Tracking a CAD-ALK gene rearrangement in urine and blood of a colorectal cancer patient treated with an ALK inhibitor.

BACKGROUND: Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. PATIENTS AND METHODS: We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying a CAD-ALK translocation during treatment with an ALK inhibitor. RESULTS: Using a custom next generation sequencing panel we identified the genomic CAD-ALK rearrangement and a TP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of the CAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient's clinical course. Detection of the CAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identified ALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. CONCLUSION: We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.

Comparison of urine specimen collection times and testing fractions for the detection of high-risk human papillomavirus and high-grade cervical precancer.

BACKGROUND: Urine testing for high-risk human papillomavirus (HR-HPV) detection could provide a non-invasive, simple method for cervical cancer screening. OBJECTIVES: We examined whether HR-HPV detection is affected by urine collection time, portion of urine stream, or urine fraction tested, and assessed the performance of HR-HPV testing in urine for detection of cervical intraepithelial neoplasia grade II or worse (CIN2+). STUDY DESIGN: A total of 37 female colposcopy clinic attendees, ≥ 30 years, provided three urine samples: "first void" urine collected at home, and "initial stream" and "mid-stream" urine samples collected at the clinic later in the day. Self- and physician-collected brush specimens were obtained at the same clinic visit. Colposcopy was performed and directed biopsies obtained if clinically indicated. For each urine sample, HR-HPV DNA testing was conducted for unfractionated, pellet, and supernatant fractions using the Trovagene test. HR-HPV mRNA testing was performed on brush specimens using the Aptima HPV assay. RESULTS: HR-HPV prevalence was similar in unfractionated and pellet fractions of all urine samples. For supernatant urine fractions, HR-HPV prevalence appeared lower in mid-stream urine (56.8%[40.8-72.7%]) than in initial stream urine (75.7%[61.9-89.5%]). Sensitivity of CIN2+ detection was identical for initial stream urine and physician-collected cervical specimen (89.9%[95%CI=62.7-99.6%]), and similar to self-collected vaginal specimen (79.1%[48.1-96.6%]). CONCLUSION: This is among the first studies to compare methodologies for collection and processing of urine for HR-HPV detection. HR-HPV prevalence was similar in first void and initial stream urine, and was highly sensitive for CIN2+ detection. Additional research in a larger and general screening population is needed.

Cancer of unknown primary: progress in the search for improved and rapid diagnosis leading toward superior patient outcomes.

This paper explores the enigma of cancer of unknown primary (CUP) in relation to rapidly improving molecular diagnostic approaches. It is based on the first global collaboration meeting on improving research and clinical outcomes in CUP organized by the CUP Foundation. We review the difficulties of classifying this widely heterogeneous disease and the available diagnostic and pathological evaluative techniques, focusing on molecular profiling. Retrospective studies in CUP patients are shown to provide indirect validation of the accuracy of several platforms of gene expression profiling assays that may identify CUP subsets that respond favorably to active chemotherapy regimens. This review concludes that the recent major improvements in pathologic and molecular diagnostics, coupled with new improved therapies for several specific advanced solid tumors, need to be harmonized with more evidence from clinical-translational trials. All patients with CUP could thus be appropriately managed without the constant uncertainty that has previously severely hampered patient care and optimal outcomes. The longer-term objective is to understand the biology of highly metastatic disease, leading to the development of future global therapeutic programs. Current clinical studies, such as CUP-ONE, will address some of these issues.

Breast Cancer Index predicts pathological complete response and eligibility for breast conserving surgery in breast cancer patients treated with neoadjuvant chemotherapy.

