Understanding homologous recombination repair deficiency in biliary tract cancers: clinical implications and correlation with platinum sensitivity.

BACKGROUND: Biliary tract cancers (BTCs) exhibit high mortality rates and significant heterogeneity in both clinical and molecular characteristics. This study aims to molecularly characterize a cohort of patients with BTC, with a specific focus on genomic alterations within homologous recombination repair (HRR) genes in a real-world setting. PATIENTS AND METHODS: We carried out a retrospective analysis on 256 patients with BTC treated at five Austrian centers and one German comprehensive cancer center between 2016 and 2023 utilizing comprehensive genomic profiling platforms to assess HRR status and its correlation with clinical outcomes after platinum-based chemotherapy. RESULTS: A total of 67 patients (27.5%) exhibited HRR gene mutations (HRRm), with the most common pathogenic alterations in BAP1 (9%), ARID1A (7.8%), and ATM (6.1%). Time to failure of the first-line strategy (TFS) between patients with HRRm and non-HRRm treated with platinum agents was 7.9 and 6.7 months, respectively [hazard ratio (HR) 0.89; P = 0.49]. The overall survival (OS) estimates at 6, 18, and 24 months were 82%, 45%, and 39% in the HRRm group (median 16.01 months) and 81%, 42%, and 22% in the HRR group (median 15.68 months), respectively (Fleming-Harrington test P = 0.0004; log-rank P = 0.022). Significance did not persist in the multivariate analysis (HR 0.72; 95% confidence interval 0.489-1.059; P = 0.095). An interaction between HRRm status and molecular-informed therapeutic strategies in later lines was noted. In the second-line treatment, OS following an irinotecan-based regimen was comparable to re-exposure to platinum-based agents (12.36 versus 10.13 months; HR 0.92; P = 0.85). No better outcome was noted for patients with HRRm versus patients with non-HRRm with second-line platinum agents (HR 1.45; P = 0.35). CONCLUSIONS: Patients with HRRm with BTC showed a potential advantage in OS following platinum-based first-line chemotherapy, presumably attributed to enhanced opportunities for targetable coalterations. Further investigation is needed to outline HRR within the scope of BTCs and detail a clinically meaningful sensitivity to platinum agents or targeted approaches with poly (ADP-ribose) polymerase (PARP) inhibitors.

Bevacizumab mitigates codon-specific effects of trifluridine/tipiracil on efficacy outcome parameters in metastatic colorectal cancer.

BACKGROUND: Molecular informed therapy changed treatment patterns of metastatic colorectal cancer (mCRC). Recently KRAS G12, the most prevalent RAS mutation in mCRC, was investigated to be a negative predictive marker for the efficacy of trifluridine/tipiracil (FTD/TPI). Whether this proposed selectivity remains when FTD/TPI is combined with bevacizumab remains elusive. We aimed to describe the efficacy of FTD/TPI + bevacizumab depending on the RAS mutational status in a real-world population. PATIENTS AND METHODS: Patients from five different cancer centers in Austria who received FTD/TPI + bevacizumab in any treatment line having available information on their molecular profile were eligible. Data were retrospectively collected by chart review. Survival data were compared using log-rank test. Multivariate Cox regression models included several established covariates. RESULTS: One hundred and twenty-three patients with mCRC were included in this study. Median overall survival (OS) was highly similar in the RAS wild type (WT) [9.63 months (95% confidence interval [CI] 8.055-13.775 months)] and the RAS mutant cohorts [8.78 months (95% CI 8.055-11.014 months)], which was confirmed in a multivariable model adjusting for potential confounders; hazard ratio (HR): 1.05 (95% CI 0.618-1.785; P = 0.857). In addition, no effect of KRAS G12 status on patient outcome was observed. In detail, OS was 8.88 months (95% CI 7.332-12.921 months) in patients with KRAS G12 mutation, compared to 9.47 months (95% CI 8.088-11.375 months) in patients with RAS WT/no-KRAS G12 disease [HR: 0.822 (95% CI 0.527-1.282; P = 0.387)]. CONCLUSION: This real-world study indicates that the efficacy of FTD/TPI + bevacizumab is independent of RAS mutational status and that bevacizumab may therefore mitigate the potentially limited efficacy of FTD/TPI monotherapy in the KRAS G12-mutated population.

