Survival benefit of neoadjuvant FOLFIRINOX for patients with borderline resectable pancreatic cancer.

Backgrounds/Aims: While patients with borderline resectable pancreatic cancer (BRPC) are a target population for neoadjuvant chemotherapy (NAC), formal guidelines for neoadjuvant therapy are lacking. We assessed the perioperative and oncological outcomes in patients with BRPC undergoing NAC with FOLFIRINOX for patients undergoing upfront surgery (US). Methods: The AHPBA criteria for borderline resectability and/or a CA19-9 level > 100 µ/mL defined borderline resectable tumors retrieved from a prospectively populated institutional registry from 2007 to 2020. The primary outcome was overall survival (OS) at 1 and 3 years. A Cox Proportional Hazard model based on intention to treat was used. A receiver-operator characteristics (ROC) curve was constructed to assess the discriminatory capability of the use of CA19-9 > 100 µ/mL to predict resectability and mortality. Results: Forty BRPC patients underwent NAC, while 46 underwent US. The median OS with NAC was 19.8 months (interquartile range [IQR], 10.3-44.24) vs. 10.6 months (IQR, 6.37-17.6) with US. At 1 year, 70% of the NAC group and 41.3% of the US group survived (p = 0.008). At 3 years, 42.5 % of the NAC group and 10.9% of the US group survived (p = 0.001). NAC significantly reduced the hazard of death (adjusted hazard ratio, 0.20; 95% confidence interval, 0.07-0.54; p = 0.001). CA19-9 > 100 µ/mL showed poor discrimination in predicting mortality, but was a moderate predictor of resectability. Conclusions: We found a survival benefit of NAC with FOLFIRINOX for BRPC. Greater pre-treatment of CA19-9 and multivessel involvement on initial imaging were associated with progression of the disease following NAC.

The utility of self-expanding metal stents in benign biliary strictures- a retrospective case series.

BACKGROUND: Benign biliary strictures can have a significant negative impact on patient quality of life. There are several modalities which can be utilized with the goal of stricture resolution. These techniques include balloon dilatation, placement of multiple plastic stents and more recently, the use of metal stents. The aim of this study was to evaluate the local success of self-expanding metal stents in successfully resolving benign biliary strictures. METHODS: This was a single institution, retrospective case series. Patients included in our study were patients who underwent endoscopic retrograde cholangiopancreatography with placement of self expanding metal stents for benign biliary strictures at our institution between 2016-2022. Patients were excluded for the following: malignant stricture, and inability to successfully place metal stent. Data was evaluated using two-sided t-test with 95% confidence interval. RESULTS: A total of 31 patients underwent placement of 43 self-expanding metal stents and met inclusion criteria. Mean age of patients was 59 ± 10 years, and were largely male (74.2% vs. 25.8%). Most strictures were anastomotic stricture post liver transplant (87.1%), while the remainder were secondary to chronic pancreatitis (12.9%). Complications of stent placement included cholangitis (18.6%), pancreatitis (2.3%), stent migration (20.9%), and inability to retrieve stent (4.7%). There was successful stricture resolution in 73.5% of patients with anastomotic stricture and 33.3% of patients with stricture secondary to pancreatitis. Resolution was more likely if stent duration was > / = 180 days (73.3% vs. 44.4%, p < 0.05). There was no demonstrated added benefit when stent duration was > / = 365 days (75% vs. 60.9%, p = 0.64). CONCLUSIONS: This study demonstrates that self expanding metal stents are a safe and effective treatment for benign biliary strictures, with outcomes comparable to plastic stents with fewer interventions. This study indicates that the optimal duration to allow for stricture resolution is 180-365 days.

PD-1 and PD-L1 inhibitors in cold colorectal cancer: challenges and strategies.

