BluePrint molecular subtypes predict response to neoadjuvant pertuzumab in HER2-positive breast cancer.

BACKGROUND: The introduction of pertuzumab has greatly improved pathological complete response (pCR) rates in HER2-positive breast cancer, yet effects on long-term survival have been limited and it is uncertain which patients derive most benefit. In this study, we determine the prognostic value of BluePrint subtyping in HER2-positive breast cancer. Additionally, we evaluate its use as a biomarker for predicting response to trastuzumab-containing neoadjuvant chemotherapy with or without pertuzumab. METHODS: From a cohort of patients with stage II-III HER2-positive breast cancer who were treated with neoadjuvant chemotherapy and trastuzumab with or without pertuzumab, 836 patients were selected for microarray gene expression analysis, followed by readout of BluePrint standard (HER2, Basal and Luminal) and dual subtypes (HER2-single, Basal-single, Luminal-single, HER2-Basal, Luminal-HER2, Luminal-HER2-Basal). The associations between subtypes and pathological complete response (pCR), overall survival (OS) and breast cancer-specific survival (BCSS) were assessed, and pertuzumab benefit was evaluated within the BluePrint subgroups. RESULTS: BluePrint results were available for 719 patients. In patients with HER2-type tumors, the pCR rate was 71.9% in patients who received pertuzumab versus 43.5% in patients who did not (adjusted Odds Ratio 3.43, 95% CI 2.36-4.96). Additionally, a significantly decreased hazard was observed for both OS (adjusted hazard ratio [aHR] 0.45, 95% CI 0.25-0.80) and BCSS (aHR 0.46, 95% CI 0.24-0.86) with pertuzumab treatment. Findings were similar in the HER2-single subgroup. No significant benefit of pertuzumab was seen in other subtypes. CONCLUSIONS: In patients with HER2-type or HER2-single-type tumors, pertuzumab significantly improved the pCR rate and decreased the risk of breast cancer mortality, which was not observed in other subtypes. BluePrint subtyping may be valuable in future studies to identify patients that are likely to be highly sensitive to HER2-targeting agents.

[A 19-year-old female patient with acute pancreatitis of unusual cause]. / 19-jährige Patientin mit akuter Pankreatitis ungewöhnlicher Ursache.

We report the case of a 19-year-old woman with abdominal pain and diarrhea. The diagnosis of acute pancreatitis could be made clinically and through laboratory tests. The cause was a duodenal duplication cyst in the area of the papilla, which was initially relieved endoscopically. Once the acute inflammation had healed, the cyst was resected endoscopically to prevent recurrence and the increased risk of malignancy. Duodenal duplication cysts in the papillary area are a very rare (congenital) cause of acute pancreatitis. If a cyst is present in the area of the duodenal wall, however, this differential diagnosis should be considered. Resection is indicated for therapy.

Limiting systemic endocrine overtreatment in postmenopausal breast cancer patients with an ultralow classification of the 70-gene signature.

PURPOSE: Guidelines recommend endocrine treatment for estrogen receptor-positive (ER+) breast cancers for up to 10 years. Earlier data suggest that the 70-gene signature (MammaPrint) has potential to select patients that have an excellent survival without chemotherapy and limited or no tamoxifen treatment. The aim was to validate the 70-gene signature ultralow-risk classification for endocrine therapy decision making. METHODS: In the IKA trial, postmenopausal patients with non-metastatic breast cancer had been randomized between no or limited adjuvant tamoxifen treatment without receiving chemotherapy. For this secondary analysis, FFPE tumor material was obtained of ER+HER2- patients with 0-3 positive lymph nodes and tested for the 70-gene signature. Distant recurrence-free interval (DRFI) long-term follow-up data were collected. Kaplan-Meier curves were used to estimate DRFI, stratified by lymph node status, for the three predefined 70-gene signature risk groups. RESULTS: A reliable 70-gene signature could be obtained for 135 patients. Of the node-negative and node-positive patients, respectively, 20% and 13% had an ultralow-risk classification. No DRFI events were observed for node-negative patients with an ultralow-risk score in the first 10 years. The 10-year DRFI was 90% and 66% in the low-risk (but not ultralow) and high-risk classified node-negative patients, respectively. CONCLUSION: These survival analyses indicate that the postmenopausal node-negative ER+HER2- patients with an ultralow-risk 70-gene signature score have an excellent 10-year DRFI after surgery with a median of 1 year of endocrine treatment. This is in line with published results of the STO-3-randomized clinical trial and supports the concept that it is possible to reduce the duration of endocrine treatment in selected patients.

