162b2 (COV-BOOST): a multicentre, blinded, phase 2, randomised trial.

BACKGROUND: Some high-income countries have deployed fourth doses of COVID-19 vaccines, but the clinical need, effectiveness, timing, and dose of a fourth dose remain uncertain. We aimed to investigate the safety, reactogenicity, and immunogenicity of fourth-dose boosters against COVID-19. METHODS: The COV-BOOST trial is a multicentre, blinded, phase 2, randomised controlled trial of seven COVID-19 vaccines given as third-dose boosters at 18 sites in the UK. This sub-study enrolled participants who had received BNT162b2 (Pfizer-BioNTech) as their third dose in COV-BOOST and randomly assigned them (1:1) to receive a fourth dose of either BNT162b2 (30 µg in 0·30 mL; full dose) or mRNA-1273 (Moderna; 50 µg in 0·25 mL; half dose) via intramuscular injection into the upper arm. The computer-generated randomisation list was created by the study statisticians with random block sizes of two or four. Participants and all study staff not delivering the vaccines were masked to treatment allocation. The coprimary outcomes were safety and reactogenicity, and immunogenicity (anti-spike protein IgG titres by ELISA and cellular immune response by ELISpot). We compared immunogenicity at 28 days after the third dose versus 14 days after the fourth dose and at day 0 versus day 14 relative to the fourth dose. Safety and reactogenicity were assessed in the per-protocol population, which comprised all participants who received a fourth-dose booster regardless of their SARS-CoV-2 serostatus. Immunogenicity was primarily analysed in a modified intention-to-treat population comprising seronegative participants who had received a fourth-dose booster and had available endpoint data. This trial is registered with ISRCTN, 73765130, and is ongoing. FINDINGS: Between Jan 11 and Jan 25, 2022, 166 participants were screened, randomly assigned, and received either full-dose BNT162b2 (n=83) or half-dose mRNA-1273 (n=83) as a fourth dose. The median age of these participants was 70·1 years (IQR 51·6-77·5) and 86 (52%) of 166 participants were female and 80 (48%) were male. The median interval between the third and fourth doses was 208·5 days (IQR 203·3-214·8). Pain was the most common local solicited adverse event and fatigue was the most common systemic solicited adverse event after BNT162b2 or mRNA-1273 booster doses. None of three serious adverse events reported after a fourth dose with BNT162b2 were related to the study vaccine. In the BNT162b2 group, geometric mean anti-spike protein IgG concentration at day 28 after the third dose was 23 325 ELISA laboratory units (ELU)/mL (95% CI 20 030-27 162), which increased to 37 460 ELU/mL (31 996-43 857) at day 14 after the fourth dose, representing a significant fold change (geometric mean 1·59, 95% CI 1·41-1·78). There was a significant increase in geometric mean anti-spike protein IgG concentration from 28 days after the third dose (25 317 ELU/mL, 95% CI 20 996-30 528) to 14 days after a fourth dose of mRNA-1273 (54 936 ELU/mL, 46 826-64 452), with a geometric mean fold change of 2·19 (1·90-2·52). The fold changes in anti-spike protein IgG titres from before (day 0) to after (day 14) the fourth dose were 12·19 (95% CI 10·37-14·32) and 15·90 (12·92-19·58) in the BNT162b2 and mRNA-1273 groups, respectively. T-cell responses were also boosted after the fourth dose (eg, the fold changes for the wild-type variant from before to after the fourth dose were 7·32 [95% CI 3·24-16·54] in the BNT162b2 group and 6·22 [3·90-9·92] in the mRNA-1273 group). INTERPRETATION: Fourth-dose COVID-19 mRNA booster vaccines are well tolerated and boost cellular and humoral immunity. Peak responses after the fourth dose were similar to, and possibly better than, peak responses after the third dose. FUNDING: UK Vaccine Task Force and National Institute for Health Research.

Persistence of immunogenicity after seven COVID-19 vaccines given as third dose boosters following two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK: Three month analyses of the COV-BOOST trial.

