Direct brain recordings suggest a causal subsequent-memory effect.

Endogenous variation in brain state and stimulus-specific evoked activity can both contribute to successful encoding. Previous studies, however, have not clearly distinguished among these components. We address this question by analysing intracranial EEG recorded from epilepsy patients as they studied and subsequently recalled lists of words. We first trained classifiers to predict recall of either single items or entire lists and found that both classifiers exhibited similar performance. We found that list-level classifier output-a biomarker of successful encoding-tracked item presentation and recall events, despite having no information about the trial structure. Across widespread brain regions, decreased low- and increased high-frequency activity (HFA) marked successful encoding of both items and lists. We found regional differences in the hippocampus and prefrontal cortex, where in the hippocampus HFA correlated more strongly with item recall, whereas, in the prefrontal cortex, HFA correlated more strongly with list performance. Despite subtle differences in item- and list-level features, the similarity in overall classification performance, spectral signatures of successful recall and fluctuations of spectral activity across the encoding period argue for a shared endogenous process that causally impacts the brain's ability to learn new information.

Refractory sphenoid wing osteomyelitis as a complication of orbital decompression.

Osteomyelitis of the sphenoid wing is a rare clinical entity that can result in significant morbidity due to cranio-orbital infection. This entity has never previously been described as a complication of orbital decompression. An elderly patient developed relapsing orbital cellulitis and cranio-orbital abscesses following orbital decompression. Multiple attempts at incision and drainage with extended antibiotic therapy failed to eliminate the infection. The patient's clinical course was indicative of sphenoid osteomyelitis at the prior decompression site. A combined craniotomy-orbitotomy with debridement of the involved portion of the sphenoid resulted in resolution of the patient's clinical symptoms. Surgeons should be aware of the possibility of this rare infection following any surgery in which bone is structurally modified or removed, including orbital decompression.

Stimulation better targets fast-ripple generating networks in super responders to the responsive neurostimulator system.

How responsive neurostimulation (RNS) decreases seizure frequency is unclear. Stimulation may alter epileptic networks during inter-ictal epochs. Definitions of the epileptic network vary but fast ripples (FRs) may be an important substrate. We, therefore, examined whether stimulation of FR-generating networks differed in RNS super responders and intermediate responders. In 10 patients, with subsequent RNS placement, we detected FRs from stereo-electroencephalography (SEEG) contacts during pre-surgical evaluation. The normalized coordinates of the SEEG contacts were compared with those of the eight RNS contacts, and RNS-stimulated SEEG contacts were defined as those within 1.5 cm3 of the RNS contacts. We compared the post-RNS placement seizure outcome to (1) the ratio of stimulated SEEG contacts in the seizure-onset zone (SOZ stimulation ratio [SR]); (2) the ratio of FR events on stimulated contacts (FR SR); and (3) the global efficiency of the FR temporal correlational network on stimulated contacts (FR SGe). We found that the SOZ SR (p = .18) and FR SR (p = .06) did not differ in the RNS super responders and intermediate responders, but the FR SGe did (p = .02). In super responders, highly active desynchronous sites of the FR network were stimulated. RNS that better targets FR networks, as compared to the SOZ, may reduce epileptogenicity more.

Temporal lobe interictal spikes disrupt encoding and retrieval of verbal memory: A subregion analysis.

OBJECTIVE: The medial temporal lobe (MTL) encodes and recalls memories and can be a predominant site for interictal spikes (IS) in patients with focal epilepsy. It is unclear whether memory deficits are due to IS in the MTL producing a transient decline. Here, we investigated whether IS in the MTL subregions and lateral temporal cortex impact episodic memory encoding and recall. METHODS: Seventy-eight participants undergoing presurgical evaluation for medically refractory focal epilepsy with depth electrodes placed in the temporal lobe participated in a verbal free recall task. IS were manually annotated during the pre-encoding, encoding, and recall epochs. We examined the effect of IS on word recall using mixed-effects logistic regression. RESULTS: IS in the left hippocampus (odds ratio [OR] = .73, 95% confidence interval [CI] = .63-.84, p < .001) and left middle temporal gyrus (OR = .46, 95% CI = .27-.78, p < .05) during word encoding decreased subsequent recall performance. Within the left hippocampus, this effect was specific for area CA1 (OR = .76, 95% CI = .66-.88, p < .01) and dentate gyrus (OR = .74, 95% CI = .62-.89, p < .05). IS in other MTL subregions or inferior and superior temporal gyrus and IS occurring during the prestimulus window did not affect word encoding (p > .05). IS during retrieval in right hippocampal (OR = .22, 95% CI = .08-.63, p = .01) and parahippocampal regions (OR = .24, 95% CI = .07-.8, p < .05) reduced the probability of recalling a word. SIGNIFICANCE: IS in medial and lateral temporal cortex contribute to transient memory decline during verbal episodic memory.