Colorectal cancer (CRC) is the second most common cause of cancer mortality, with mismatch repair proficient (pMMR) and/or microsatellite stable (MSS) CRC making up more than 80% of metastatic CRC. Programmed death-ligand 1 (PD-L1) and programmed death 1 (PD-1) immune checkpoint inhibitors (ICIs) are approved as monotherapy in many cancers including a subset of advanced or metastatic colorectal cancer (CRC) with deficiency in mismatch repair (dMMR) and/or high microsatellite instability (MSI-H). However, proficient mismatch repair and microsatellite stable (pMMR/MSS) cold CRCs have not shown clinical response to ICIs alone. To potentiate the anti-tumor response of PD-L1/PD-1 inhibitors in patients with MSS cold cancer, combination strategies currently being investigated include dual ICI, and PD-L1/PD-1 inhibitors in combination with chemotherapy, radiotherapy, vascular endothelial growth factor (VEGF) /VEGF receptor (VEGFR) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, and signal transducer and activation of transcription 3 (STAT3) inhibitors. This paper will review the mechanisms of PD-1/PD-L1 ICI resistance in pMMR/MSS CRC and potential combination strategies to overcome this resistance, summarize the published clinical experience with different combination therapies, and make recommendations for future avenues of research.

Liver transplantation with donation after medical assistance in dying: Case series and systematic review of the literature.

Medical assistance in dying (MAiD) has been a legally approved practice in Canada since 2016. Only recently have patients undergoing MAiD also been considered as donors for liver transplantation (LT). This study aimed to evaluate a case series of LT outcomes for recipients with MAiD donors and was paired with a systematic literature review of studies assessing the efficacy of MAiD-associated liver donation. A retrospective chart review of patients registered within the LT Registry at London Health Sciences Centre (LHSC) in London, Ontario, Canada, that had received MAiD donor LT was conducted to develop a case series. Descriptive statistics were produced based on available patient outcomes information. The systematic review included euthanasia due to MAiD being a term exclusive to Canada. Case series had a 100% 1-year graft survival rate, with 50% of patients experiencing early allograft dysfunction but having no significant clinical outcome. A single case of postoperative biliary complication was reported. Median warm ischemic time ranged from 7.8-13 minutes among case series and literature reviews. Utilization of donation after circulatory death allografts procured after MAiD appears to be promising. Mechanisms associated with potential impact in postoperative outcomes include relatively lower warm ischemic time relative to donation after circulatory death Maastricht III graft recipients.

Reducing length of stay in patients following liver transplantation using the model for continuous improvement.

Length of stay (LOS) is a significant contributor to overall patient outcomes for patients undergoing liver transplantation. This study documents a quality improvement project aiming to reduce the median post-transplant LOS for liver transplant patients. We instituted five Plan-Do-Study-Act cycles with the goal of reducing LOS by 3 days from a baseline median of 18.4 days over 1 year. Balancing measures such as readmission rates ensured any decrease in stay was not associated with significantly increased patient complications. Over the 28-month intervention period and 24-month follow-up period, there were 193 patients discharged from hospital with a median LOS of 9 days. The changes appreciated during quality improvement interventions carried over to sustained improvements, with no significant variability in LOS postintervention. Discharge within 10 days increased from 18.4% to 60% over the study period, with intensive care unit stay decreasing from a median of 3.4-1.9 days. Thus, the development of a multidisciplinary care pathway, with patient engagement, led to improved and sustained discharge rates with no significant differences in readmission rates.

Cost analysis of indocyanine green fluorescence angiography for prevention of anastomotic leakage in colorectal surgery.

BACKGROUND: Indocyanine green, near infrared, fluorescence angiography (ICG-FA) is increasingly adopted in colorectal surgery for intraoperative tissue perfusion assessment to reduce anastomotic leakage rates. However, the economic impact of this intervention has not been investigated. This study is a cost analysis of the routine use of ICG-FA in colorectal surgery from the hospital payer perspective. METHODS: A decision analysis model was developed for colorectal resections considering two scenarios: resection without using ICG-FA and resection with intraoperative ICG-FA for anastomotic perfusion assessment. Incorporated into the model were the costs of ICG agent, fluorescence angiography equipment, surgery, anastomotic leak, and the leak rates with and without ICG-FA. All input data were derived from recent publications. RESULTS: The routine use of ICG-FA for colorectal anastomosis is cost saving when cost analysis is performed using the following base case assumptions: 8.6% leak rate without ICG-FA, odds ratio of 0.46 for reduction of leakage with ICG-FA (4.8% leak rate relative to 8.6% base case), cost of ICG-FA of $250, and incremental cost of leak, not requiring reoperation, of $9,934.50. In one-way sensitivity analyses, routine use of ICG-FA was cost saving if the cost of an anastomotic leak is more than $5616.29, the cost of ICG-FA is less than $634.44, the leak rate (without ICG-FA) is higher than 4.9%, or the odds ratio for reduction of leak with ICG-FA is less than 0.69. There is a per-case saving of $192.22 with the use of ICG-FA. CONCLUSION: Using the best available evidence and most conservative base case values, routine use of ICG-FA in colorectal surgery was found to be cost saving. Since the evidence suggests there is a reduction in leak rate, the routine use of ICG-FA is a dominating strategy. However, the overall quality of evidence is low and there is a clear need for prospective, randomized controlled trials.