Technical note: Automatic evaluation of infrared thermal images by computerized active shape modeling of bovine udders challenged with Escherichia coli.

Mastitis causes substantial economic losses and animal suffering in the dairy industry. The trend toward larger herd sizes complicates the monitoring of udder health in individual animals. Infrared thermography has successfully been used for early mastitis detection. However, manual thermogram analysis is time consuming and requires a skilled examiner, and automated image processing has not been tested. The aim of this study was to determine whether automatic evaluation of thermograms showed results comparable to those of manual evaluation of thermograms. Five healthy cows underwent an intramammary challenge with Escherichia coli to induce clinical mastitis. Multiple udder thermograms were taken every 2 h for 24 h before and after the challenge, resulting in 4,143 images in total. All images were evaluated using image recognition software (automatically) and a polygon tool (manually) to calculate the average and maximum surface temperatures. Because of the slightly different regions of interest, temperatures ascertained from the thermograms using the automatic method were consistently lower than those ascertained using the manual method. However, average udder surface temperatures evaluated using both methods were strongly correlated (r = 0.98 in the left hindquarter, and r = 0.99 in the right hindquarter) and showed maximum temperature peaks at the same time, 13 and 15 h after intramammary challenge. In the receiver operating characteristic analysis, both methods provided good results for sensitivity and specificity in detecting clinical E. coli-induced mastitis at different threshold values. For automatically evaluated maximum right hindquarter temperature, sensitivity was 93.75% and specificity was 94.96%, and for manually evaluated maximum right hindquarter temperature, sensitivity was 93.75% and specificity was 96.40%. Thus, automatic thermogram evaluation is a promising tool for automated mastitis detection.

Targeting ß-catenin dependent Wnt signaling via peptidomimetic inhibitors in murine chondrocytes and OA cartilage.

OBJECTIVE: The canonical Wnt signaling pathway has been shown to be involved in regulating chondrocyte hypertrophic differentiation during Osteoarthritis (OA). The aim of this study was to test the therapeutic potential of two stapled peptide canonical Wnt inhibitors - SAH-Bcl9 and StAx-35R - in preventing Wnt induced cartilage changes in OA. METHODS: Primary neonatal murine chondrocytes and cartilage explants from OA patients undergoing total joint replacement for knee OA, were used for microscopy to determine matrix and cell penetrating capacity of fluorescein isothiocyanate FITC-tagged SAH-Bcl9 and StAx-35R peptides. T cell factor/lymphoid enhancer-binding factor (TCF/LEF) reporter assays were used to monitor the inhibition of Wnt3a induced ß-catenin signaling by each peptide. Changes in chondrocyte phenotypic marker gene expression were analyzed by qRT PCR. RESULTS: Both peptides localized intercellular in primary murine chondrocytes and cartilage explants. They inhibited Wnt3a induced TCF/LEF promoter activity in primary murine chondrocytes. Both inhibitors did not rescue Wnt3a altered expression of chondrocyte phenotypic genes (Sox9, Col2a1, Acan) and hypertrophy marker gene (Col10a1) at high doses (100 ng/ml). Upon application of 10 ng/ml Wnt3a, StAx-35R partially reversed the Wnt effect on Sox9 and Col2a1 gene expression. Both peptides, however, reversed the downregulation of SOX9 and aggrecan (ACAN), and decrease of COL10A1 gene expression in preserved human OA cartilage explants. CONCLUSION: These data indicate that blockade of canonical Wnt signaling might be a therapeutic strategy to treat early OA cases and protect further cartilage degradation by preventing chondrocyte hypertrophic differentiation.