Prognostic value of metastatic pattern in colorectal cancer: a multicenter retrospective analysis in a real-life cohort.

BACKGROUND: Metastatic patterns have been linked with prognosis in colorectal cancer. We aim to determine the distribution of metastases, their dynamics during disease and their prognostic impact for specific clinical treatment scenarios (resection of metastasis and/or systemic treatment, best supportive care). MATERIAL AND METHODS: 978 patients diagnosed with metastatic colorectal adenocarcinoma treated at three oncological centers from 2006 to 2018 were included. Overall survival was assessed depending on tumor load, distribution of metastases and treatment of the patients. RESULTS: Most patients had single site metastasis (n = 684; 69.9%): 398 patients had liver (n = 398; 40.7%) and 103 patients had lung only metastasis (10.6%). The number of organs involved in metastases at diagnosis was highly prognostic (HR 0.77; CI 0.65, 0.90), whereas the additional gain of metastases during progression of the disease was not. The majority of patients (62.9-74.2%) with initial lung, liver or both metastases retained their initial metastatic status. In the overall population, lung only metastases were associated with the most favorable outcome (HR 0.64; CI 0.50, 0.81). This was also observed in patients receiving best supportive care (HR 0.45; CI 0.27, 0.75). Resection of lung only metastases resulted in longer median survival (102.2 months). A relevant survival difference in patients treated by systemic therapy alone was not observed. Lung only metastasis was associated with rectal cancer (p < .001) and RAS-mutation (p = .01); both, lung and liver metastasis were associated with time from diagnosis to first metastasis (p < .001). CONCLUSION: The number of organs involved in metastasis at diagnosis but not the total cumulative number of involved organs is of prognostic relevance in colorectal adenocarcinoma. This prognostic relevant initial metastasis distribution remains unchanged in the majority of patients during the disease. However, the prognostic impact of the metastatic pattern is potentially altered by treatment modality.

The liver graft as Trojan horse-multilineage donor-derived hematopoiesis after liver transplantation: case report.

Hematopoietic macrochimerism, which is rarely seen after orthotopic liver transplantation (OLT), has been linked to the development of graft versus host disease (GvHD). We report on a patient with GvHD after OLT in whom full engraftment of donor-derived, multilineage hematopoiesis occurred, indicating that the liver contains pluripotent hematopoietic progenitor cells (HPC) capable to restore hematopoiesis in recipients. Although preventing graft rejection, standard immunosuppressive therapy may be under certain immunological conditions not sufficient to prevent GvHD. Age-, disease-, and treatment-related variables might be critical determinants for the development of an effective alloreactive T-cell response leading to the establishment of full hematopoietic chimerism.

Ovarian cancer stem cells.

Because of its semi-solid character in dissemination and growth, advanced ovarian cancer with its hundreds of peritoneal tumor nodules and plaques appears to be an excellent in vivo model for studying the cancer stem cell hypothesis. The most important obstacle, however, is to adequately define and isolate these tumor-initiating cells endowed with the properties of anoikis-resistance and unlimited self-renewal. Until now, no universal single marker or marker constellation has been found to faithfully isolate (ovarian) cancer stem cells. As these multipotent cells are known to possess highly elaborated efflux systems for cytotoxic agents, these pump systems have been exploited to outline putative stem cells as a side-population (SP) via dye exclusion analysis. Furthermore, the cells in question have been isolated via flow cytometry on the basis of cell surface markers thought to be characteristic for stem cells.In the Vienna variant of the ovarian cancer cell line A2780 a proof-of-principle model with both a stable SP and a stable ALDH1A1+ cell population was established. Double staining clearly revealed that both cell fractions were not identical. Of note, A2780V cells were negative for expression of surface markers CD44 and CD117 (c-kit). When cultured on monolayers of healthy human mesothelial cells, green-fluorescence-protein (GFP)-transfected SP of A2780V exhibited spheroid-formation, whereas non-side-population (NSP) developed a spare monolayer growing over the healthy mesothelium. Furthermore, A2780V SP was found to be partially resistant to platinum. However, this resistance could not be explained by over-expression of the "excision repair cross-complementation group 1" (ERCC1) gene, which is essentially involved in the repair of platinated DNA damage. ERCC1 was, nonetheless, over-expressed in A2780V cells grown as spheres under stem cell-selective conditions as compared to adherent monolayers cultured under differentiating conditions. The same was true for the primary ovarian cancer cells B-57.In summary our investigations indicate that even in multi-passaged cancer cell lines hierarchic government of growth and differentiation is conserved and that the key cancer stem cell population may be composed of small overlapping cell fractions defined by various arbitrary markers.