BACKGROUND: The aim of neoadjuvant chemotherapy is to increase the likelihood of successful breast conservation surgery (BCS). Accurate identification of BCS candidates is a diagnostic challenge. Breast Cancer Index (BCI) predicts recurrence risk in estrogen receptor+lymph node-breast cancer. Performance of BCI to predict chemosensitivity based on pathological complete response (pCR) and BCS was assessed. METHODS: Real-time RT-PCR BCI assay was conducted using tumor samples from 150 breast cancer patients treated with neoadjuvant chemotherapy. Logistical regression and c-index were used to assess predictive strength and additive accuracy of BCI beyond clinicopathologic factors. RESULTS: BCI classified 42% of patients as low, 35% as intermediate and 23% as high risk. Low BCI risk group had 98.4% negative predictive value (NPV) for pCR and 86% NPV for BCS. High versus low BCI group had a 34 and 5.8 greater likelihood of achieving pCR and BCS, respectively (P=0.0055; P=0.0022). BCI increased c-index for pCR (0.875-0.924; P=0.017) and BCS prediction (0.788-0.843; P=0.027) beyond clinicopathologic factors. CONCLUSIONS: BCI significantly predicted pCR and BCS beyond clinicopathologic factors. High NPVs indicate that BCI could be a useful tool to identify breast cancer patients who are not eligible for neoadjuvant chemotherapy. These results suggest that BCI could be used to assess both chemosensitivity and eligibility for BCS.

Prognostic utility of HOXB13:IL17BR and molecular grade index in early-stage breast cancer patients from the Stockholm trial.

BACKGROUND: A dichotomous index combining two gene expression assays, HOXB13:IL17BR (H:I) and molecular grade index (MGI), was developed to assess risk of recurrence in breast cancer patients. The study objective was to demonstrate the prognostic utility of the combined index in early-stage breast cancer. METHODS: In a blinded retrospective analysis of 588 ER-positive tamoxifen-treated and untreated breast cancer patients from the randomised prospective Stockholm trial, H:I and MGI were measured using real-time RT-PCR. Association with patient outcome was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression. A continuous risk index was developed using Cox modelling. RESULTS: The dichotomous H:I+MGI was significantly associated with distant recurrence and breast cancer death. The >50% of tamoxifen-treated patients categorised as low-risk had <3% 10-year distant recurrence risk. A continuous risk model (Breast Cancer Index (BCI)) was developed with the tamoxifen-treated group and the prognostic performance tested in the untreated group was 53% of patients categorised as low risk with an 8.3% 10-year distant recurrence risk. CONCLUSION: Retrospective analysis of this randomised, prospective trial cohort validated the prognostic utility of H:I+MGI and was used to develop and test a continuous risk model that enables prediction of distant recurrence risk at the patient level.

Cloning of rat histamine H(3) receptor reveals distinct species pharmacological profiles.

The recent cloning and characterization of the human histamine H(3) receptor cDNA marked a significant step toward a greater understanding of the role of this receptor in the central nervous system. We now report the cloning of the rat histamine H(3) receptor cDNA and demonstrate distinct pharmacological species differences. The rat cDNA clone encodes a putative 445-amino acid protein with 93% identity to the human H(3) receptor. Northern blot analysis revealed a major single entity of 2.7-kb in length expressed only in brain. Transfection of the rat receptor cDNA into SK-N-MC cells conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine and other H(3) agonists. N-[(3)H]methylhistamine saturably bound to transfected cells with high affinity (K(d) = 0.8 nM). All agonists tested had low or subnanomolar K(i) values similar to that for the human recombinant receptor. The antagonists thioperamide and clobenpropit also bound with high affinity (K(i) = 4 and 0.4 nM, respectively). This is in contrast to the antagonist profile obtained for the human recombinant receptor that showed K(i) values of 58 and 0.6 nM for thioperamide and clobenpropit, respectively. These data suggest that the low affinity of thioperamide for the human H(3) receptor represents a species difference in pharmacology and not a unique pharmacological subtype. It also was found that chloroproxyfan behaved as a full agonist at the rat recombinant receptor. These findings highlight the significance of validating the pharmacology of experimental compounds at both the rat and human H(3) receptors.

TOGA: an automated parsing technology for analyzing expression of nearly all genes.

We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3' ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tissue extracts to determine simultaneously the presence and relative concentration of nearly every mRNA in the extracts, regardless of whether the mRNA has been discovered previously. Visualization of the electrophoretically separated fluorescent assay products from different extracts displayed via a Netscape browser-based graphical user interface allows the status of each mRNA to be compared among samples and its identity to be matched with sequences of known mRNAs compiled in databases.

Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery.