Kidney transplant outcomes after medical assistance in dying.

INTRODUCTION: After nearly four years of Canadian experience with medical assistance in dying (MAID), the clinical volume of organ transplantation following MAID remains low. This is the first Canadian report evaluating recipient outcomes from kidney transplantation following MAID. METHODS: This was a retrospective review of the first nine cases of kidney transplants following MAID at a Canadian transplant center. RESULTS: Nine patients underwent MAID followed by kidney retrieval during the study period. Their diagnoses were largely neuromuscular diseases. The mean warm ischemic time was 20 minutes (standard deviation [SD] 7). The nine recipients had a mean age of 60 (SD 19.7). The mean cold ischemic time was 525 minutes (SD 126). Delayed graft function occurred in only one patient out of nine. The mean 30-day creatinine was 124 umol/L (SD 52). The mean three-month creatinine was 115 umol/L (SD 29). CONCLUSIONS: We report nine cases of kidney transplantation following MAID. The process minimized warm ischemia, resulting in low delayed graft function rates, and acceptable post-transplant outcomes. Further large-scale research is necessary to optimize processes and outcomes in this novel clinical pathway.

Germinal center reactions in tertiary lymphoid structures associate with neoantigen burden, humoral immunity and long-term survivorship in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has traditionally been thought of as an immunologically quiescent tumor type presumably because of a relatively low tumor mutational burden (TMB) and poor responses to checkpoint blockade therapy. However, many PDAC tumors exhibit T cell inflamed phenotypes. The presence of tertiary lymphoid structures (TLS) has recently been shown to be predictive of checkpoint blockade response in melanomas and sarcomas, and are prognostic for survival in PDAC. In order to more comprehensively understand tumor immunity in PDAC patients with TLS, we performed RNA-seq, single and multiplex IHC, flow cytometry and predictive genomic analysis on treatment naïve, PDAC surgical specimens. Forty-six percent of tumors contained distinct T and B cell aggregates reflective of "early-stage TLS" (ES-TLS), which correlated with longer overall and progression-free survival. These tumors had greater CD8+ T cell infiltration but were not defined by previously published TLS gene-expression signatures. ES-TLS+ tumors were enriched for IgG1 class-switched memory B cells and memory CD4+ T cells, suggesting durable immunological memory persisted in these patients. We also observed the presence of active germinal centers (mature-TLS) in 31% of tumors with lymphocyte clusters, whose patients had long-term survival (median 56 months). M-TLS-positive tumors had equivalent overall T cell infiltration to ES-TLS, but were enriched for activated CD4+ memory cells, naive B cells and NK cells. Finally, using a TCGA-PDAC dataset, ES-TLS+ tumors harbored a decreased TMB, but M-TLS with germinal centers expressed significantly more MHCI-restricted neoantigens as determined by an in silico neoantigen prediction method. Interestingly, M-TLS+ tumors also had evidence of increased rates of B cell somatic hypermutation, suggesting that germinal centers form in the presence of high-quality tumor neoantigens leading to increased humoral immunity that confers improved survival for PDAC patients. AbbreviationsTLS: tertiary lymphoid structures; GC: germinal center(s); PDAC: pancreatic ductal adenocarcinoma; RNA-seq: RNA sequencing; BCRseq: B cell receptor sequencing; HEV: high endothelial venule; PNAd: peripheral node addressin; TMB: tumor mutational burden; TCGA: the cancer genome atlas; PAAD: pancreatic adenocarcinoma; FFPE: formalin fixed paraffin embedded; TIME: tumor immune microenvironment.

Liver transplantation in adult polycystic liver disease: the Ontario experience.