Elevated tryptase levels selectively cluster in myeloid neoplasms: a novel diagnostic approach and screen marker in clinical haematology.

BACKGROUND: Recent data suggest that tryptase, a mast cell enzyme, is expressed in neoplastic cells in myeloid leukaemias. In several of these patients, increased serum tryptase levels are detectable. MATERIALS AND METHODS: We have determined serum tryptase levels in 914 patients with haematological malignancies, including myeloproliferative disorders (n = 156), myelodysplastic syndromes (MDS, n = 241), acute myeloid leukaemia (AML, n = 317), systemic mastocytosis (SM, n = 81), non-Hodgkin's lymphoma (n = 59) and acute lymphoblastic leukaemia (n = 26). Moreover, tryptase was measured in 136 patients with non-neoplastic haematological disorders, 102 with non-haematological disorders and 164 healthy subjects. RESULTS: In healthy subjects, the median serum tryptase was 5.2 ng mL(-1). Elevated serum tryptase levels were found to cluster in myeloid neoplasm, whereas almost all patients with lymphoid neoplasms exhibited normal tryptase. Among myeloid neoplasms, elevated tryptase levels (> 15 ng mL(-1)) were recorded in > 90% of patients with SM, 38% with AML, 34% with CML and 25% with MDS. The highest tryptase levels, often > 1000 ng mL(-1), were found in advanced SM and core-binding-factor leukaemias. In most patients with non-neoplastic haematological disorders and non-haematological disorders analysed in our study, tryptase levels were normal, the exception being a few patients with end-stage kidney disease and helminth infections, in whom a slightly elevated tryptase was found. CONCLUSIONS: In summary, tryptase is a new diagnostic marker of myeloid neoplasms and a useful test in clinical haematology.

The kinase inhibitor imatinib--an immunosuppressive drug?

The phenylaminopyrimidine-derivate Imatinib mesylate has been developed for targeted inhibition of the Abelson kinase (c-ABL), which is constitutively activated when translocated to the genetic locus of the breakpoint cluster region (leading to the BCR/ABL fusion gene), thereby forming the causative pathogenetic event for the development of chronic myeloid leukemia (CML). Of note, due to its physico-chemical properties, kinase specificity of Imatinib is limited. Despite of its well documented clinical efficacy mediated by inhibition of constitutively activated tyrosine kinases such as BCR/ABL in CML, PDGF-RA in HES and mutated c-kit in GIST patients, other tyrosine kinases such as Flt-3, Lck and mitogen-activated kinases (MAPK) are affected as well. Accordingly, it has recently been shown that therapeutic doses of Imatinib also target a variety of immune cells, e.g. by modulating the differentiation of dendritic cells (DC) as well as by impeding proper T-cell and macrophage function. In contrast, combining Imatinib with Interleukin 2 (IL-2) potently activates NK-cells and led to the description of a new subclass of DC, so-called IK-DC. The latter mediate Imatinib/IL-2-induced regression of tumors in pre-clinical animal models via production of high amounts of IFN-gamma and the death receptor ligand TRAIL. Thus, Imatinib exerts potent immuno-modulatory effects in vitro and in vivo, which will be discussed together with their clinical relevance in detail throughout this review.

Association of low-grade inflammation with nephropathy in type 2 diabetic patients: role of elevated CRP-levels and 2 different gene-polymorphisms of proinflammatory cytokines.