A recently identified neuropeptide with PRL-releasing capabilities binds to and activates a previously known orphan G protein-coupled receptor, GPR10. We initiated a study to define the pharmacology of the peptide/receptor interaction and to identify the distribution of the peptide and its receptor in the central nervous system to elucidate sites of action of the peptide. The PRL-releasing peptide (PrRP) is a C-terminally amidated, 31-amino acid peptide derived from a 98-amino acid precursor. Radioiodinated PrRP-(1-31) binds to its receptor with high affinity (1 nM) and stimulates calcium mobilization in CHOK1 cells stably transfected with the receptor. A series of N-terminal deletions reveals that the PrRP-(12-31) amino acid is equipotent to PrRP-(1-31). Further N-terminal deletions reduce the affinity of the ligand considerably, although PrRP-(25-31) is still able to compete for binding and behaves as an agonist. The arginine residues at position 26 and 30 are critical for binding, as substitution with either lysine or citrulline reduces the affinity substantially. In situ hybridization reveals a distinct tissue distribution for both the peptide and receptor messenger RNAs. The receptor is expressed abundantly in the reticular thalamic nucleus, periventricular hypothalamus, dorsomedial hypothalamus, nucleus of the solitary tract, area postrema, anterior pituitary, and adrenal medulla. The peptide messenger RNA is expressed in the dorsomedial hypothalamus, nucleus of the solitary tract, ventrolateral reticular nucleus, and intestine. This tissue distribution suggests an alternative function of PrRP than its purported hypophysiotropic function, such as a potential role for PrRP in the central feedback control of neuroendocrine and autonomic homeostasis. Further work using selective agonists and antagonists should help define additional physiological roles of this novel mammalian neuropeptide.

The pharmacological and functional characteristics of the serotonin 5-HT(3A) receptor are specifically modified by a 5-HT(3B) receptor subunit.

While homomers containing 5-HT(3A) subunits form functional ligand-gated serotonin (5-HT) receptors in heterologous expression systems (Jackson, M. B., and Yakel, J. L. (1995) Annu. Rev. Physiol. 57, 447-468; Lambert, J. J., Peters, J. A., and Hope, A. G. (1995) in Ligand-Voltage-Gated Ion Channels (North, R., ed) pp. 177-211, CRC Press, Inc., Boca Raton, FL), it has been proposed that native receptors may exist as heteromers (Fletcher, S., and Barnes, N. M. (1998) Trends Pharmacol. Sci. 19, 212-215). We report the cloning of a subunit 5-HT(3B) with approximately 44% amino acid identity to 5-HT(3A) that specifically modified 5-HT(3A) receptor kinetics, voltage dependence, and pharmacology. Co-expression of 5-HT(3B) with 5-HT(3A) modified the duration of 5-HT(3) receptor agonist-induced responses, linearized the current-voltage relationship, increased agonist and antagonist affinity, and reduced cooperativity between subunits. Reverse transcriptase-polymerase chain reaction in situ hybridization revealed co-localization of both 5-HT(3B) and 5-HT(3A) in a population of neurons in the amygdala, telencephalon, and entorhinal cortex. Furthermore, 5-HT(3A) and 5-HT(3B) mRNAs were expressed in spleen and intestine. Our data suggest that 5-HT(3B) might contribute to tissue-specific functional changes in 5-HT(3)-mediated signaling and/or modulation.

Immune-associated nucleotide-1 (IAN-1) is a thymic selection marker and defines a novel gene family conserved in plants.

Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

Cloning and functional expression of the human histamine H3 receptor.

Histamine regulates neurotransmitter release in the central and peripheral nervous systems through H3 presynaptic receptors. The existence of the histamine H3 receptor was demonstrated pharmacologically 15 years ago, yet despite intensive efforts, its molecular identity has remained elusive. As part of a directed effort to discover novel G protein-coupled receptors through homology searching of expressed sequence tag databases, we identified a partial clone (GPCR97) that had significant homology to biogenic amine receptors. The GPCR97 clone was used to probe a human thalamus library, which resulted in the isolation of a full-length clone encoding a putative G protein-coupled receptor. Homology analysis showed the highest similarity to M2 muscarinic acetylcholine receptors and overall low homology to all other biogenic amine receptors. Transfection of GPCR97 into a variety of cell lines conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine, but not to acetylcholine or any other biogenic amine. Subsequent analysis revealed a pharmacological profile practically indistinguishable from that for the histamine H3 receptor. In situ hybridization in rat brain revealed high levels of mRNA in all neuronal systems (such as the cerebral cortex, the thalamus, and the caudate nucleus) previously associated with H3 receptor function. Its widespread and abundant neuronal expression in the brain highlights the significance of histamine as a general neurotransmitter modulator. The availability of the human H3 receptor cDNA should greatly aid in the development of chemical and biological reagents, allowing a greater appreciation of the role of histamine in brain function.