Contribution of left supramarginal and angular gyri to episodic memory encoding: An intracranial EEG study.

The role of the left ventral lateral parietal cortex (VPC) in episodic memory is hypothesized to include bottom-up attentional orienting to recalled items, according to the dual-attention model (Cabeza et al., 2008). However, its role in memory encoding could be further clarified, with studies showing both positive and negative subsequent memory effects (SMEs). Furthermore, few studies have compared the relative contributions of sub-regions in this functionally heterogeneous area, specifically the anterior VPC (supramarginal gyrus/BA40) and the posterior VPC (angular gyrus/BA39), on a within-subject basis. To elucidate the role of the VPC in episodic encoding, we compared SMEs in the intracranial EEG across multiple frequency bands in the supramarginal gyrus (SmG) and angular gyrus (AnG), as twenty-four epilepsy patients with indwelling electrodes performed a free recall task. We found a significant SME of decreased theta power and increased high gamma power in the VPC overall, and specifically in the SmG. Furthermore, SmG exhibited significantly greater spectral tilt SME from 0.5 to 1.6 s post-stimulus, in which power spectra slope differences between recalled and unrecalled words were greater than in the AnG (p = 0.04). These results affirm the contribution of VPC to episodic memory encoding, and suggest an anterior-posterior dissociation within VPC with respect to its electrophysiological underpinnings.

In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies.

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

Sequence Variation Associated with SLC12A5 Gene Expression Is Linked to Brain Structure and Function in Healthy Adults.

A single-nucleotide polymorphism in the promoter region of the Matrix Metalloproteinase-9 (MMP9) gene, rs3918242, has been shown to affect MMP9 expression in macrophages and was associated with schizophrenia by two independent groups. However, rs3918242's effects on MMP9 expression were not replicable in cell lines or brain tissue. Additionally, publically available data indicate that rs3918242 genotype is related not to MMP9 expression, but rather to expression of SLC12A5, a nearby gene coding for a K+/Cl- cotransporter, whose expression has also been related to schizophrenia. Here, we studied brain structure and function in healthy participants stratified by rs3918242 genotype using structural MRI (N = 298), functional MRI during an N-back working memory task (N = 554), and magnetoencephalography (MEG) during the same task (N = 190). We found rs3918242 was associated with gray matter volume (GMV) in the insula and dorsolateral prefrontal cortex bilaterally, closely replicated in discovery and replication samples; and with inferior parietal lobule (IPL) GMV when the samples were meta-analytically combined. Additionally, using both fMRI and MEG, rs3918242 was associated with right IPL working memory-related activation, replicated in two cohorts and across imaging modalities. These convergent results provide further impetus for examinations of the relationship of SLC12A5 with brain structure and function in neuropsychiatric disease.

Intravascular papillary endothelial hyperplasia of the periocular region.

Intravascular papillary endothelial hyperplasia (IPEH) is a benign vascular lesion, typically found in the skin, consisting of a papillary proliferation of endothelial cells intimately related to a thrombus; it appears to be a rare variant of an organizing process. IPEH very rarely develops in the periocular region, with fewer than 20 such cases currently reported in the English language literature. We describe one of the largest ever case series of this uncommon lesion occurring in the periocular region and discuss its clinical features, imaging characteristics, and surgical management.

Clinical and Magnetic Resonance Imaging Characteristics of Postfenestration Optic Nerve Sheath Pseudomeningoceles.

