Phase 1 study of twice weekly pulse dose and daily low-dose erlotinib as initial treatment for patients with EGFR-mutant lung cancers.

Background: Patients with EGFR-mutant lung cancers treated with EGFR tyrosine kinase inhibitors (TKIs) develop clinical resistance, most commonly with acquisition of EGFR T790M. Evolutionary modeling suggests that a schedule of twice weekly pulse and daily low-dose erlotinib may delay emergence of EGFR T790M. Pulse dose erlotinib has superior central nervous system (CNS) penetration and may result in superior CNS disease control. Methods: We evaluated toxicity, pharmacokinetics, and efficacy of twice weekly pulse and daily low-dose erlotinib. We assessed six escalating pulse doses of erlotinib. Results: We enrolled 34 patients; 11 patients (32%) had brain metastases at study entry. We observed 3 dose-limiting toxicities in dose escalation: transaminitis, mucositis, and rash. The MTD was erlotinib 1200 mg days 1-2 and 50 mg days 3-7 weekly. The most frequent toxicities (any grade) were rash, diarrhea, nausea, fatigue, and mucositis. 1 complete and 24 partial responses were observed (74%, 95% CI 60-84%). Median progression-free survival was 9.9 months (95% CI 5.8-15.4 months). No patient had progression of an untreated CNS metastasis or developed a new CNS lesion while on study (0%, 95% CI 0-13%). Of the 18 patients with biopsies at progression, EGFR T790M was identified in 78% (95% CI 54-91%). Conclusion: This is the first clinical implementation of an anti-cancer TKI regimen combining pulse and daily low-dose administration. This evolutionary modeling-based dosing schedule was well-tolerated but did not improve progression-free survival or prevent emergence of EGFR T790M, likely due to insufficient peak serum concentrations of erlotinib. This dosing schedule prevented progression of untreated or any new central nervous system metastases in all patients.

Nomogram to predict the presence of EGFR activating mutation in lung adenocarcinoma.

Epidermal growth factor receptor (EGFR) tumour genotyping is crucial to guide treatment decisions regarding the use of EGFR tyrosine kinase inhibitors in nonsmall cell lung cancer (NSCLC). However, some patients may not be able to obtain tumour testing, either because tissue is limited and/or tests are not routinely offered. Here, we aimed to build a model-based nomogram to allow for prediction of the presence of EGFR mutations in NSCLC. We retrospectively collected clinical and pathological data on 3,006 patients with NSCLC who had their tumours genotyped for EGFR mutations at five institutions worldwide. Variables of interest were integrated in a multivariate logistic regression model. In the 2,392 non-Asian patients with lung adenocarcinomas, the most important predictors of harbouring EGFR mutation were: lower tobacco smoking exposure (OR 0.41, 95% CI 0.37-0.46), longer time interval between smoking cessation and diagnosis (OR 2.19, 95% CI 1.71-2.80), advanced stage (OR 1.58, 95% CI 1.18-2.13), and papillary (OR 4.57, 95% CI 3.14-6.66) or bronchioloalveolar (OR 2.84, 95% CI 1.98-4.06) histologically predominant subtype. A nomogram was established and showed excellent discriminating accuracy: the concordance index on an independent validation dataset was 0.84. As clinical practices transition to incorporating genotyping as part of routine care, this nomogram could be highly useful to predict the presence of EGFR mutations in lung adenocarcinoma in non-Asian patients when mutational profiling is not available or possible.

Inclusion of RBD improves the diagnostic classification of dementia with Lewy bodies.

OBJECTIVE: To determine whether adding REM sleep behavior disorder (RBD) to the dementia with Lewy bodies (DLB) diagnostic criteria improves classification accuracy of autopsy-confirmed DLB. METHODS: We followed 234 consecutive patients with dementia until autopsy with a mean of 4 annual visits. Clinical diagnoses included DLB, Alzheimer disease (AD), corticobasal syndrome, and frontotemporal dementia. Pathologic diagnoses used the 2005 DLB consensus criteria and included no/low likelihood DLB (non-DLB; n = 136) and intermediate/high likelihood DLB (DLB; n = 98). Regression modeling and sensitivity/specificity analyses were used to evaluate the diagnostic role of RBD. RESULTS: Each of the 3 core features increased the odds of autopsy-confirmed DLB up to 2-fold, and RBD increased the odds by 6-fold. When clinically probable DLB reflected dementia and 2 or more of the 3 core features, sensitivity was 85%, and specificity was 73%. When RBD was added and clinically probable DLB reflected 2 or more of 4 features, sensitivity improved to 88%. When dementia and RBD were also designated as probable DLB, sensitivity increased to 90% while specificity remained at 73%. The VH, parkinsonism, RBD model lowered sensitivity to 83%, but improved specificity to 85%. CONCLUSIONS: Inclusion of RBD as a core clinical feature improves the diagnostic accuracy of autopsy-confirmed DLB.

