Evaluation of TERT mRNA expression using RNAscope®: A potential histopathologic diagnostic and prognostic tool.

BACKGROUND: Telomerase reverse transcriptase (TERT) activation has been shown to be an important cancer hallmark; the activation and expression of TERT has been documented in >90% of tumors and TERT activation has been touted as a prognostic marker in many cancers. However, there is currently no simple testing modality to detect TERT mRNA expression in surgical pathology specimens. In this study we aim to evaluate and validate the utility and reliability of the TERT RNAscope® in-situ hybridization (ISH) assay for the detection of TERT mRNA expression in formalin-fixed, paraffin embedded tissue. METHODS AND MATERIALS: RNAscope® detection for TERT was performed on a Leica Biosystems BOND III research staining robot using the Hs-TERT-O1 (ACD, 481968) probe. Twenty three samples containing 48 tissue types were assessed. TERT genomic alterations were determined by targeted next generation sequencing (NGS), while TERT mRNA expression was determined by both targeted RNA-sequencing and TERT RNAscope® and the results compared. Manual vs automated TERT expression quantification methodologies were evaluated for the ISH assay. The expression levels in normal vs. neoplastic tissues were also compared. RESULTS: The RNAscope® assay showed high TERT expression in neoplastic tissues, while most normal tissues have no or very low expression levels (p-value= 0.0001, AUC: 0.99). In addition, there was good correlation of TERT expression between the RNAscope® assay and RNA-sequencing. For RNAscope® quantification, manual calculation of TERT signal/cell ratio based on a count of 100 cells was superior compared to automated signal detection. CONCLUSION: TERT RNAscope® assay is a simple and reliable tool for the evaluation of TERT mRNA expression. TERT signal/cell ratio based on a count of 100 cells is a reproducible and accurate interpretation approach for evaluation of TERT expression.

Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

Comparison of In Situ Hybridization, Immunohistochemistry, and Reverse Transcription-Droplet Digital Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Testing in Tissue.

CONTEXT.­: Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription-polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). OBJECTIVE.­: To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. DESIGN.­: Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. RESULTS.­: Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (κ = 0.75). Interobserver reliability was almost perfect for IHC (κ = 0.91) and fair to moderate for RNAish (κ = 0.38-0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P = .03, Pearson correlation = -0.65) and were more likely to be positive by RNAish (P = .02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. CONCLUSIONS.­: Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

Evaluation of Human Kidney Injury Molecule 1 (hKIM-1) Expression in Tumors From Various Organs by Messenger RNA In Situ Hybridization.

OBJECTIVES: Human kidney injury molecule 1 (hKIM-1) is a sensitive and specific marker for detection of clear cell renal cell carcinoma (CRCC), papillary renal cell carcinoma (PRCC), and ovarian clear cell carcinoma (OCCC). Its use was limited to a few surgical pathology laboratories because this specific antibody to hKIM-1 was not commercially available. We investigated the diagnostic utility of RNA in situ hybridization/RNAscope in the detection of hKIM-1 in tumors from various organs. METHODS: RNAscope for hKIM-1 was performed on 1,252 cases on tissue microarray sections, including CRCC (n = 185), PRCC (n = 59), chromophobe renal cell carcinoma (n = 18), oncocytoma (n = 12), OCCC (n = 27), and metastatic CRCC (n = 46). RESULTS: Fifty-nine (100%) of 59 PRCCs, 94 (95%) of 99 low-grade CRCCs, 83 (96%) of 86 high-grade CRCCs, and 24 (89%) of 27 OCCCs, and 44 (96%) of 46 metastatic CRCCs were positive for hKIM-1. In contrast, hKIM-1 expression was not seen in normal renal tubules or in most nonrenal tumors. Low-level expression could be seen in a small percentage of urothelial, hepatocellular, and colon carcinomas. CONCLUSIONS: hKIM-1 is a sensitive and relatively specific marker (1) for diagnosing PRCC, CRCC, and OCCC when working on a tumor of unknown origin and (2) for differentiating CRCC from chromophobe renal cell carcinoma and oncocytoma.

Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection.

The relationship of SARS-CoV-2 pulmonary infection and severity of disease is not fully understood. Here we show analysis of autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter-patient and intra-patient heterogeneity of pulmonary virus infection. There is a spectrum of high and low virus cases associated with duration of disease. High viral cases have high activation of interferon pathway genes and a predominant M1-like macrophage infiltrate. Low viral cases are more heterogeneous likely reflecting inherent patient differences in the evolution of host response, but there is consistent indication of pulmonary epithelial cell recovery based on napsin A immunohistochemistry and RNA expression of surfactant and mucin genes. Using a digital spatial profiling platform, we find the virus corresponds to distinct spatial expression of interferon response genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

Temporal and Spatial Heterogeneity of Host Response to SARS-CoV-2 Pulmonary Infection.