PURPOSE: The clinical significance of postoperative pseudomeningocele formation following optic nerve sheath fenestration (ONSF) has not been fully characterized. A literature review identifies 9 previously published cases the authors believe demonstrate pseudomeningocele formation and approximately 19 other similar findings that were either transient or less defined blebs. This study was undertaken to more clearly define the clinical, radiographic, and histopathologic features associated with this entity. METHODS: Sixteen-year, single-center, retrospective chart review of all ONSF cases performed by 2 surgeons. Clinical data, intracranial pressure, radiographic imaging, and histopathology of clinically detected pseudomeningoceles after ONSF were reviewed. RESULTS: Eighty-six eyes in 57 patients underwent ONSF (28 unilateral, 12 bilateral sequential, 17 bilateral simultaneous). Forty-nine of 57 patients had elevated intracranial pressure preoperatively (41 idiopathic intracranial hypertension, 4 venous thrombosis, 2 meningitis, 1 arteriovenous malformation, and 1 sarcoid). In 32 patients undergoing postoperative imaging, 4 eyes (4.7%) in 4 patients developed well-defined pseudomeningoceles, of which 3 were symptomatic and 2 required surgical revision. Each pseudomeningocele developed in the setting of elevated preoperative intracranial pressure (350, 360, 430, 500 mm H20). Magnetic resonance imaging and/or computed tomography revealed sharply demarcated fluid-filled sacs adjacent to the optic nerve. The contents of these sacs were hypointense on T1-weighted imaging, hyperintense on T2-weighting, variably enhanced with contrast, and hypointense on fluid attenuated inversion recovery, and were thus consistent with cerebrospinal fluid. Histopathologic analysis of one of these outpouchings demonstrated an acellular, fibrocollagenized lining consistent with pseudomeningocele. Three eyes in 3 additional patients had less well-defined findings on imaging interpreted as bleb-like or cyst-like change. CONCLUSIONS: Pseudomeningoceles following ONSF may be asymptomatic or may cause symptomatic orbital mass effect and rarely visual loss, amendable to surgical excision. Post-ONSF pseudomeningoceles are identified on computed tomography or magnetic resonance imaging to occur at the locations of fenestration sites and contain cerebrospinal fluid communicating with the subdural space that may act as a "filtration" bleb in some cases. Imaging findings may represent a spectrum spanning intraorbital cerebrospinal fluid leakage, partial walling off of bleb, or fully developed cysts. Resection of optic nerve pseudomeningoceles is considered in symptomatic cysts or eyes with papilledema that fails to improve.

High-gamma and beta bursts in the Left Supramarginal Gyrus can accurately differentiate verbal memory states and performance.

The left supramarginal gyrus (LSMG) may mediate attention to memory, and gauge memory state and performance. We performed a secondary analysis of 142 verbal delayed free recall experiments, in patients with medically-refractory epilepsy with electrode contacts implanted in the LSMG. In 14 of 142 experiments (in 14 of 113 patients), the cross-validated convolutional neural networks (CNNs) that used 1-dimensional(1-D) pairs of convolved high-gamma and beta tensors, derived from the LSMG recordings, could label recalled words with an area under the receiver operating curve (AUROC) of greater than 60% [range: 60-90%]. These 14 patients were distinguished by: 1) higher amplitudes of high-gamma bursts; 2) distinct electrode placement within the LSMG; and 3) superior performance compared with a CNN that used a 1-D tensor of the broadband recordings in the LSMG. In a pilot study of 7 of these patients, we also cross-validated CNNs using paired 1-D convolved high-gamma and beta tensors, from the LSMG, to: a) distinguish word encoding epochs from free recall epochs [AUC 0.6-1]; and distinguish better performance from poor performance during delayed free recall [AUC 0.5-0.86]. These experiments show that bursts of high-gamma and beta generated in the LSMG are biomarkers of verbal memory state and performance.

Graph theoretical measures of fast ripple networks improve the accuracy of post-operative seizure outcome prediction.

Fast ripples (FR) are a biomarker of epileptogenic brain, but when larger portions of FR generating regions are resected seizure freedom is not always achieved. To evaluate and improve the diagnostic accuracy of FR resection for predicting seizure freedom we compared the FR resection ratio (RR) with FR network graph theoretical measures. In 23 patients FR were semi-automatically detected and quantified in stereo EEG recordings during sleep. MRI normalization and co-registration localized contacts and relation to resection margins. The number of FR, and graph theoretical measures, which were spatial (i.e., FR rate-distance radius) or temporal correlational (i.e., FR mutual information), were compared with the resection margins and with seizure outcome We found that the FR RR did not correlate with seizure-outcome (p > 0.05). In contrast, the FR rate-distance radius resected difference and the FR MI mean characteristic path length RR did correlate with seizure-outcome (p < 0.05). Retesting of positive FR RR patients using either FR rate-distance radius resected difference or the FR MI mean characteristic path length RR reduced seizure-free misclassifications from 44 to 22% and 17%, respectively. These results indicate that graph theoretical measures of FR networks can improve the diagnostic accuracy of the resection of FR events for predicting seizure freedom.