OBJECTIVES: To evaluate the persistence of immunogenicity three months after third dose boosters. METHODS: COV-BOOST is a multicentre, randomised, controlled, phase 2 trial of seven COVID-19 vaccines used as a third booster dose. The analysis was conducted using all randomised participants who were SARS-CoV-2 naïve during the study. RESULTS: Amongst the 2883 participants randomised, there were 2422 SARS-CoV-2 naïve participants until D84 visit included in the analysis with median age of 70 (IQR: 30-94) years. In the participants who had two initial doses of ChAdOx1 nCov-19 (Oxford-AstraZeneca; hereafter referred to as ChAd), schedules using mRNA vaccines as third dose have the highest anti-spike IgG at D84 (e.g. geometric mean concentration of 8674 ELU/ml (95% CI: 7461-10,085) following ChAd/ChAd/BNT162b2 (Pfizer-BioNtech, hearafter referred to as BNT)). However, in people who had two initial doses of BNT there was no significant difference at D84 in people given ChAd versus BNT (geometric mean ratio (GMR) of 0.95 (95%CI: 0.78, 1.15). Also, people given Ad26.COV2.S (Janssen; hereafter referred to as Ad26) as a third dose had significantly higher anti-spike IgG at D84 than BNT (GMR of 1.20, 95%CI: 1.01,1.43). Responses at D84 between people who received BNT (15 µg) or BNT (30 µg) after ChAd/ChAd or BNT/BNT were similar, with anti-spike IgG GMRs of half-BNT (15 µg) versus BNT (30 µg) ranging between 0.74-0.86. The decay rate of cellular responses were similar between all the vaccine schedules and doses. CONCLUSIONS: 84 days after a third dose of COVID-19 vaccine the decay rates of humoral response were different between vaccines. Adenoviral vector vaccine anti-spike IgG concentrations at D84 following BNT/BNT initial doses were similar to or even higher than for a three dose (BNT/BNT/BNT) schedule. Half dose BNT immune responses were similar to full dose responses. While high antibody tires are desirable in situations of high transmission of new variants of concern, the maintenance of immune responses that confer long-lasting protection against severe disease or death is also of critical importance. Policymakers may also consider adenoviral vector, fractional dose of mRNA, or other non-mRNA vaccines as third doses.

Clinical impact of molecular point-of-care testing for suspected COVID-19 in hospital (COV-19POC): a prospective, interventional, non-randomised, controlled study.

BACKGROUND: The management of the COVID-19 pandemic is hampered by long delays associated with centralised laboratory PCR testing. In hospitals, these delays lead to poor patient flow and nosocomial transmission. Rapid, accurate tests are therefore urgently needed in preparation for the next wave of the pandemic. METHODS: We did a prospective, interventional, non-randomised, controlled study of molecular point-of-care testing in patients aged 18 years or older presenting with suspected COVID-19 to the emergency department or other acute areas of Southampton General Hospital during the first wave of the pandemic in the UK. Nose and throat swab samples taken at admission from patients in the point-of-care testing group were tested with the QIAstat-Dx Respiratory SARS-CoV-2 Panel. Samples taken from patients in a contemporaneous control group were tested by laboratory PCR. The primary outcome was time to results in the full cohort. This study is registered with ISRCTN (ISRCTN14966673) and is completed. FINDINGS: Between March 20 and April 29, 2020, 517 patients were assessed for eligibility, of whom 499 were recruited to the point-of-care testing group and tested by the QIAstat-Dx Respiratory SARS-CoV-2 Panel. 555 contemporaneously identified patients were included in the control group and tested by laboratory PCR. The two groups were similar with regard to the distribution of sex, age, and ethnicity. 197 (39%) patients in the point-of-care testing group and 155 (28%) in the control group tested positive for COVID-19 (difference 11·5% [95% CI 5·8-17·2], p=0·0001). Median time to results was 1·7 h (IQR 1·6-1·9) in the point-of-care testing group and 21·3 h (16·0-27·9) in the control group (difference 19·6 h [19·0-20·3], p<0·0001). A Cox proportional hazards regression model controlling for age, sex, time of presentation, and severity of illness also showed that time to results was significantly shorter in the point-of-care testing group than in the control group (hazard ratio 4023 [95% CI 545-29 696], p<0·0001). INTERPRETATION: Point-of-care testing is associated with large reductions in time to results and could lead to improvements in infection control measures and patient flow compared with centralised laboratory PCR testing. FUNDING: University Hospitals Southampton NHS Foundation Trust.

CD27 in defining memory B-cell origins in Waldenström's macroglobulinemia.