Immunohistochemical versus molecular (BluePrint and MammaPrint) subtyping of breast carcinoma. Outcome results from the EORTC 10041/BIG 3-04 MINDACT trial.

PURPOSE: This study compares immunohistochemical (IHC) versus molecular subtyping (BluePrint and MammaPrint) in the population of patients enrolled in MINDACT and outcome based on molecular subtyping (MS) versus surrogate pathological subtyping (PS) as defined by the 2013 St. Gallen guidelines. METHODS: MS classified patients in the following subtypes: Luminal A, Luminal B, HER-2-, and Basal-type. IHC/FISH for pathological subtyping (ER, PgR, HER-2, and Ki67) was centrally assessed in the European Institute of Oncology (n = 5806). Hazard ratios for distant-metastasis-free survival (DMFS) by subtype were adjusted for chemotherapy and endocrine therapy administration and thus independent of adjuvant treatment allocation. RESULTS: PS Luminal cancers classified as HER-2+ or Basal-type by MS did not have a significantly lower DMFS than the Luminal-type cancers by MS (95.9%): HR = 1.40, 95% CI 0.75-2.60 (p = 0.294). More patients were identified with Luminal A disease by MS (63%) as compared with PS (47%) with comparable 5-year DMFS (≥96.0%). Among the 500 patients with PS TN cancers, MS identified 24 (5%) patients as Luminal-type with 5-year DMFS estimated at 100% versus 71.4% for MS HER-2+ or 90.1% for MS Basal-type. CONCLUSIONS: MS was able to re-stratify 54% of patients with a Luminal-B PS subtype to a low-risk Luminal A-type group with comparable outcome. Among TN EBC, 5% were classified as Luminal by MS with Luminal-like outcome. Molecular classification can help to identify a larger group of patients with low risk of recurrence compared with the more contemporarily used classification methodology including high-quality assessed Ki67.

Characterisation of multifocal breast cancer using the 70-gene signature in clinical low-risk patients enrolled in the EORTC 10041/BIG 03-04 MINDACT trial.

BACKGROUND: In multifocal breast cancer, guidelines recommend basing adjuvant systemic treatment decisions on characteristics of the largest lesion, disregarding multifocality as an independent prognosticator. We assessed the association between multifocal disease and both the 70-gene signature (70-GS), and distant metastasis-free survival (DMFS) in clinical low-risk breast cancer patients enrolled in the European Organisation for Research and Treatment of Cancer 10041/BIG 03-04 Microarray In Node-negative and 1 to 3 positive lymph node Disease may Avoid ChemoTherapy (MINDACT) trial. PATIENTS AND METHODS: The analysed population consisted of enrolled patients in the MINDACT trial with clinical low-risk disease, defined by a modified Adjuvant! Online cut-off for the 10-year risk of recurrent disease or death. Eligibility criteria of MINDACT dictate that patients with multifocal disease could be included if the different lesions had similar pathological characteristics. The presence of multifocal disease was deducted from the case report form (CRF)-question for sum of diameter for all invasive tumour foci. Clinicopathological characteristics and gene expression of patients with unifocal and multifocal (largest lesion) disease were compared. Subsequently, the association between multifocal disease and the 70-GS was evaluated as well as the association between multifocality and 5-year DMFS. RESULTS: The study included 3090 clinical low-risk patients with unifocal and 238 patients with multifocal disease. Apart from a higher prevalence of lobular tumours (21.8% versus 10.8%, by local pathology), we did not observe differences in baseline characteristics between multifocal and unifocal tumours. Patients with multifocal tumours were more likely to be at high genomic risk as compared to patients with unifocal tumours (22.7% versus 17.3%, odds ratio [OR] 1.45, 95% confidence interval [CI] 1.02-2.07, P = 0.038). We did not find a significant association between tumour focality and DMFS (97.1% for unifocal versus 96.9% for multifocal, hazard ratio [HR] = 1.55, 95% CI 0.68-3.46, P = 0.172), nor a signal for a potential interaction between the prognostic effect of the 70-GS and focality of the tumour regarding DMFS. CONCLUSION: In the group of clinical low-risk MINDACT patients, multifocal tumours were more likely to have a high-risk 70-GS profile compared to unifocal tumours. We did not observe a significant interaction between multifocality and the 70-GS with respect to survival without distant metastasis in these patients.