Spatiotemporal Alterations in Working Memory-Related Beta Band Neuromagnetic Activity of Patients With Schizophrenia On and Off Antipsychotic Medication: Investigation With MEG.

BACKGROUND AND HYPOTHESIS: We used the uniquely high combined spatial and temporal resolution of magnetoencephalography to characterize working memory (WM)-related modulation of beta band activity in neuroleptic-free patients with schizophrenia in comparison to a large sample of performance-matched healthy controls. We also tested for effects of antipsychotic medication on identified differences in these same patients. STUDY DESIGN: Inpatients with schizophrenia (n = 21) or psychotic disorder not otherwise specified (n = 4) completed N-back and control tasks during magnetoencephalography while on placebo and during antipsychotic medication treatment, in a blinded, randomized, counterbalanced manner. Healthy, performance-matched controls (N = 100) completed the same tasks. WM-related neural activation was estimated as beta band (14-30 Hz) desynchronization throughout the brain in successive 400 ms time windows. Voxel-wise statistical comparisons were performed between controls and patients while off-medication at each time window. Significant clusters resulting from this between-groups analysis were then used as regions-of-interest, the activations of which were compared between on- and off-medication conditions in patients. STUDY RESULTS: Controls showed beta-band desynchronization (activation) of a fronto-parietal network immediately preceding correct button press responses-the time associated with WM updating and task execution. Altered activation in medication-free patients occurred largely during this time, in prefrontal, parietal, and visual cortices. Medication altered patients' neural responses such that the activation time courses in these regions-of-interest more closely resembled those of controls. CONCLUSIONS: These findings demonstrate that WM-related beta band alterations in schizophrenia are time-specific and associated with neural systems targeted by antipsychotic medications. Future studies may investigate this association by examining its potential neurochemical basis.

Graph theoretical measures of fast ripples support the epileptic network hypothesis.

The epileptic network hypothesis and epileptogenic zone hypothesis are two theories of ictogenesis. The network hypothesis posits that coordinated activity among interconnected nodes produces seizures. The epileptogenic zone hypothesis posits that distinct regions are necessary and sufficient for seizure generation. High-frequency oscillations, and particularly fast ripples, are thought to be biomarkers of the epileptogenic zone. We sought to test these theories by comparing high-frequency oscillation rates and networks in surgical responders and non-responders, with no appreciable change in seizure frequency or severity, within a retrospective cohort of 48 patients implanted with stereo-EEG electrodes. We recorded inter-ictal activity during non-rapid eye movement sleep and semi-automatically detected and quantified high-frequency oscillations. Each electrode contact was localized in normalized coordinates. We found that the accuracy of seizure onset zone electrode contact classification using high-frequency oscillation rates was not significantly different in surgical responders and non-responders, suggesting that in non-responders the epileptogenic zone partially encompassed the seizure onset zone(s) (P > 0.05). We also found that in the responders, fast ripple on oscillations exhibited a higher spectral content in the seizure onset zone compared with the non-seizure onset zone (P < 1 × 10-5). By contrast, in the non-responders, fast ripple had a lower spectral content in the seizure onset zone (P < 1 × 10-5). We constructed two different networks of fast ripple with a spectral content >350 Hz. The first was a rate-distance network that multiplied the Euclidian distance between fast ripple-generating contacts by the average rate of fast ripple in the two contacts. The radius of the rate-distance network, which excluded seizure onset zone nodes, discriminated non-responders, including patients not offered resection or responsive neurostimulation due to diffuse multifocal onsets, with an accuracy of 0.77 [95% confidence interval (CI) 0.56-0.98]. The second fast ripple network was constructed using the mutual information between the timing of the events to measure functional connectivity. For most non-responders, this network had a longer characteristic path length, lower mean local efficiency in the non-seizure onset zone, and a higher nodal strength among non-seizure onset zone nodes relative to seizure onset zone nodes. The graphical theoretical measures from the rate-distance and mutual information networks of 22 non- responsive neurostimulation treated patients was used to train a support vector machine, which when tested on 13 distinct patients classified non-responders with an accuracy of 0.92 (95% CI 0.75-1). These results indicate patients who do not respond to surgery or those not selected for resection or responsive neurostimulation can be explained by the epileptic network hypothesis that is a decentralized network consisting of widely distributed, hyperexcitable fast ripple-generating nodes.