BACKGROUND: Liver transplantation (LT) remains the curative treatment for symptomatic Polycystic Liver Disease (PCLD) patients and is associated with excellent survival rates. The aim of the study is to review the Ontario experience in LT for PCLD. METHODS: A retrospective study was performed from pre-existing LT databases from the LT Units at Toronto General Hospital and London Health Sciences Center, which are the two LT programs in Ontario, Canada. This database contains demographic, clinical parameters and follow-up of all patients transplanted for PCLD. Data was extracted for patients who underwent LT between January 2000-April 2017 and included follow up until December 31st, 2018. RESULTS: A total of 3560 patients underwent LT, of whom 51 (1.4%) had PCLD and met inclusion criteria. 43 (84%) of these patients were female. The median physiologic Model for End Stage Liver Disease (MELD-Na) score at time of referral was 13 (IQR = 7-22), however all patients required MELD-Na exception points to receive LT. The median age of transplant was 62 years (IQR = 59-64) for male vs. 52 (IQR = 45-56) for female patients. 33 (65%) of our cohort had PCLD while 9 (17.5%) had ADPKD and 9 (17.5%) had both diseases. 39 (76%) had LT due to symptoms of mass effect, while 8 (16%) had portal hypertensive complications. After a median follow-up of 6.3 (IQR = 2.9-12.5) years, the probability of survival was 96% (95% CI: 90%, 100%). Log-rank test, comparing survival analysis between males and females did not show a statistically significant difference (p = 0.26). CONCLUSION: Most patients underwent LT for PCLD due to symptoms of mass effect with women being more likely than men to undergo LT. LT for PCLD had excellent long-term survival.

Transcriptional and immunohistological assessment of immune infiltration in pancreatic cancer.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes.

Liver Transplant Tourism.

Liver transplant tourism is travel for transplantation involving organ trafficking and/or transplant commercialism. Various medical, financial, and organizational factors play a role in transplant care including waiting lists, Model for End-Stage Liver Disease scores, and financial aid. We outline the international experiences with transplant tourism (TT) and its effect on their medical communities and patients. For clinicians providing care to patients involved in TT, we also discuss pretransplant counseling and posttransplant care.

Predictors and Cumulative Frequency of Hepatocellular Carcinoma in High and Intermediate LI-RADS Lesions: A Cohort Study from a Canadian Academic Institution.

BACKGROUND: The frequency and predictors of hepatocellular carcinoma (HCC) within each liver imaging reporting and data system (LI-RADS) category remains unclear. We sought to estimate the cumulative frequency of HCC in LI-RADS observations of high/intermediate category and identify clinical/radiographic features associated with HCC. METHODS: Our diagnostic imaging database was searched for computed tomography/magnetic resonance imaging reports of patients with evidence of cirrhosis and liver observations. LI-RADS categories were determined by imaging review, while demographic and clinical outcomes were assigned by chart review. A composite outcome of clinical/radiographic confirmation of HCC was used. We used multivariable analysis to identify features associated with HCC, and competing risks regression to estimate the cumulative frequency of HCC in each category. RESULTS: Our search returned 95 patients with 137 observations (LR2 = 4, LR3 = 53, LR4 = 37, and LR5 = 43). On multivariable analysis, increasing age (hazard ratio [HR] 1.76 per 10 years, p = 0.049), washout (HR 5.34, p < 0.002), and increasing size (size < 10 mm reference, 10-20 mm, HR 3.93, p = 0.014; size > 20 mm, HR 21.69, p < 0.001) were associated with HCC. Median time to diagnosis was 6.13 months (interquartile range [IQR] 4.6-13.1), 4.7 months (IQR 2.5-14.5), and 3.6 months (IQR 1.9-6.6) for LR3, 4, and 5 category observations, respectively. The cumulative frequency of HCC was 59.8% in LR3, 84.62% in LR4, and 99.84% in LR5, at last follow-up. CONCLUSION: The frequency of HCC within each LI-RADS category reflects the intended purpose, intermediate probability for LR3, probable HCC for LR4, and definite HCC for LR5.

Pulmonary Metastasectomy for Colorectal Cancer: Predictors of Survival in Routine Surgical Practice.