MRI-imaging and clinical findings of eleven children with tick-borne encephalitis and review of the literature.

OBJECTIVES: The incidence of tick-borne encephalitis (TBE) is increasing in many countries. Magnetic resonance imaging (MRI) in the course of TBE is not regularly performed in children. The aim of our study was evaluating MRI-findings of children and adolescents with TBE. PATIENTS AND METHODS: Retrospective evaluation of the charts and MRIs of patients who had been treated for TBE in the four participating hospitals in the last twenty years. RESULTS: 11 patients (5 male; age at TBE 3 weeks-15 9/12 years; mean 104.9 months) were included. MRI (within the first week after admission) revealed symmetric or asymmetric T2-hyperintensities in both thalami in 7/11 patients with additional bilateral lesions in putamen and/or caudate nucleus in 3 patients, and additional cortical lesions in 2 patients. Our youngest patient presented with T2-hyperintensities affecting the whole left cerebral hemisphere including white and grey matter and both cerebellar hemispheres. One patient had a minimal reversible T2-hyperintensity in the splenium of the corpus callosum (RHSCC). 3/11 patients had a normal MRI. 4/11 patients showed complete neurological recovery (2/4 with a normal MRI, RHSCC patient). 6/11 children survived with significant sequelae: hemiparesis (n = 4); cognitive deficits (n = 4); pharmacoresistant epilepsy (n = 2). One patient died of a malignant brain edema. DISCUSSION: A spectrum of MRI findings can be found in children with TBE, often showing involvement of the subcortical deep grey matter structures. In children presenting with a meningoencephalitis and bilateral thalamic involvement TBE should be included in the differential diagnosis.

High concordance of protein (by IHC), gene (by FISH; HER2 only), and microarray readout (by TargetPrint) of ER, PgR, and HER2: results from the EORTC 10041/BIG 03-04 MINDACT trial.

BACKGROUND: To investigate the correlation of TargetPrint with local and central immunohistochemistry/fluorescence in situ hybridization assessment of estrogen (ER), progesterone (PgR), and human epidermal growth factor receptor 2 (HER2) in the first 800 patients enrolled in the MINDACT trial. PATIENTS AND METHODS: Data from local (N = 800) and central (N = 626) assessments of receptor status were collected and compared with TargetPrint results. RESULTS: For ER, the positive agreement (the percentage of central pathology positive assessments that were also TargetPrint/local laboratory positive) for TargetPrint in comparison to centralized assessment was 98% with a negative agreement (the percentage of central pathology negative assessments that were also TargetPrint/local laboratory negative) of 96%. For PgR, the positive agreement was 83% with a negative agreement of 92%. For HER2, the positive agreement was 75% with a negative agreement of 99%. Even though the local assessment showed higher positive agreement for PgR (89%) and higher positive agreement for HER2 (85%), the range of discordant local versus central assessments were as high as 6.7% for ER, 12.9% for PgR, and 4.3% for HER2. CONCLUSION: TargetPrint and local assessment of ER, PgR, and HER2 show high concordance with central assessment in the first 800 MINDACT patients. However, there are concerns about the higher discordance rates for some local sites. TargetPrint can improve the reliability of hormone receptor and HER2 testing for those centers with a lower rate of concordance with the reference laboratory, with the limitation of a positive agreement of 75% for HER2. TargetPrint consequently has important implications for treatment decisions in clinical practice and is a reliable alternative to local assessment for ER. CLINICAL TRIALS NUMBER: NCT00433589.