BACKGROUND: Chronic inflammatory processes are thought to play a key role in the development of micro- and macrovascular complications in type 2 diabetes mellitus. An association between low -grade inflammation and type 2 diabetes has been described in some studies. We assayed the association of two frequent polymorphisms in proinflammatory cytokines: the interleukin 6 G(-174)C promoter polymorphism [IL-6G(-174)C], the exon 2 interleukin receptor antagonist insertion deletion polymorphism [IL1RA]) and serum CRP levels with the prevalence of diabetic nephropathy in patients suffering from type 2 diabetes mellitus. SUBJECTS AND METHODS: A total of 141 patients with type 2 diabetes mellitus, with and without diabetic nephropathy was genotyped for the above mentioned polymorphisms: 66 with normoalbuminuria, 31 with microalbuminuria and 44 with macroalbuminuria. CRP levels were analysed by a high sensitivity - immunnephelometric assay. RESULTS: While a significant association be-tween macroalbuminuria and CRP could be observed (p<0,015), no associations were found between IL-6G(-174)C or IL1RA genotype and any stage of nephropathy. CRP-levels were similar in the 3 different IL-6G(-174)C genotypes as well as in the 2 IL1RA genotypes. CONCLUSIONS: In type 2 diabetic subjects elevated CRP levels are associated with an increased prevalence of albuminuria. The two investigated proinflammatory polymorphisms do not seem to contribute to initiation of nephropathy in type 2 diabetic patients but we cannot exclude effects of these polymorphisms on course of nephropathy.

The antiapoptotic effect of IL-6 autocrine loop in a cellular model of advanced prostate cancer is mediated by Mcl-1.

Levels of the proinflammatory cytokine interleukin-6 (IL-6) are increased in therapy-resistant prostate cancer. IL-6 has been considered a positive growth factor in late-stage prostate cancer cells and a potential target for therapeutic interference. Effects of inhibition of IL-6 on cell survival were studied in LNCaP-IL6+ cells, a model system for advanced prostate cancer, which produce IL-6. We show that the autocrine IL-6 loop is responsible for resistance to apoptosis and increased cellular levels of myeloid cell leukemia-1 (Mcl-1) protein, an antiapoptotic member of the Bcl-2 family. Treatment of cells with a chimeric anti-IL-6 antibody (CNTO 328) led to the induction of apoptosis and downregulation of Mcl-1 protein levels. Specific knockdown of Mcl-1 gene expression by small interfering RNA also yielded an increase in apoptosis of LNCaP-IL-6+ cells. Vice versa, inactivation of IL-6 autocrine loop had no influence on apoptosis levels in the absence of Mcl-1, thus suggesting this molecule as a mediator of the survival action of IL-6. Mcl-1 protein regulation by the endogenous cytokine directly involved the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway. Our data support the concept of anti-IL-6 targeted therapy in therapy-resistant prostate cancer.

Skin cholesterol: test performance, evaluation of potential determinants and correlation analysis with cardiovascular risk factors and circulating markers of inflammation.

BACKGROUND: Skin cholesterol (SkC) has been suggested to be an additional risk predictor, so we evaluated the test performance, potential determinants of this marker as well as a potential correlation of SkC with markers of inflammation and the history of cardiovascular events. PATIENTS AND METHODS: SkC, determined by the non-invasive PREVU POC Skin Sterol test, as well as serum lipids, the body fat status, high-sensitive CRP (hs-CRP) and serum amyloid A (SAA) were evaluated in consecutive patients with and without documented atherosclerotic disease. RESULTS: SkC was assessed in 201 patients. The within-day precision (CV) was 3.8%, the day-to-day CV of the right hand was 8.6% and 4.3% for the left hand, respectively. Neither univariate analysis nor multiple regressions identified a significant influence of age, sex, serum lipids, body fat status, smoking or diabetes mellitus on SkC, corresponding results were observed in a further analysis including 174 of these patients concerning hs-CRP and SAA (all p > 0.05). T-test analyses detected no significant differences between patients with and without a history of coronary, peripheral vascular and cerebrovascular events (all p > 0.05). CONCLUSIONS: The PREVU POC Skin Sterol test for the assessment of SkC proved an acceptable test performance. SkC is independent from serum lipids, traditional cardiovascular risk factors, two sensitive markers of systemic inflammation as well as the history of cardiovascular events indicating that the perception of this parameter as an established marker of vascular disease is premature.

[Complete remission of an idiopathic hypereosinophilic syndrome while using imatinib]. / Komplette Remission eines idiopathischen hypereosinophilen Syndroms unter Imatinib.