The relationship of SARS-CoV-2 lung infection and severity of pulmonary disease is not fully understood. We analyzed autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter- and intra- patient heterogeneity of pulmonary virus infection. There was a spectrum of high and low virus cases that was associated with duration of disease and activation of interferon pathway genes. Using a digital spatial profiling platform, the virus corresponded to distinct spatial expression of interferon response genes and immune checkpoint genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

Detection of Albumin Expression by RNA In Situ Hybridization Is a Sensitive and Specific Method for Identification of Hepatocellular Carcinomas and Intrahepatic Cholangiocarcinomas.

OBJECTIVES: Inconsistent data on detection of albumin expression by ribonucleic acid (RNA) in situ hybridization have been reported. We investigated the utility of RNAscope (Advanced Cell Diagnostics, Hayward, CA) in detection of albumin in hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (ICCs), and carcinomas from various organs using manual and automated staining. METHODS: RNAscope for albumin detection was performed on 482 cases on tissue microarray sections and on 22 cases of ICC, including 14 surgical resection and eight core biopsy specimens. RESULTS: Thirty-six of 37 (97%) HCCs had detectable mRNA, whereas all non-HCC and non-ICC cases, except one lung adenocarcinoma, were negative for albumin. Fourteen of 22 ICCs (64%) were positive for albumin. CONCLUSIONS: RNAscope for albumin is highly sensitive and specific for identifying HCCs and is highly specific and moderately sensitive for detection of ICCs; however, rare carcinomas (non-HCC, non-ICC, and those with no hepatoid histomorphology) can also have aberrant expression of albumin.

Analytical Performance of a Gene Expression Classifier for Medullary Thyroid Carcinoma.

BACKGROUND: The aim of this study was to demonstrate the analytical validity of an RNA classifier for medullary thyroid carcinoma (MTC). METHODS: Fresh-frozen tissue specimens were obtained from commercial sources, and MTC diagnoses were confirmed by histopathology review. De-identified patient fine-needle aspiration biopsies (FNABs) and whole blood from normal donors were obtained. Total RNA was extracted, amplified, and hybridized to custom microarrays for gene expression analysis. Gene expression data were normalized and classified via a machine learning algorithm. Positive control materials were produced from MTC tissues and tested across multiple experiments and laboratories. Twenty-seven MTC tissue specimens were used to evaluate the sensitivity of the MTC classifier. Gene expression data from tissues and FNABs were used to model classifier response to mixtures of MTC samples with normal thyroid tissue, a benign thyroid nodule, a Hürthle cell adenoma, and whole blood. Select mixture conditions were confirmed in vitro. Assay tolerance to RNA input variation (5-25 ng) and genomic DNA contamination (30% by mass) was evaluated. The intra- and inter-run reproducibility and inter-laboratory accuracy of MTC classifier results were characterized. RESULTS: The MTC classifier sensitivity of 96.3% [confidence interval 81.0-99.9%] was determined retrospectively using 27 MTC confirmed tissue specimens. One false-negative result in a necrotic tissue implicated sample necrosis in reduced classifier sensitivity. Dilution modeling of MTC samples with normal or benign tissues showed consistent detection of MTC down to 20% sample proportions, with in vitro confirmation of 20% analytical sensitivity. Classifier tolerance to RNA input variation (5-25 ng), genomic DNA contamination (30% by mass), and an interfering substance (blood) was demonstrated with 100% accurate classifier results under all tested conditions. The maximum observed run-to-run score difference for a single FNAB sample was ∼1 unit compared with the average score difference between 38 MTC and non-MTC FNABs of ∼32 units. MTC classifier results for 20 tissues processed from total RNA in two different laboratories showed 100% concordance. CONCLUSIONS: The MTC classifier, offered as part of the routine molecular testing of cytology-indeterminate thyroid nodules, demonstrates robust analytical sensitivity, specificity, accuracy, and reproducibility.

A Genomic Alternative to Identify Medullary Thyroid Cancer Preoperatively in Thyroid Nodules with Indeterminate Cytology.