Additional prognostic value of the 70-gene signature (MammaPrint(®)) among breast cancer patients with 4-9 positive lymph nodes.

BACKGROUND: The 70 gene-signature (MammaPrint(®)) is a prognostic profile of distant recurrence and survival of primary breast cancer (BC). BC patients with 4-9 positive nodes (LN 4-9) are considered clinically at high-risk. Herein we examined MammaPrint(®) added prognostic value in this group. PATIENTS AND METHODS: MammaPrint(®) profiles were generated from frozen tumours of patients operated from primary BC. Samples were classified as genomic Low Risk (GLR) or genomic High Risk (GHR). RESULTS: Among the 173 samples, 70 (40%) were classified as GLR and 103 (60%) as GHR. Tumours in the GHR group were significantly more often ductal carcinomas (93%), grade 3 (60%), oestrogen and progesterone-negative, Her2 positive (25%). In the GLR category, the 5-year overall survival was 97% vs. 76% for in the GHR group (p < 0.01); Distant Metastasis Free Survival (DMFS) at 5 years was 87% for GLR patients and 63% for GHR patients (p < 0.01). In the Luminal A subgroup, the genomic profile was the only independent risk factor for DM and BC specific death. CONCLUSION: In the Luminal A subgroup, MammaPrint(®) is an independent prognostic marker in BC patients with LN 4-9 and may be integrated in a selection strategy of patients candidate for more aggressive therapeutic approaches.

The 70-gene prognosis signature predicts early metastasis in breast cancer patients between 55 and 70 years of age.

BACKGROUND: The majority of breast cancer patients are postmenopausal women who are increasingly being offered adjuvant chemotherapy. Since the beneficial effect of chemotherapy in postmenopausal patients predominantly occurs in the first 5 years after diagnosis, a prognostic marker for early events can be of use for adjuvant treatment decision making. The aim of this study was to evaluate the prognostic value of the 70-gene prognosis signature for early events in postmenopausal patients. METHODS: Frozen tumor samples from 148 patients aged 55-70 years were selected (T1-2, N0) and classified by the 70-gene prognosis signature (MammaPrint) into good or poor prognosis. Eighteen percent received hormonal therapy. RESULTS: Breast cancer-specific survival (BCSS) at 5 years was 99% for the good-prognosis signature versus 80% for the poor-prognosis signature group (P = 0.036). The 70-gene prognosis signature was a significant and independent predictor of BCCS during the first 5 years of follow-up with an adjusted hazard ratio of 14.4 (95% confidence interval 1.7-122.2; P = 0.01) at 5 years. CONCLUSION: The 70-gene prognosis signature can accurately select postmenopausal patients at low risk of breast cancer-related death within 5 years of diagnosis and can be of clinical use in selecting postmenopausal women for adjuvant chemotherapy.

Validation of 70-gene prognosis signature in node-negative breast cancer.