Accuracy of high-frequency oscillations recorded intraoperatively for classification of epileptogenic regions.

To see whether acute intraoperative recordings using stereo EEG (SEEG) electrodes can replace prolonged interictal intracranial EEG (iEEG) recording, making the process more efficient and safer, 10 min of iEEG were recorded following electrode implantation in 16 anesthetized patients, and 1-2 days later during non-rapid eye movement (REM) sleep. Ripples on oscillations (RonO, 80-250 Hz), ripples on spikes (RonS), sharp-spikes, fast RonO (fRonO, 250-600 Hz), and fast RonS (fRonS) were semi-automatically detected. HFO power and frequency were compared between the conditions using a generalized linear mixed-effects model. HFO rates were compared using a two-way repeated measures ANOVA with anesthesia type and SOZ as factors. A receiver-operating characteristic (ROC) curve analysis quantified seizure onset zone (SOZ) classification accuracy, and the scalar product was used to assess spatial reliability. Resection of contacts with the highest rate of events was compared with outcome. During sleep, all HFOs, except fRonO, were larger in amplitude compared to intraoperatively (p < 0.01). HFO frequency was also affected (p < 0.01). Anesthesia selection affected HFO and sharp-spike rates. In both conditions combined, sharp-spikes and all HFO subtypes were increased in the SOZ (p < 0.01). However, the increases were larger during the sleep recordings (p < 0.05). The area under the ROC curves for SOZ classification were significantly smaller for intraoperative sharp-spikes, fRonO, and fRonS rates (p < 0.05). HFOs and spikes were only significantly spatially reliable for a subset of the patients (p < 0.05). A failure to resect fRonO areas in the sleep recordings trended the most sensitive and accurate for predicting failure. In summary, HFO morphology is altered by anesthesia. Intraoperative SEEG recordings exhibit increased rates of HFOs in the SOZ, but their spatial distribution can differ from sleep recordings. Recording these biomarkers during non-REM sleep offers a more accurate delineation of the SOZ and possibly the epileptogenic zone.

The MUC1 oncoprotein as a functional target: immunotoxin binding to alpha/beta junction mediates cell killing.

MUC1, a heavily glycosylated mucin, has generated considerable interest as a target for tumor killing because of its overexpression in malignancies. Full-length MUC1 (MUC1/TM) is proteolytically cleaved after synthesis generating alpha and beta subunits, which specifically bind in a noncovalent interaction. Although the beta chain remains on the cell surface, the alpha chain binds in an on-and-off interaction. Most anti-MUC1 antibodies (Abs) described to date recognize epitopes within the highly immunogenic alpha-chain tandem repeat. Because the alpha-chain is shed, such Abs are sequestered and fail to reach MUC1-expressing cells. Immunizing with cDNA encoding MUC1/TM and the spliced MUC1/X isoform from which the tandem repeat has been deleted yielded antibodies to the MUC1 alpha/beta junction. Pseudomonas toxin PE38 linked to polyclonal anti-MUC1 alpha/beta junction Abs both bound and killed MUC1-positive malignant cells. Monoclonal DMC209 binds the MUC1 alpha/beta junction in both MUC1/X and MUC1/TM. When injected into SCID mice xenotransplanted with human breast cancer MDA-MB-231, monoclonal DMC209 showed significant in vivo tumor-suppressive activity. The MUC1/X alpha/beta junction presents a biologically-significant target in MUC1-expressing malignancies because (i) antibodies directed against cell-bound alpha/beta junction epitopes reach the intended cellular target, (ii) antibodies to junction epitope are internalized into cells, (iii) anti alpha/beta junction antibodies can effectively kill high MUC1-expressing cancer cells as antibody-toxin conjugates and (iv) antibodies targeting the MUC1 cell-bound alpha/beta junction results in tumor suppression in vivo. Our results indicate that cell-bound MUC1 alpha/beta junction, unlike shed alpha chain, represents a highly effective moiety for targeting and killing MUC1-expressing malignancies.

Targeting cell-bound MUC1 on myelomonocytic, monocytic leukemias and phenotypically defined leukemic stem cells with anti-SEA module antibodies.