High expression of stanniocalcin in differentiated brain neurons.

Stanniocalcin (STC) is a glycoprotein hormone first found in fish, in which it regulates calcium homeostasis and protects against hypercalcemia. Human and mouse stc cDNA were recently cloned. We found a dramatically upregulated expression of STC during induced neural differentiation in a human neural crest-derived cell line, Paju. Immunohistochemical staining of sections from human and adult mouse brain revealed abundant presence of STC in the neurons with no activity in the glial cells. STC expression was not seen in immature brain neurons of fetal or newborn mice. Given that STC has been found to regulate calcium/phosphate metabolism in some mammalian epithelia, we suggest that STC may act as a regulator of calcium homeostasis in terminally differentiated brain neurons.

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells.

We studied gene expression in relation to induced neural differentiation in a human neural crest-derived cell line, Paju. Messenger RNA isolated before and after treatment with phorbol 12-myristate 13-acetate was analyzed by differential display reverse transcription PCR. A strongly up-regulated expression of decay-accelerating factor (DAF, CD55) was found to parallel the induced neural sprouting while the expression of two other complement regulatory proteins (CD59/protectin, CD46/membrane cofactor protein) remained unaltered during neural differentiation. The increased membrane expression of DAF, which was also seen on neural processes and growth cones, conferred elevated resistance to complement-mediated lysis. Our findings suggest that in sprouting neurons DAF expression is up-regulated to provide additional complement resistance to pathfinding axons/dendrites invading new environment. It is also suggested that membrane expression of DAF may constitute a marker of growing and regenerating neurons.

Postdifferential display: parallel processing of candidates using small amounts of RNA.

The necessity of screening differentially expressed candidate genes has imposed a limit on the application of differential display to large-scale analysis of gene expression patterns. Screening candidates has indeed proven a burden because traditional screening methods require the purification of large amounts of RNA. In this article we describe an assay that allows the screening of 240 candidate genes with only 5 microg of total RNA. This assay consists of using cDNA probes synthesized from amplified RNA in differential screening and can be performed in a 96-well plate format.

Screening differentially expressed cDNA clones obtained by differential display using amplified RNA.

The major obstacle of differential display is not the technique itself but rather the post-differential display issueof discriminating between false positives and the truly differentially expressed mRNAs. This process is arduous and requires large amounts of RNA. We present and validate a method which allows one to screen putative positives from differential display analysis using only micrograms of total RNA. More importantly, we demonstrate that cDNA probes generated from amplified RNA are representative of the starting mRNA population and can be used for differential screening of mRNA species at a detectable limit of sensitivity of>/=1/40 000.

Cloning differentially expressed mRNAs.

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

In vivo characterization of the proteasome regulator PA28.

A proteasome regulator, termed PA28, has been shown to modulate peptidase activities of the proteasomes in vitro. Two different but homologous PA28 molecules, designated as PA28alpha and PA28beta, have been cloned. Both alpha and beta polypeptides of PA28 are found in PA28 complexes isolated from cells, indicating that both are constituents of functional PA28 complexes. Using antisera specific to PA28alpha, PA28beta, and epitope-tagged PA28 molecules, we show that expression of PA28alpha and PA28beta is coordinately induced by various cytokines in different cell lines and that PA28 subunits and proteasomes have almost identical half-lives. In addition, we show that PA28 complexes are associated with 20 S but not 26 S proteasomes in vivo. Moreover, we demonstrate that PA28 complex is a heterohexamer composed of both alpha and beta subunits with a stoichiometry of alpha3beta3 in an alternating order.