An integrated genomic analysis of lung cancer reveals loss of DUSP4 in EGFR-mutant tumors.

To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This showed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with underexpression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling, and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of a reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers.

Oncogenes come of age.

Mutations of proto-oncogenes are common events in the pathogenesis of cancers, as shown in a wide range of studies during the 30 years since the discovery of these genes. The benefits of novel therapies that target the products of mutant alleles in human cancers, and the demonstrated dependence of cancers in mouse models on continued expression of initiating oncogenes, are especially promising signs that revolutionary improvements in cancer care are possible. Full realization of the promise of targeted therapies, however, will require better definitions of the genotypes of human cancers, new approaches to interrupt the biochemical consequences of oncogenic mutations, and a greater understanding of drug resistance and tumor progression. In this paper, we summarize recent efforts toward these goals in our laboratory and others.

Single vs. dual vessel porcine extracorporeal liver perfusion.

BACKGROUND: The use of porcine extracorporeal liver perfusion (PECLP) to provide temporary hepatic support for patients in fulminant hepatic failure has been limited by the fact that individual perfusions can be sustained for only a few hours. Inadequate liver function and/or hemodynamic instability are the major contributing factors for early interruption of PECLP. Recent reports suggest that the choice of single (portal vein only) vs dual (portal vein and hepatic artery) vessel perfusion may influence the duration of perfusion. We hypothesize that PECLP with single vessel perfusion (SVP) is associated with worse liver function and greater hemodynamic instability than PECLP with dual vessel perfusion (DVP). MATERIALS AND METHODS: To eliminate the potentially confounding influences of liver failure and xenograft rejection, liver isografts procured from White-Landrace pig donors were perfused by either SVP or DVP via an extracorporeal circuit established with normal White-Landrace pig recipients. The function of perfused livers was evaluated by measuring production of bile and Factors V and VIII, clearance of ammonia and lactate, and extraction of O(2) at baseline and at 0, 1, 3, 6, 12, and 24 h after initiation of PECLP. The impact of PECLP on recipient hemodynamic status was assessed by monitoring BP, heart rate, urine output, O(2) saturation, etc. Among other parameters evaluated were serum albumin and total protein and hepatic release of IL-1beta and nitric oxide to assess their possible contributions to hemodynamic instability. RESULTS: DVP and SVP livers cleared ammonia and lactate similarly. Both approaches were associated with progressive hypoalbuminemia and hypoproteinemia. DVP livers produced more bile and Factor V and were associated with less recipient hypotension and IL-1beta and NO release than SVP livers. CONCLUSIONS: Livers with DVP function better than livers with SVP. The duration of PECLP can be limited by recipient hypotension, although this complication is less severe with DVP than with SVP.

Epidural abscess and vertebral osteomyelitis caused by Listeria monocytogenes: case report and literature review.

A 69-y-old male with an epidural abscess and concomitant vertebral osteomyelitis caused by Listeria monocytogenes is presented, together with a brief review of the literature. To our knowledge, no cases of epidural abscess and only 3 cases of osteomyelitis have previously been reported in association with this organism.

Simple purification methods for an alphagalactose-specific antibody from chicken eggs.

For many applications avian antibody from egg yolk (IgY) offers advantages over the well-known mammalian antibodies. Different experimental techniques for the purification of IgY from chickens immunized with an alphagalactose-containing antigen (alphaGal-trisaccharide) were compared. These included ammonium sulfate precipitation, filtration with diatomaceous earth, treatment with deoxycholate, and thiophilic and affinity chromatography. Samples were tested for overall purity, protein and lipid content, and specific activity. Evaluated on the basis of these results and the simplicity of the process, the favored purification method is ammonium sulfate precipitation of diluted egg yolk directly followed by affinity chromatography. The high lipid content of IgY preparations is greatly reduced by either thiophilic or affinity chromatography. Affinity purification of ammonium sulfate precipitated material resulted in anti-alphaGal-trisaccharide IgY preparations with approximately 1% of the original protein content but approximately 100-fold higher specific activity for the alphaGal-trisaccharide epitope.