Cell surface molecules aberrantly expressed or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1 is a polymorphic glycoprotein, the cleavage of which yields two unequal chains: a large extracellular α subunit containing a tandem repeat array bound in a strong noncovalent interaction to a smaller ß subunit containing the transmembrane and cytoplasmic domains. Because the α-chain can be released from the cell-bound domains of MUC1, agents directed against the α-chain will not effectively target MUC1+ cells. The MUC1 SEA (a highly conserved protein module so called from its initial identification in a sea urchin sperm protein, in enterokinase, and in agrin) domain formed by the binding of the α and ß chains  represents a stable structure fixed to the cell surface at all times. DMB-5F3, a partially humanized murine anti-MUC1 SEA domain monoclonal antibody, was used to examine MUC1 expression in acute myeloid leukemia (AML) and was found to bind acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5) cell lines. We also examined monocytic neoplasms freshly obtained from patients including chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, which were found to uniformly express MUC1. CD34+/lin-/CD38- or CD38+ presumed leukemic stem cell populations from CD34+ AML and CD34-CD38- or CD38+ populations from CD34- AML were also found to express MUC1, although at low percentages. Based on these studies, we generated an anti-MUC1 immunotoxin to directly gauge the cytotoxic efficacy of targeting AML-bound MUC1. Using single-chain DMB-5F3 fused to recombinant gelonin toxin, the degree of AML cytotoxicity was found to correlate with MUC1 expression. Our data support the use of an anti-MUC1 SEA module-drug conjugates to selectively target and inhibit MUC1-expressing myelomonocytic leukemic cells.

MUC1/X protein immunization enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target malignant cells.

MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most antibodies against MUC1 recognize epitopes within the highly immunogenic alpha chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 alpha chain is shed into the peripheral circulation, sequesters circulating antitandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recognizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 alpha subunit binding the membrane-tethered beta subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding full-length MUC1 (MUC1/TM) followed by boosting with the alternatively spliced MUC1/X isoform from which the tandem repeat array has been deleted, we generated monoclonal antibodies, designated DMC209, which specifically bind the MUC1 alpha/beta junction. DMC209 is exquisitely unique for this site; amino acid mutations, which abrogate MUC1 cleavage, also abrogate DMC209 binding. Additionally, DMC209 specifically binds the MUC1 alpha/beta junction on full-length MUC1/TM expressed by breast and ovarian cancer cell lines and on freshly obtained, unmanipulated MUC1-positive malignant plasma cells of multiple myeloma. DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly and as an immunotoxin conjugate. Moreover, the novel immunization procedure used in generating DMC209 can be used to generate additional anti-MUC1 alpha/beta junction antibodies, which may, analogously to Herceptin, have cytotoxic activity. Lastly, sequential immunization with MUC1/TM cDNA acting as a nonspecific adjuvant followed by protein of interest may prove to be a generalizable method to yield high-titer specific antibodies.

Overexpression of DNA-binding protein B gene product in breast cancer as detected by in vitro-generated combinatorial human immunoglobulin libraries.

Molecules differentially expressed or overexpressed by malignant cells can serve in detecting and tracking of tumor. Additionally, they potentially can be applied in histologic-specific antitumor therapy. Few breast cancer-associated candidate molecules have been identified. Here we describe the use of combinatorial immunoglobulin [antigen-binding fragment of immunoglobulin molecule (Fab) fragment] phage libraries generated from patients with breast carcinoma to identify cancer-associated gene expression. The libraries were enriched for tumor-binding Fab by 3 logs and yielded a group of antibodies against DNA-binding protein B (DbpB), a 35-kDa thrombin-inducible nuclear factor and member of the Y-box family of proteins, which are known to act both negatively in selective gene suppression and positively as promoters of gene transcription. Sequencing of the anti-DbpB showed a degree of heterogeneity and bp substitutions suggesting that the Fabs selected from the combinatorial library represented a varied anti-DbpB immune response and did not simply arise from in vitro amplification by PCR of a single or limited numbers of immunoglobulin genes. Sequencing of the DbpB molecule expressed in malignant breast cancer showed no evidence of tumor-specific mutations. Evaluation of levels of DbpB gene product expression however showed the molecule to be constitutively expressed in normal nonmalignant breast tissues but to have consistently differentially higher expression in breast cancer. Immunohistological staining revealed DbpB to be present both intracellularly and on the cell surface, which suggests it may be a means whereby malignant cells repair and replicate DNA in a selectively advantageous manner as compared with nonmalignant cells. DbpB expression in breast cancer may advance the basic understanding of the role of Y-box binding proteins as regulatory agents, and in defining malignant cell phenotypes. In addition, DbpB and the antibodies generated against it may have direct application in tumor detection and in molecule-targeted immunotherapy.