PURPOSE: The 70-gene prognosis signature (van't Veer et al., Nature 415(6871):530-536, 2002) may improve the selection of lymph node-negative breast cancer patients for adjuvant systemic therapy. Optimal validation of prognostic classifiers is of great importance and we therefore wished to evaluate the prognostic value of the 70-gene prognosis signature in a series of relatively recently diagnosed lymph node negative breast cancer patients. METHODS: We evaluated the 70-gene prognosis signature in an independent representative series of patients with invasive breast cancer (N = 123; <55 years; pT1-2N0; diagnosed between 1996 and 1999; median follow-up 5.8 years) by classifying these patients as having a good or poor prognosis signature. In addition, we updated the follow-up of the node-negative patients of the previously published validation-series (Van de Vijver et al., N Engl J Med 347(25):1999-2009, 2002; N = 151; median follow-up 10.2 years). The prognostic value of the 70-gene prognosis signature was compared with that of four commonly used clinicopathological risk indexes. The endpoints were distant metastasis (as first event) free percentage (DMFP) and overall survival (OS). RESULTS: The 5-year OS was 82 +/- 5% in poor (48%) and 97 +/- 2% in good prognosis signature (52%) patients (HR 3.4; 95% CI 1.2-9.6; P = 0.021). The 5-years DMFP was 78 +/- 6% in poor and 98 +/- 2% in good prognosis signature patients (HR 5.7; 95% CI 1.6-20; P = 0.007). In the updated series (N = 151; 60% poor vs. 40% good), the 10-year OS was 51 +/- 5% and 94 +/- 3% (HR 10.7; 95% CI 3.9-30; P < 0.01), respectively. The DMFP was 50 +/- 6% in poor and 86 +/- 5% in good prognosis signature patients (HR 5.5; 95% CI 2.5-12; P < 0.01). In multivariate analysis, the prognosis signature was a strong independent prognostic factor in both series, outperforming the clinicopathological risk indexes. CONCLUSION: The 70-gene prognosis signature is also an independent prognostic factor in node-negative breast cancer patients for women diagnosed in recent years.

PRAME expression and clinical outcome of breast cancer.

The tumour antigen PReferentially expressed Antigen of MElanoma (PRAME) is expressed in a variety of malignancies, including breast cancer. We have analysed PRAME gene expression in relation to clinical outcome for 295 primary breast cancer patients. Kaplan-Meier survival curves show a correlation of PRAME expression levels with increased rates of distant metastases and decreased overall patient survival. This correlation existed both for the entire patient group (n=295) and for the subgroup of patients (n=185) who did not receive adjuvant chemotherapy. Multivariable analysis indicated that PRAME is an independent marker of shortened metastasis-free interval in patients who did not receive adjuvant chemotherapy. PRAME expression was associated with tumour grade and negative oestrogen receptor status. We conclude that PRAME expression is a prognostic marker for clinical outcome of breast cancer, independent of traditional clinicopathological markers.

Primary testicular diffuse large B-cell lymphomas have activated B-cell-like subtype characteristics.

Diffuse large B-cell lymphomas (DLBCLs) constitute a heterogeneous group of lymphomas in which germinal centre B-cell-like and activated B-cell-like subtypes can be discerned based on pathology, clinical presentation, and gene expression patterns. Testicular DLBCLs form an immune-privileged site-related subgroup of DLBCLs with an unfavourable prognosis. In the present study, cDNA microarray analysis, immunohistochemistry for CD10, Bcl6 and MUM1, and somatic hypermutation analysis of the immunoglobulin heavy chain gene rearrangements were used to determine the subtype of primary testicular DLBCL. Immunohistochemistry revealed 14/22 testicular DLBCLs with an activated B-cell-like immunophenotype and 8/22 with an ambiguous immunophenotype co-expressing CD10 and high levels of MUM1. cDNA microarray analysis of these 22 and four additional cases showed a uniform activated B-cell-like gene expression pattern for both immunophenotypes. Somatic hypermutation analysis of immunoglobulin heavy chain genes showed a very high mutation load in seven cases tested, but intraclonal heterogeneity was found at low level in only one of these cases. It is concluded that primary testicular DLBCLs have uniform activated B-cell-like subtype characteristics despite a number of cases showing an ambiguous immunophenotype.

No common denominator for breast cancer lymph node metastasis.