Distribution of, and immune response to, chicken anti-alpha Gal immunoglobulin Y antibodies in wild-type and alpha Gal knockout mice.

Chicken antibodies (immunoglobulin Y; IgY) to the alpha Gal epitope (galactose alpha-1,3-galactose) bind to alpha Gal antigens of mouse and porcine tissues and endothelial cells in vitro and block human anti-alpha Gal antibody binding, complement activation and antibody-dependent cell-mediated lysis mechanisms. The activities and toxicity of anti-alpha Gal IgY have not been tested in vivo. In this study, we tested the effects of multiple injections of affinity-purified anti-alpha Gal IgY (AP-IgY) in both wild-type (WT) and alpha-1,3-galactosyltransferase knockout (Gal KO) mice. WT and Gal KO mice were injected once, twice, three, or four times intravenously (i.v.) with AP-IgY and killed at 1 hr or 24 hr. Mice displayed no toxicity to four injections of AP-IgY. Heart, lung, liver, kidney, spleen and pancreatic tissue were evaluated using immunohistochemical techniques for the presence of the alpha Gal epitope using the GSI-B4 lectin, and for bound IgY, as well as mouse IgM and IgG. The binding of AP-IgY antibodies to the endothelium of WT mouse tissues was essentially identical to the pattern of binding of the GSI-B4 lectin after injection of WT mice and death at 1 hr. WT mice killed 24 hr after i.v. injection of AP-IgY showed little remaining bound IgY in their endothelia, indicating that IgY is cleared over that time period. We also evaluated the blood drawn at the time of death for the presence of anti-alpha Gal IgY, anti-IgY IgM and anti-IgY IgG by enzyme-linked immunosorbent assay. Anti-alpha Gal IgY was almost undetectable in WT mouse sera at all injection and killing times. In contrast, Gal KO mouse sera showed increasing anti-alpha Gal IgY levels until 24 hr after the fourth injection, when anti-alpha Gal IgY levels were almost undetectable. Anti-IgY IgM and IgG levels in WT and Gal KO mouse sera showed a typical increase in anti-IgY IgM 24 hr after the second injection (3 days after the first injection) and an increase in anti-IgY IgG 24 hr after the third injection (5 days after the first injection). These results show that IgY binds to alpha Gal epitopes in the WT mice and is cleared sometime over a 24-hr time period and that IgY is an expected immunogen in mice eliciting a rather typical anti-IgY IgM and IgG response.

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival.

BACKGROUND: Acute vascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. METHODS: CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. RESULTS: CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. CONCLUSIONS: The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.

IgY antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding.

Avian IgY antibodies are structurally different from mammalian IgGs and do not fix mammalian complement components or bind human Fc receptors. As these antibody-mediated interactions are believed to play significant roles in both hyperacute rejection (HAR) and acute vascular xenograft rejection (AVXR), IgY antibodies to xenoantigen target epitopes may inhibit these rejection processes. In this report, we show that chicken IgY antibodies to alpha-Gal antigen epitopes and to other porcine aortic endothelial cell (PAEC) antigens block human xenoreactive natural antibody binding to both porcine and rat cardiac tissues and porcine kidney tissues. Chicken IgY antibodies blocked complement-mediated lysis of PAECs by human serum, and inhibited antibody-dependent cell-mediated lysis of PAECs by heat-inactivated human serum plus peripheral blood leukocytes. Binding of IgY to porcine endothelial cells did not affect cell morphology nor expression of E-selectin. These results suggest that avian IgYs could be of potential use in inhibiting pig-to-human xenograft rejection.

Numerous eruptive lesions of panniculitis associated with group A streptococcus bacteremia in an immunocompetent child.

A previously healthy 13-month-old boy developed group A beta-hemolytic streptococcus bacteremia coinciding with numerous eruptive subcutaneous lesions primarily on his extremities. Skin biopsy revealed infectious panniculitis; gram-positive cocci were present within both fat lobules and septa. Molecular genetic analysis of an isolate from the patient's blood revealed an emm type 4 organism displaying the emm chromosomal pattern E that is characteristic of opacity factor-producing strains; the organism also harbored the gene encoding for streptococcal pyrogenic exotoxin C (speC). To our knowledge, this clinical presentation has not yet been described in the spectrum of infections directly caused by group A beta-hemolytic streptococci.

Primary gamma delta cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation.