BACKGROUND: The use of calcitonin screening for the rare medullary thyroid cancer (MTC) is controversial due to questions of efficacy, accuracy, and cost-effectiveness. This study reports the results of a large prospective validation using a machine-trained algorithm (MTC Classifier) to preoperatively identify MTC in fine-needle aspiration biopsies in lieu of calcitonin measurements. METHODS: Cytology analysis on a prospective consecutive series of 50,430 thyroid nodule biopsies yielded a total of 7815 indeterminate (Bethesda categories III/IV) cases, which were tested with the MTC classifier. A prospective, consecutively submitted series of 2673 Bethesda III-VI cases with cytology determined locally was also evaluated. RNA was isolated and tested for the MTC Classifier using microarrays. RESULTS: Forty-three cases were positive by the MTC Classifier among 10,488 tested nodules (0.4%), consistent with the low prevalence of MTC. Of these, all but one was histologically or biochemically confirmed as MTC, yielding a positive predictive value (PPV) of 98%. Of the positive cases, only 19 (44%) had been specifically suspected of MTC by cytology, highlighting the limitations of light microscopy to detect this disease. Three surgically confirmed MTC cases that were detected by the MTC Classifier had low basal serum calcitonin values, indicating these would have been missed by traditional calcitonin screening methods. A pooled analysis of three independent validation sets demonstrates high test sensitivity (97.9%), specificity (99.8%), PPV (97.9%), and negative predictive value (99.8%). CONCLUSIONS: A clinical paradigm is proposed, whereby cytologically indeterminate thyroid nodules being tested for common malignancies using gene expression can be simultaneously tested for MTC using the same genomic assay at no added cost.

Analytical performance of a bronchial genomic classifier.

BACKGROUND: The current standard practice of lung lesion diagnosis often leads to inconclusive results, requiring additional diagnostic follow up procedures that are invasive and often unnecessary due to the high benign rate in such lesions (Chest 143:e78S-e92, 2013). The Percepta bronchial genomic classifier was developed and clinically validated to provide more accurate classification of lung nodules and lesions that are inconclusive by bronchoscopy, using bronchial brushing specimens (N Engl J Med 373:243-51, 2015, BMC Med Genomics 8:18, 2015). The analytical performance of the Percepta test is reported here. METHODS: Analytical performance studies were designed to characterize the stability of RNA in bronchial brushing specimens during collection and shipment; analytical sensitivity defined as input RNA mass; analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA; and assay performance studies including intra-run, inter-run, and inter-laboratory reproducibility. RESULTS: RNA content within bronchial brushing specimens preserved in RNAprotect is stable for up to 20 days at 4 °C with no changes in RNA yield or integrity. Analytical sensitivity studies demonstrated tolerance to variation in RNA input (157 ng to 243 ng). Analytical specificity studies utilizing cancer positive and cancer negative samples mixed with either blood (up to 10 % input mass) or genomic DNA (up to 10 % input mass) demonstrated no assay interference. The test is reproducible from RNA extraction through to Percepta test result, including variation across operators, runs, reagent lots, and laboratories (standard deviation of 0.26 for scores on > 6 unit scale). CONCLUSIONS: Analytical sensitivity, analytical specificity and robustness of the Percepta test were successfully verified, supporting its suitability for clinical use.

The diagnostic application of RNA sequencing in patients with thyroid cancer: an analysis of 851 variants and 133 fusions in 524 genes.

BACKGROUND: Thyroid carcinomas are known to harbor oncogenic driver mutations and advances in sequencing technology now allow the detection of these in fine needle aspiration biopsies (FNA). Recent work by The Cancer Genome Atlas (TCGA) Research Network has expanded the number of genetic alterations detected in papillary thyroid carcinomas (PTC). We sought to investigate the prevalence of these and other genetic alterations in diverse subtypes of thyroid nodules beyond PTC, including a variety of samples with benign histopathology. This is the first clinical evaluation of a large panel of TCGA-reported genomic alterations in thyroid FNAs. RESULTS: In FNAs, genetic alterations were detected in 19/44 malignant samples (43% sensitivity) and in 7/44 histopathology benign samples (84% specificity). Overall, after adding a cohort of tissue samples, 38/76 (50%) of histopathology malignant samples were found to harbor a genetic alteration, while 15/75 (20%) of benign samples were also mutated. The most frequently mutated malignant subtypes were medullary thyroid carcinoma (9/12, 75%) and PTC (14/30, 47%). Additionally, follicular adenoma, a benign subtype of thyroid neoplasm, was also found to harbor mutations (12/29, 41%). Frequently mutated genes in malignant samples included BRAF (20/76, 26%) and RAS (9/76, 12%). Of the TSHR variants detected, (6/7, 86%) were in benign nodules. In a direct comparison of the same FNA also tested by an RNA-based gene expression classifier (GEC), the sensitivity of genetic alterations alone was 42%, compared to the 91% sensitivity achieved by the GEC. The specificity based only on genetic alterations was 84%, compared to 77% specificity with the GEC. CONCLUSIONS: While the genomic landscape of all thyroid neoplasm subtypes will inevitably be elucidated, caution should be used in the early adoption of published mutations as the sole predictor of malignancy in thyroid. The largest set of such mutations known to date detects only a portion of thyroid carcinomas in preoperative FNAs in our cohort and thus is not sufficient to rule out cancer. Due to the finding that variants are also found in benign nodules, testing only GEC suspicious nodules may be helpful in avoiding false positives and altering the extent of treatment when selected mutations are found.