HISTORY AND ADMISSION FINDINGS: A 47-year-old man with a hypereosinophilic syndrome (HES), which has been known for 20 years, was admitted to our department due to insufficient therapeutic response to hydroxyurea. In general, the patient felt well, but reported increasing neurological problems, such as ataxia, memory deficits and dysarthria. INVESTIGATIONS: Bone marrow assessments corroborated the diagnosis of a HES. However, we were not able to detect the insertional deletion 4q12 with concomitant fusion of the FIP1L1 to the PDGFRA locus. Magnetic resonance imaging (MRI) indicated a granulomatous vasculitis, which was most likely due to the hematologic malignancy. TREATMENT AND COURSE: : Despite negativity for the FIP1L1-PDGFRA fusion gene, therapy was started with the tyrosine kinase inhibitor Imatinib. This led to a rapid normalization of eosinophilic granulocytes in the peripheral blood as well as in the bone marrow. In addition, the neurologic symptoms substantially improved. CONCLUSION: Imatinib provides a potent therapeutic option in FIP1L1-PDGFRA negative patients suffering from HES.

TGF-beta cytokines increase senescence-associated beta-galactosidase activity in human prostate basal cells by supporting differentiation processes, but not cellular senescence.

The family of transforming growth factors betas (TGF-betas) comprises molecules involved in growth inhibition, stress-induced premature senescence, epithelial mesenchymal transition and differentiation processes. The aim of this study was to clarify the effect of long term exposure of human prostate basal cells to TGF-betas, which are found in high concentrations in prostatic fluid and areas of benign prostatic hyperplasia (BPH). Basal cell cultures established from prostate explants (n=3) were either grown into cellular senescence, or stimulated with TGF-beta1, beta2 and beta3. Similar to cellular senescence, TGF-beta stimulation resulted in an increase of SA-beta galactosidase (SA-beta-gal) activity, flattened and enlarged cell morphology, and down-regulation of the inhibitor of differentiation Id-1. TGF-beta-treated prostate epithelial cells neither showed terminal growth arrest nor induction of important senescence-relevant genes, such as p16(INK4A), IFI-6-16, IGFBP-3 or Dkk-3. Cells stained positive for cytokeratins 8/18, but did not express other lumenal markers, such as prostate-specific antigen and androgen-receptors. TGF-betas increased also the expression of the mesenchymal marker vimentin, indicating that basal epithelial cells underwent differentiation with lumenal and mesenchymal features. In contrast, in vitro-differentiated neuroendocrine-like cells from prostate organoide cultures, expressing chromogranin A and cytokeratin 18, strongly stained positive for SA-beta-gal. Thus, SA-beta-gal activity is not only a marker for senescence, but also for differentiation of human prostate epithelial cells. With regard to the in vivo situation, in addition to cellular senescence, TGF-beta could contribute to the increased number of SA-beta-gal positive epithelial cells in BPH.

Seminal plasma factors induce in vitro PRL secretion in smooth muscle cells of the human prostate.

Next to the sex steroid hormone T, PRL has been shown to influence prostatic function and development. Transgenic mice overexpressing the rat PRL gene develop dramatic enlargements of the prostate gland. Proliferation and secretory activities of epithelial cells are stimulated by PRL in rodents and men. Low concentrations of human PRL (hPRL) and hPRL receptors have been observed in human prostatic epithelial cells (ECs). The aim of this study was to compare regulation of the in vitro hPRL secretion in prostatic ECs and stromal smooth muscle cells (SMCs) after stimulation with seminal plasma (SMP), containing a variety of prostatic factors. SMCs released up to 1 ng hPRL/ml (i.e., approximately 500-fold more than unstimulated SMCs and ECs). Quantification of PRL mRNA by highly sensitive quantitative RT-PCR revealed that hPRL gene expression increased 5-fold within 24 h of SMP incubation. Sex steroids (dihydrotestosterone, progesterone, 17beta-estradiol), prostaglandins (PGE-1, PGE-2), and cAMP-stimulating substances (forskolin) were not responsible for induction of hPRL. Compared with endometrial SMCs, regulation of prostatic hPRL secretion was independent of progesterone and cAMP. HPLC analysis of human SMP revealed that the common action of at least two different proteins and a low molecular cofactor is required. We concluded that prostatic ECs secrete proteins acting synergistically with low-molecular-weight cofactors to induce differentiation and hPRL release in SMCs. Age-related increases in SMC-derived hPRL might contribute to the development of benign hyperplasia of the prostate.