The division of CD4+ alpha beta T cells into Th1 and Th2 subsets has become an established and important paradigm. The respective activities of these subsets appear to have profound effects on the course of infectious and autoimmune diseases. It is believed that specific programs of differentiation induce the commitment of an uncommitted Th0 precursor cell to Th1 or Th2. A component of these programs is hypothesized to be the nature of MHC-peptide antigen presentation to the alpha beta T cell. It has heretofore remained uncertain whether a Th1/Th2 classification likewise defines, at the clonal level, gamma delta T cells. Such cells do not, as a general rule, express either CD4 or CD8 alpha beta, and they do not commonly recognize peptide-MHC. In this report, gamma delta cell clones are described that conform strikingly to the Th1/Th2 classification, both by cytokine expression and by functional activities of the clones in vitro and in vivo. Provocatively, both the gamma delta cell clones and primary gamma delta cells in vivo showed a strong association of the Th2 phenotype with CD4 expression. These results are discussed with regard to the immunoregulatory role that is increasingly emerging for gamma delta cells.

Persistent infection with Theiler's virus leads to CNS autoimmunity via epitope spreading.

Multiple sclerosis (MS) is a T cell-mediated autoimmune demyelinating disease, which may be initiated by a virus infection. Theiler's murine encephalomyelitis virus (TMEV), a natural mouse pathogen, is a picornavirus that induces a chronic, CD4+ T cell-mediated demyelinating disease with a clinical course and histopathology similar to that of chronic progressive MS (ref. 3). Demyelination in TMEV-infected mice is initiated by a mononuclear inflammatory response mediated by virus-specific CD4+ T cells targeting virus, which chronically persists in the CNS (ref. 4-6). We show that beginning 3-4 weeks after disease onset, T-cell responses to multiple myelin autoepitopes arise in an ordered progression and may play a pathologic role in chronic disease. Kinetic and functional studies show that T-cell responses to the immunodominant myelin proteolipid protein epitope (PLP139-151) did not arise because of cross-reactivity between TMEV and self epitopes (that is, molecular mimicry), but because of de novo priming of self-reactive T cells to sequestered autoantigens released secondary to virus-specific T cell-mediated demyelination (that is, epitope spreading). Epitope spreading is an important alternate mechanism to explain the etiology of virus-induced organ-specific autoimmune diseases.

Intrathymic delta selection events in gammadelta cell development.

The major pathway of gammadelta cell development is shown to be regulated by in-frame rearrangements at the T cell receptor (TCR) delta locus. Such "delta selection" occurs at or around the same point in thymocyte development as selection for in-frame rearrangements at the TCRbeta locus. However, there are at least two major differences with beta selection: first, delta selection commonly involves selection on the cognate TCR chain, gamma, suggesting that there is no "preTgamma" chain of major biological significance; second, most gammadelta-selected thymocytes differentiate rather than proliferate. Nonetheless, some delta selection events seemingly facilitate thymocyte expansion, similar to alphabeta T cell development. In these cases, TCRgamma selection is less obvious. Furthermore, the capacity of individual gamma chains to facilitate gammadelta selection is shown to vary with developmental age. The results further clarify early T cell development at the beta selection/delta selection stage and place clear constraints on models of cell fate determination.

Gamma delta T cell help of B cells is induced by repeated parasitic infection, in the absence of other T cells.

BACKGROUND: gamma delta T cells, like alpha beta T cells, are components of all well-studied vertebrate immune systems. Yet, the contribution of gamma delta T cells to immune responses is poorly characterized. In particular, it has not been resolved whether gamma delta cells, independent of any other T cells, can help B cells produce immunoglobulin and form germinal centers, anatomical foci of specialized T cell-B cell collaboration. RESULTS: TCR beta-/- mice, which lack all T cells except gamma delta T cells, routinely displayed higher levels of antibody than fully T cell-deficient mice. Repeated parasitic infection of TCR beta-/- mice, but not of T cell-deficient mice, increased antibody levels and induced germinal centers that contained B cells and monoclonal gamma delta cells in close juxtaposition. However, antibody specificities were more commonly against self than against the challenging pathogen. gamma delta T cell-B cell help was not induced by repeated inoculation of TCR beta-/- mice with mycobacterial antigens. CONCLUSIONS: In the absence of any other T cells, gamma delta T cell-B cell collaboration can be significantly enhanced by repeated infection. However, the lack of obvious enrichment for antibodies against the challenging pathogen distinguishes gamma delta T cell help from alpha beta T cell help induced under analogous circumstances. The increased production of generalized antibodies may be particularly relevant to the development of autoimmunity, which commonly occurs in patients suffering from alpha beta T cell deficiencies, such as AIDS.