CD27 is a tumor necrosis factor receptor family glycoprotein, identified in seminal studies as an apparently robust marker for normal memory B cells. Somatic hypermutation (SHM) in immunoglobulin variable (V) region genes, however, remains the definitive memory imprint. In Waldenström's macroglobulinemia (WM), SHM defines a predominant mutated (MUT) subset and a minor unmutated subset indicative of naive B-cell origin. In MUT-WM, tumor cells can lack CD27 expression, raising suggestions of unusual memory B-cell origins. We recently identified such normal IgM+D+CD27-ve memory B-cells, with low levels of SHM in VH genes. While these could seed WM, the possibility remains that WM could derive from classical memory B cells that shed CD27. The utility of CD27 expression in defining memory in MUT-WM origins, then, is uncertain, but SHM unequivocally defines memory B-cell derivation in most WM. Patterns of SHM and additional IgH locus events furthermore reveal ongoing intra-tumoral diversification in WM.

Immunoglobulin heavy chain locus events and expression of activation-induced cytidine deaminase in epithelial breast cancer cell lines.

When cells transform, phenotypic and genetic profiles can be dramatically altered. Nevertheless, a recent report identifying IgG in breast cancer cells was unexpected, revealing differentiation features normally associated with B lymphocytes. To extend these findings, we focused on immunoglobulin variable (V) region gene analysis using well-defined breast cancer cell lines expressing the epithelial marker, epithelial cell adhesion molecule (EpCAM). V(H) gene transcripts were identifiable by nested reverse transcription-PCR either as single or dual V, diversity (D), and joining (J) rearrangements in four of six lines, most being potentially functional. V(D)J transcripts were observed in sequential cultures, indicating stable expression. To exclude coexisting lymphocytes, each cell line was shown to be EBV negative, with CD19/CD20 and cytoplasmic/surface immunoglobulin also absent by flow cytometry. Identified V(H) transcripts were then sought in individual tumor cells, isolated as EpCAM+ single cells by flow cytometry. Importantly, in three of three selected cell lines, V(H) genes were identifiable in a significant fraction (approximately 32%) of single cells. In five of six identified V(H) genes, somatic mutations were apparent with no intraclonal variation, indicating cessation of mutational activity. V(H) transcripts were pre- and post-isotype switch, with activation of switch events evident from expressed germ-line switch transcripts in two of six lines. Strikingly, six of six cell lines expressed activation-induced cytidine deaminase (AID) essential for mutational and switch activity. These data suggest either a de novo rearrangement and modification of V(H) genes in epithelial tumor cells or assimilation of lymphocyte-derived chromatin. Constitutive AID activation in malignant epithelial cells further raises a potential for inducing aberrant mutational activity.

Immunoglobulin M myeloma: evaluation of molecular features and cytokine expression.

Immunoglobulin (Ig) M myeloma is a distinct entity with features of multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM). The malignant cells in IgM myeloma have a distinctive chromosomal translocation that differentiates them from WM. These cells are postgerminal-center in origin with isotype-switch transcripts. They appear to be arrested at a point of maturation between that of WM and MM. Preliminary data indicate that a pattern of osteoclast-activating factor and osteoprotegerin expression similar to that observed in classic MM is present in IgM myeloma. Additional studies on patients with this rare tumor may provide further insight into the pathogenesis of bone disease in plasma cell dyscrasias.

Determining mutational status of immunoglobulin v genes in chronic lymphocytic leukemia: a useful prognostic indicator.

Determining the clonal origins of malignant B-cells will have an impact on disease understanding and management. In this regard, immunoglobulin variable (V) region gene analysis already is having a significant impact in delineating the tumor cell of origin. It can identify, among other features whether such a cell has undergone somatic mutation, which usually occurs within germinal centres. Remarkably, in chronic lymphocytic leukemia (CLL), the mutational status of V genes has allowed researchers to identify two subsets of disease, one originating from an unmutated B-cell with a markedly poorer disease outcome and the other from a mutated B-cell, which associates with long-term survival. The V gene status in CLL thus provides a robust indicator of disease outcome, which is beginning to shape clinical treatment. This chapter describes in detail the methodology for determining V gene usage in CLL, from acquisition of patient sample to generating the V-gene readout.

Mantle cell lymphoma with t(11;14) and unmutated or mutated VH genes expresses AID and undergoes isotype switch events.