BACKGROUND: Resection of lung metastases is considered standard treatment for patients with metastatic colorectal cancer. We describe surgical management, prognostic factors, and outcomes in routine clinical practice. METHODS: All cases of colorectal cancer lung metastases in Ontario, Canada, resected during 2002 to 2009 were identified using the Ontario Cancer Registry and linked electronic records. Pathology reports were reviewed to identify extent of disease. RESULTS: The study population included 420 patients (60% male). Median age was 64 years. A solitary metastasis was present in 61% (256 of 420). Mean size of the largest metastasis was 2.4 cm. Lymph nodes were resected in 63% (263 of 420) of patients. The 5-year cancer-specific survival (CSS) and overall survival (OS) was 42% (95% confidence interval [CI], 37% to 47%) and 40% (95% CI, 35% to 45%), respectively. On adjusted analyses, greater number (p < 0.001) and size (p = 0.001) of lesions and lymph node involvement (p < 0.001) were associated with inferior CSS and OS. Lymph node positivity was strongly associated with survival (adjusted CSS hazard ratio, 2.19 [95% CI, 1.48 to 3.25]; adjusted OS hazard ratio, 2.08 [95% CI, 1.41 to 3.07]). Unadjusted 5-year CSS/OS was 49%/47% for node-negative disease and 19%/19% for node-positive disease. The negative prognostic effect of size (>2 cm) and number (>1) of lesions was additive: 5-year CSS/OS ranged from 57%/55% (single lesion <2 cm) to 24%/20% (multiple lesions, largest lesion>2 cm). CONCLUSIONS: Long-term survival of patients with resected colorectal cancer lung metastases in routine practice is comparable to outcomes reported in institutional case series. Lymph node positivity is strongly associated with reduced survival. Combining size and number of metastatic lesions in advance of the operation may facilitate treatment decision making.

Resection of the largest reported hepatic small vessel neoplasm.

Hepatic small vessel neoplasm (HSVN) is a recently described vascular neoplasm of the adult liver. The neoplastic cells are positive for markers of vascular lineage (CD31, CD34, FLI-1). The distinctive morphology and infiltrative borders separate HSVN from benign vascular tumors such as cavernous hemangioma, while lack of atypical morphologic features, low to absent mitotic activity and low proliferation index distinguish it from malignant vascular tumors such as epithelioid hemangioendothelioma and angiosarcoma. Due to its infiltrative nature and lack of adequate follow-up information, the benign versus low-grade nature of this tumor is currently uncertain. We present a patient with resected HSVN involving all but the right posterior section of the liver, making this case the largest reported in the current literature.

Comparing the tensile strength of square and reversing half-hitch alternating post knots.

BACKGROUND: Square knots are the gold standard in hand-tie wound closure, but are difficult to reproduce in deep cavities, inadvertently resulting in slipknots. The reversing half-hitch alternating post (RHAP) knot has been suggested as an alternative owing to its nonslip nature and reproducibility in limited spaces. We explored whether the RHAP knot is noninferior to the square knot by assessing tensile strength. METHODS: We conducted 10 trials for each baseline and knot configuration, using 3-0 silk and 3-0 polyglactin 910 sutures. We compared tensile strength between knot configurations at the point of knot failure between slippage and breakage. RESULTS: Maximal failure strength (mean ± SD) in square knots was reached with 4-throw in both silk (30 ± 1.5 N) and polyglactin 910 (39 ± 12 N). For RHAP knots, maximal failure strength was reached at 5-throw for both silk (31 ± 1.5 N) and polyglactin 910 (41 ± 13 N). In both sutures, there were no strength differences between 3-throw square and 4-throw RHAP, between 4-throw square and 5-throw RHAP, or between 5-throw square and 6-throw RHAP knots. Polyglactin 910 sutures, in all knot configurations, were more prone to slippage than silk sutures (p < 0.001). CONCLUSION: The difference in mean tensile strength could be attributed to the proportion of knot slippage versus breakage, which is material-dependent. Future studies can re-evaluate findings in monofilament sutures and objectively assess the reproducibility of square and RHAP knots in deep cavities. Our results indicate that RHAP knots composed of 1 extra throw provide equivalent strength to square knots and may be an alternative when performing hand-ties in limited cavities with either silk or polyglactin 910 sutures.