A low-molecular-weight fraction of human seminal plasma activates adenylyl cyclase and induces caspase 3-independent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential and Bcl-2/Bax ratio.

The majority of elderly men are affected by benign and malign diseases of the prostate that are governed by endocrine factors and local stromal/epithelial and luminal/epithelial interactions. Prostate epithelial cells secrete numerous factors into the seminal plasma (SMP) that are thought to be responsible for nutrition, accurate pH, and ionic environment of sperm. Our hypothesis assumes that prostatic factors responsible for optimal fertility might have retrograde influences on epithelial cell growth, differentiation, and function. SMP was analyzed for proteins and other biologically active substances by size exclusion high-performance liquid chromatography. Each fraction was investigated for its effect on cell growth and death. A low molecular mass fraction (2-4 kDa) was responsible for inducing apoptosis in proliferating prostate epithelial cells. Signal transduction was mediated by the production of cAMP; no significant changes in tyrosine phosphorylation of membrane receptors were observed. Mechanisms of apoptosis, i.e., caspase- and mitochondria-dependent pathways, were investigated in prostate epithelial cells by caspase activity assays, annexin/propidium iodide staining, changes in mitochondrial potential, p53, Par-4, Bax, and Bcl-2 protein levels. SMP induced p53- and Bcl-2-dependent apoptosis without activation of caspase-3. Obviously, SMP contains protective factors that help eliminate degenerated cells and control epithelial renewal. Age-related changes in the composition of SMP or the susceptibility of epithelial cells might, therefore, contribute to proliferative prostatic diseases

High levels of zinc ions induce loss of mitochondrial potential and degradation of antiapoptotic Bcl-2 protein in in vitro cultivated human prostate epithelial cells.

Prostate epithelial cells contain the highest levels of zinc among all organs and tissues in the human body. Zinc is accumulated primarily in the mitochondria, where it is responsible for inhibition of mitochondrial aconitase activity, thereby increasing citrate production. The present study was designed to clarify the role of zinc for human prostate epithelial cell growth and apoptosis. Apoptosis of in vitro cultivated human prostate epithelial cells exposed to ZnCl(2) was analyzed by determination of phospholipid membrane asymmetry, nuclear fragmentation, DNA strand breaks, changes of mitochondrial potential and cellular pro/antiapoptotic proteins. Zinc induced apoptosis without involvement of p53 by decreasing mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 protein levels in proliferating epithelial cells. Thus, the high local concentrations of zinc ions in the prostatic lumen seem to be necessary to regulate proliferative activities and to enforce epithelial differentiation processes.

pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens.

We have previously identified a birch pollen profilin hexadecapeptide (Bp36/51), which was recognized by a monoclonal antibody (moAb 4A6) with high affinity. Here, we report the construction of a T7 RNA polymerase-driven high-level plasmid expression system, pET-prof, capable of producing proteins and peptides containing the Bp36/51 birch profilin-derived peptide fused to their N-terminus. As examples, the cDNAs coding for two major timothy grass (Phleum pratense) pollen allergens, Phl p 2 and Phl p 6, as well as for an alder (Alnus glutinosa) pollen allergen, Aln g 4, were overexpressed in Escherichia coli as BP36/51-tagged proteins. All three recombinant allergens were readily detected in nitrocellulose-blotted E. coli extracts by the Bp36/51-specific moAb 4A6. We demonstrate comparable IgE recognition of Bp36/51-tagged and untagged recombinant allergens by immunoblotting. A sandwich ELISA was developed using plate-bound moAb 4A6 to immobilize and present Bp36/51-tagged recombinant allergens to IgE antibodies of allergic patients. Using immunoelectronmicroscopy, we demonstrate that even under harsh fixation conditions, tagged allergens can be localized simultaneously in situ by moAb 4A6 and allergen-specific antisera. We suggest the use of the pET-prof system for the high-level expression of Bp36/51-tagged polypeptides that can be rapidly detected in total protein extracts, immunolocalized in situ, immobilized and presented to other antigen-specific antibodies (e.g. IgE), even when they occur in minute concentrations.