Isotype switch commonly follows onset of somatic hypermutation in the germinal center (GC), with activation-induced cytidine deaminase (AID) as a prerequisite. Mantle cell lymphoma (MCL) with t(11;14) includes a subset with unmutated (UM) and a minor subset with mutated (MUT) VH genes. Here, we investigated whether switch events and AID expression occur in MCL. In 4 of 6 UM and 4 of 7 MUT MCLs, alternative tumor-derived Cgamma,alpha,epsilon transcripts were identified. AID transcripts, including a splice variant, were common to both subsets. AID expression correlated with switch in 8 of 8 cases, but in 3 of 5 cases it occurred with switch absent. Circle transcripts (Igamma-Cmicro/Ialpha-Cmu) were identified in 5 of 7 evaluated cases. In 1 of 12 cases, 12% of tumor cells expressed immunoglobulin L-restricted surface IgA. Ongoing switch recombination events appear to be a feature of MCL, likely restricted to a minor tumor subpopulation, with occasional variant sIg expression. UM MCLs implicate origins from pre-GC B cells and reveal switch events at ectopic sites.

Hairy cell leukemia: at the crossroad of somatic mutation and isotype switch.

Hairy cell leukemia (HCL) commonly expresses multiple immunoglobulin isotypes, a feature rare in other B-cell malignancies or in normal B cells. In HCL, there is no phenotypic evidence for subpopulations, and single cells from one previous case contained transcripts for several isotypes. This raises the questions of the differentiation status of the cell of origin and of posttransformation events. We have investigated 9 cases, all expressing multiple immunoglobulin isotypes. Multiple tumor-derived variable-(diversity)-joining-constant mu delta, gamma, alpha (V(D)J-Cmu, delta, gamma, alpha) transcripts were confirmed in single cells of a further case. All cases were negative for germinal center (GC)-associated markers CD27 and CD38. Seven of 9 cases had mutated V(H) genes, with low levels of intraclonal heterogeneity, but 2 of 9 were unmutated, indicative of pre-GC origin. Eight of 9 cases expressed activation-induced cytidine deaminase (AID), a molecule essential for somatic mutation and isotype switch. All cases expressed germ line heavy-chain I exon (I(H))-C(H) transcripts which paralleled surface immunoglobulin (sIg) isotype. Significantly, no circle transcripts indicative of deletional recombination of switched isotypes were detectable in 9 of 9 cases. These data indicate heterogeneity in the cell of origin in terms of mutational status, but reveal common features of AID expression and isotype-switching events occurring prior to deletional recombination. Both mutational and switching events may be influenced by environmental factors at extrafollicular sites.

A subset of t(11;14) lymphoma with mantle cell features displays mutated IgVH genes and includes patients with good prognosis, nonnodal disease.

We analyzed lymphocyte morphology, histology, immunophenotype, immunoglobulin heavy chain (IgVH) gene mutations, and clinical course in 80 unselected patients presenting with circulating t(11;14) lymphocytes. Of the 80 patients, 43 had peripheral lymphadenopathy (nodal group), and histology confirmed mantle cell lymphoma (MCL) in all. There were 37 patients with no lymphadenopathy (nonnodal group); 13 of 37 had histology, all showing MCL. IgVH genes were unmutated in 28 (90%) of 31 nodal and 15 (44%) of 34 nonnodal cases (P =.0001); CD38 was positive in 32 (94%) of 34 nodal and 16 (48%) of 33 nonnodal cases (P <.001); 41 (95%) of 43 nodal patients required immediate treatment compared with 18 (49%) of 37 nonnodal patients who had indolent disease (P <.0001). Median survival (95% confidence interval) was 30 months (10-50) in the nodal group and 79 months (22-136) in the nonnodal group (P =.005). Mutation status did not statistically affect survival, but of 6 long-term survivors (> 90 months) all were nonnodal and 5 of 5 had mutated IgVH genes. Lymphocyte morphology was heterogeneous in both groups: typical MCL in 56 cases (34 nodal, 22 nonnodal), blastoid MCL in 8 cases (3 nodal, 5 nonnodal), and small-cell MCL in 16 cases (6 nodal, 10 nonnodal, P =.12). Matutes immunophenotyping score was 1 in 65 cases and 2 in 15 (8 nodal, 7 nonnodal). We find no evidence against a diagnosis of MCL in the nonnodal group and suggest that mutated IgVH genes may help identify patients with indolent disease.