CONTEXTE: Les nœuds plats sont la norme en matière de points de suture manuels, mais ils sont difficiles à reproduire dans des cavités profondes et deviennent par inadvertance des nœuds coulants. Le nœud de type demi-clé inversée alternée a été proposé comme solution de rechange en raison de sa nature non glissante et de sa reproductibilité dans des espaces restreints. Nous avons voulu vérifier la non-infériorité de la demi-clé inversée alternée par rapport au nœud plat en évaluant sa résistance à la traction. MÉTHODES: Nous avons réalisé 10 essais de référence et autant d'essais pour chaque type de nœud, en utilisant des fils de soie de taille 3-0 et de polyglactine 910 de taille 3-0. Nous avons comparé la résistance à la traction des différentes configurations de nœuds, c'est-à-dire le point de défaillance entre le glissement du nœud et la rupture de celui-ci. RÉSULTATS: La résistance maximale à la traction (moyenne ± É.-T.) des nœuds plats a été atteinte avec 4 boucles, tant pour le fil de soie (30 ± 1,5 N) que le fil de polyglactine 910 (39 ± 12 N). Pour les demi-clés inversées alternées, la résistance maximale à la traction a été atteinte avec 5 boucles pour le fil de soie (31 ± 1,5 N) et de polyglactine 910 (41 ± 13 N). Dans les 2 types de sutures, on n'a noté aucune différence de résistance entre le nœud plat à 3 boucles et la demi-clé inversée alternée à 4 boucles, entre le nœud plat à 4 boucles et la demi-clé inversée alternée à 5 boucles, ni entre le nœud plat à 5 boucles et la demi-clé inversée alternée à 6 boucles. Dans tous les types de nœuds, les sutures de polyglactine 910 ont été plus sujettes au glissement que les sutures de soie (p < 0,001). CONCLUSION: La différence de résistance moyenne à la traction a pu être attribuée à la proportion de glissement c. la rupture des nœuds, qui dépend du matériau. D'autres études pourraient réévaluer les résultats obtenus avec des sutures monofilaments et mesurer objectivement la reproductibilité des nœuds plats et des demi-clés inversées alternées dans des cavités profondes. Selon nos résultats, les demi-clés inversées alternées comportant une boucle supplémentaire offrent une résistance équivalente à celle des nœuds plats et seraient une solution de rechange lors de sutures manuelles dans des cavités restreintes, avec la soie ou la polyglactine 910.

Immunoglobulin gene conversion: synthesizing antibody diversification and DNA repair.

Recent developments in the field of antibody (Ab) diversification have rapidly advanced our understanding of the molecular mechanism underlying these events. Key to these developments was the identification of activation-induced cytidine deaminase (AID) as the central regulator of secondary Ab diversification, and the elucidation of its primary function as a DNA deaminase. Incredibly, current literature suggests the existence of a shared pathway, common to all secondary diversification processes, from which the separate outcomes branch outwards at various points. Immunoglobulin gene conversion (IGC) is one of these mechanisms and is used by a number of vertebrate species in both the development of the pre-immune repertoire and in affinity maturation. In a manner similar to other Ab diversification mechanisms, IGC has managed to co-opt a normal DNA repair pathway for the generation of receptor diversity. In the case of IGC specifically, that pathway is homologous recombination (HR). A burgeoning wealth of genetic, biochemical and structural data has clarified the roles of many key HR factors, allowing new insight into its molecular mechanism. These insights, combined with those from the common mechanism of AID action, synergize to develop an emerging picture of the mechanism underlying IGC.

NHEJ-deficient DT40 cells have increased levels of immunoglobulin gene conversion: evidence for a double strand break intermediate.

Activation-induced cytidine deaminase (AID) likely initiates immunoglobulin gene-conversion (GC) by deaminating cytidines within the V-region of chicken B-cells. However, the intervening DNA lesion required to initiate GC remains elusive. GC could be initiated by a single strand break or a double strand break (DSB). To distinguish between these possibilities, we examined GC in the chicken DT40 B cell line deficient in non-homologous end joining (NHEJ). It is known that the NHEJ and homologous recombination DNA repair pathways compete for DSBs. In light of this, if a DSB is the major intermediate, deficiency in NHEJ should result in increased levels of GC. Here we show that DNA-PKcs(-/-/-) and Ku70(-/-) DT40 cells had 5- to 10-fold higher levels of GC relative to wildtype DT40 as measured by surface IgM reversion and sequencing of the V-region. These data suggest that a DSB is the major DNA lesion that initiates GC.