Machine learning from concept to clinic: reliable detection of BRAF V600E DNA mutations in thyroid nodules using high-dimensional RNA expression data.

The promise of personalized medicine will require rigorously validated molecular diagnostics developed on minimally invasive, clinically relevant samples. Measurement of DNA mutations is increasingly common in clinical settings but only higher-prevalence mutations are cost-effective. Patients with rare variants are at best ignored or, at worst, misdiagnosed. Mutations result in downstream impacts on transcription, offering the possibility of broader diagnosis for patients with rare variants causing similar downstream changes. Use of such signatures in clinical settings is rare as these algorithms are difficult to validate for commercial use. Validation on a test set (against a clinical gold standard) is necessary but not sufficient: accuracy must be maintained amidst interfering substances, across reagent lots and across operators. Here we report the development, clinical validation, and diagnostic accuracy of a pre-operative molecular test (Afirma BRAF) to identify BRAF V600E mutations using mRNA expression in thyroid fine needle aspirate biopsies (FNABs). FNABs were obtained prospectively from 716 nodules and more than 3,000 features measured using microarrays. BRAF V600E labels for training (n=181) and independent test (n=535) sets were established using a sensitive quantitative PCR (qPCR) assay. The resulting 128-gene linear support vector machine was compared to qPCR in the independent test set. Clinical sensitivity and specificity for malignancy were evaluated in a subset of test set samples (n=213) with expert-derived histopathology. We observed high positive- (PPA, 90.4%) and negative (NPA, 99.0%) percent agreement with qPCR on the test set. Clinical sensitivity for malignancy was 43.8% (consistent with published prevalence of BRAF V600E in this neoplasm) and specificity was 100%, identical to qPCR on the same samples. Classification was accurate in up to 60% blood. A double-mutant still resulting in the V600E amino acid change was negative by qPCR but correctly positive by Afirma BRAF. Non-diagnostic rates were lower (7.6%) for Afirma BRAF than for qPCR (24.5%), a further advantage of using RNA in small sample biopsies. Afirma BRAF accurately determined the presence or absence of the BRAF V600E DNA mutation in FNABs, a collection method directly relevant to solid tumor assessment, with performance equal to that of an established, highly sensitive DNA-based assay and with a lower non-diagnostic rate. This is the first such test in thyroid cancer to undergo sufficient analytical and clinical validation for real-world use in a personalized medicine context to frame individual patient risk and inform surgical choice.

Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules.

OBJECTIVE: Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs). DESIGN: Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility. RESULTS: RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale). CONCLUSIONS: Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.

Impaired V(D)J recombination and lymphocyte development in core RAG1-expressing mice.

RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated "core" RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1(c/c)) mice. Significant B and T cell numbers are generated in RAG1(c/c) mice, showing that core RAG1, despite missing approximately 40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1(c/c) mice. Correspondingly, there are reduced numbers of peripheral RAG1(c/c) B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1(c/c) mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1(c/c) B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1(c/c) B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.

T cell receptor (TCR) alpha/delta locus enhancer identity and position are critical for the assembly of TCR delta and alpha variable region genes.

T cell receptor (TCR) delta and alpha variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCR delta segments are situated between TCR alpha segments. The TCR alpha enhancer (E alpha) located at the 3' end of the TCR alpha/delta locus functions over a long chromosomal distance to promote TCR alpha rearrangement and maximal TCR delta expression; whereas the TCR delta enhancer (E delta) is located among the TCR delta segments and functions with additional element(s) to mediate TCR delta rearrangement. We used gene-targeted mutation to evaluate whether the identity of E alpha and the position of E delta are critical for the developmental stage-specific assembly of TCR delta and alpha variable region genes. Specific replacement of E alpha with E delta, the core E alpha element (E alpha C), or the Ig heavy chain intronic enhancer (iE mu), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCR alpha rearrangement beyond that observed in the absence of E alpha. Therefore, the identity and full complement of E alpha-binding sites are critical for promoting accessibility within the TCR alpha locus. In the absence of the endogenous E delta element, specific replacement of E alpha with E delta also did not promote TCR delta rearrangement. However, deletion of intervening TCR alpha/delta locus sequences to restore the inserted E delta to its normal chromosomal position relative to 5' sequences rescued TCR delta rearrangement. Therefore, unlike E alpha, E delta lacks ability to function over the large intervening TCR alpha locus and or E delta function requires proximity to additional upstream element(s) to promote TCR delta accessibility.

Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice.

The recombination-activating gene (RAG)1 and RAG2 proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 protein ("core" RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombination in vitro and in transfected cell lines. Here we examine the effect of replacing the endogenous RAG2 locus in mice with core RAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 protein retains significant in vivo function. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.