The axillary lymph node status is the most powerful prognostic factor for breast cancer patients to date. The molecular mechanisms that control lymph node metastasis, however, remain poorly understood. To define patterns of genes or gene regulatory pathways that drive breast cancer lymph node metastasis, we compared the gene expression profiles of 15 primary breast carcinomas and their matching lymph node metastases using microarrays. In general, primary breast carcinomas and lymph node metastases do not differ at the transcriptional level by a common subset of genes. No classifier or single gene discriminating the group of primary tumours from those of the lymph node metastases could be identified. Also, in a series of 295 breast tumours, no classifier predicting lymph node metastasis could be developed. However, subtle differences in the expression of genes involved in extracellular-matrix organisation and growth factor signalling are detected in individual pairs of matching primary and metastatic tumours. Surprisingly, however, different sets of these genes are either up- or downregulated in lymph node metastases. Our data suggest that breast carcinomas do not use a shared gene set to accomplish lymph node metastasis.

Surgical treatment of recurrent pleomorphic adenoma of the parotid gland: a clinical analysis of 52 patients.

BACKGROUND: Surgical management of recurrent pleomorphic adenoma of the parotid gland has a considerable risk of facial nerve injury and a high re-recurrence rate. To obtain more insight into this issue we evaluated our experiences. METHODS: Medical records and histologic material of all these patients (31 women and 21 men), who had been treated from 1976-1995 were reviewed. Median interval between initial treatment and commencement of recurrences was 3 (0.8-18) years. Last surgery consisted of parotidectomy in 48 patients (92%), including 19 (40%) total procedures and wide local excision with involved skin in four patients. RESULTS: At a median follow-up of 9 years, eight patients (8/52; 15%) had re-recurrences develop, including 4 of 21 patients (19%) after a previous parotidectomy (group I) and 4 of 31 patients (13%) without prior parotidectomy (group II). The chance of re-recurrence in a group of patients with a minimal follow-up of 10 years after salvage surgery was 17% (4 of 24). The risk of a new relapse was, respectively, 4% and 8% at 1 and 5 years after treatment of recurrent disease. Acceptable N.VII function was preserved in 45 of the 49 (92%) nerves at risk. The risk of N.VII injuy was higher and more serious in group I (29% vs 10% in group II). The function of four of the five (80%) reconstructed zygomaticotemporal branches of the N.VII was adequate. CONCLUSION: Surgical treatment of recurrent pleomorphic adenoma of the parotid gland, usually consisting of a parotidectomy with wide extent and eventually facial nerve reconstruction, demonstrates favorable results with acceptable morbidity. The risk of new relapse and N.VII injury was higher after previous parotidectomy.

B-cell-autonomous somatic mutation deficit following bone marrow transplant.

Hematopoietic stem cell transplantation is characterized by a prolonged period of humoral immunodeficiency. We have previously shown that the deficiencies are probably not due to the failure to utilize the appropriate V regions in the pre-immune repertoire. However, a striking observation, which correlated with the absence of immunoglobulin IgD(-) cells and was consistent with a defect in antigen-driven responses, was that rearrangements in bone marrow transplant (BMT) recipients exhibited much less somatic mutation than did rearrangements obtained from healthy subjects. In this paper, we present evidence suggesting that naive B cells obtained from BMT recipients lack the capacity to accumulate somatic mutations in a T-cell-dependent manner compared with healthy subjects. This appears to be a B-cell-autonomous deficit because T cells from some patients, which were not able to support the accumulation of mutations in autologous naive B cells, were able to support accumulation of mutations in heterologous healthy-subject naive B cells.

Motif-specific probes identify individual genes and detect somatic mutations.

This report describes the correlation between motif-specific hybridization and nucleotide sequence as an approach to the identification of individual human V(H) genes using motif-specific oligonucleotide probes, complementary to specific motifs within individual V(H) genes. The sensitivity of the hybridization and post washing processes permits discrimination of single nucleotide differences between probe and target. This feature is used both to identify individual genes, as well as to detect mutations in genes by sequential hybridization with multiple probes. In addition to the general strategy, specific details are provided for the identification of 12 V(H)3 genes and 14 V(H)4 genes.