Predicting response to neoadjuvant chemotherapy with liquid biopsies and multiparametric MRI in patients with breast cancer.

Accurate prediction of response to neoadjuvant chemotherapy (NAC) can help tailor treatment to individual patients' needs. Little is known about the combination of liquid biopsies and computer extracted features from multiparametric magnetic resonance imaging (MRI) for the prediction of NAC response in breast cancer. Here, we report on a prospective study with the aim to explore the predictive potential of this combination in adjunct to standard clinical and pathological information before, during and after NAC. The study was performed in four Dutch hospitals. Patients without metastases treated with NAC underwent 3 T multiparametric MRI scans before, during and after NAC. Liquid biopsies were obtained before every chemotherapy cycle and before surgery. Prediction models were developed using penalized linear regression to forecast residual cancer burden after NAC and evaluated for pathologic complete response (pCR) using leave-one-out-cross-validation (LOOCV). Sixty-one patients were included. Twenty-three patients (38%) achieved pCR. Most prediction models yielded the highest estimated LOOCV area under the curve (AUC) at the post-treatment timepoint. A clinical-only model including tumor grade, nodal status and receptor subtype yielded an estimated LOOCV AUC for pCR of 0.76, which increased to 0.82 by incorporating post-treatment radiological MRI assessment (i.e., the "clinical-radiological" model). The estimated LOOCV AUC was 0.84 after incorporation of computer-extracted MRI features, and 0.85 when liquid biopsy information was added instead of the radiological MRI assessment. Adding liquid biopsy information to the clinical-radiological resulted in an estimated LOOCV AUC of 0.86. In conclusion, inclusion of liquid biopsy-derived markers in clinical-radiological prediction models may have potential to improve prediction of pCR after NAC in breast cancer.

Evaluation of strained alkynes for Cu-free click reaction in live mice.

INTRODUCTION: We report on our evaluation of the strain-promoted cyclooctyne-azide cycloaddition reaction for use in tumor pretargeting, comprising a side-by-side comparison of probes 1-3 bearing three distinct cyclooctyne moieties based respectively on the 1st and 2nd generation difluorinated cyclooctyne and the 1st generation dibenzocyclooctyne. METHODS: The probes were synthesized and labeled with (177)Lu with high yields. The probe stability and reactivity towards azides were evaluated in PBS and mouse serum, and their blood clearance, biodistribution and in vivo reactivity were evaluated in tumor-free mice. RESULTS: In serum the three probes exhibited sufficient stability for a pretargeting application with half-lives of 12-19h. In PBS, probes 2 and 3 were more reactive towards azido-conjugated Rituximab (Rtx-N3) than 1, but in contrast to 1, their reactivity decreased in mouse serum and mouse serum albumin solutions, as a result of covalent and non-covalent interactions with albumin. Biodistribution data confirmed the interactions with serum proteins in circulation: (177)Lu-1 showed a fast elimination from blood (t1/2,ß = 0.31h), while (177)Lu-2 and (177)Lu-3 were retained in blood for longer periods of time (t1/2,ß = 1.08 and 3.58h, respectively). Dual isotope biodistribution experiments assessing the reaction between (125)I-Rtx-N3 and (177)Lu-1-3 in circulation in mice showed a very limited retention of 2 and 3 in blood rich organs, indicating a minimal reactivity, while no such retention was observed for 1. CONCLUSION: The low reactivity of the studied cyclooctynes, and their serum interactions preclude their use at the low in vivo concentrations typical for pretargeting applications.

Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype.

Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.

Neuroendocrine effects of dehydration in mice lacking the angiotensin AT1a receptor.

Angiotensin (Ang) type 1a (AT1a) receptors are critical in the control of blood pressure and water balance. Experiments were performed to determine the influence of dehydration on brain Ang receptors and plasma vasopressin (VP) in mice lacking this receptor. Control or AT1a knockout (AT1aKO) male mice were give water ad libitum or deprived of water for 48 hours. Animals were anesthetized with halothane, blood samples were collected by heart puncture, and brains were processed for Ang-receptor autoradiography with 125I-sarthran (0.4 nmol/L). Dehydration produced an increase in AT1 receptors in the paraventricular nucleus (PVN) and anterior pituitary (AP) in control mice (PVN: 70+/-16 versus 146+/-10 fmol/mg protein; AP: 41+/-7 versus 86+/-15 fmol/mg protein). No changes were noted in the median preoptic nucleus. The majority of the brain receptors were of the AT1 subtype. There was little or no specific Ang binding in AT1aKO mice and no effect of dehydration. Plasma VP levels were elevated in the halothane-anesthetized animals (>200 pg/mL) with no significant effect of dehydration. A separate experiment was performed with decapitated mice anesthetized with pentobarbital. Dehydration increased plasma VP in control mice, from 3.3+/-0.6 to 13.3+/-4.7 pg/mL, whereas no change was noted in the AT1aKO mice, 5.1+/-0.3 versus 6.1+/-0.7 pg/mL (water versus dehydration). These results demonstrate a differential response to dehydration in mice lacking AT1a receptors. There was no evidence for AT1 receptors of any subtype in the brain regions examined and no effect of dehydration on VP secretion or brain Ang receptors.

NK1 receptor antagonist blocks angiotensin II responses in renin transgenic rat medulla oblongata.

Angiotensin (Ang) II increases substance P (SP) efflux from perfused slices of medulla oblongata, and a peptide antagonist of SP, [Leu11,psiCH2NH10-11]SP, blocks the acute hypotension and bradycardia caused by Ang II injected into the nucleus tractus solitarii (nTS) of Harlan Sprague-Dawley (SD) rats. We investigated whether the same relationships exist in (mRen2)27 renin transgenic (TG) rats, which have chronic elevations of medullary tissue Ang II levels. Ang II increased SP efflux (48% above control; P<0.01) from slices of medulla prepared from 8- to 12-week old male TG rats. Injections of Ang II (250 fmol in 30 nL) into the nTS of chloralose-urethane anesthetized TG rats produced a significant increase in pressure of 7+/-2 mm Hg before a 13+/-3 mm Hg fall in pressure. Ang II induced similar depressor responses in Hannover SD rats but no increase in pressure. After nTS injection of the NK1-selective SP antagonist CP-96,345 (30 pmol in 60 nL), Ang II-induced hypotension was blocked in both groups, as was the pressor component in hypertensive rats. Hypotensive and bradycardic effects of glutamate (0.6 nmol in 30 nL) injected into the nTS were not altered by CP-96,345. In vitro receptor autoradiography showed that the SP antagonist (10 or 100 microM) did not compete for 125I-Ang II binding in the dorsal medulla, a result suggesting that it did not interact directly with Ang II receptors. Thus, the nTS cardiovascular effects of Ang II are mediated by SP in both normotensive rats and a model of hypertension with altered endogenous levels of Ang II. These findings link Ang II-induced effects on SP release from brain slices of the medulla oblongata to acute cardiovascular actions of the peptide through an NK1 receptor.

Acute depressor actions of angiotensin II in the nucleus of the solitary tract are mediated by substance P.

Angiotensin II stimulates release of substance P from medulla oblongata slices, and low doses of substance P or angiotensin II injected into the nucleus of the solitary tract (NTS) decrease heart rate and mean arterial pressure. In this study, angiotensin II (250 fmol in 30 nl) was injected into the NTS of halothane-anesthetized male Sprague-Dawley rats before and after NTS injections of the substance P antagonist [Leu11, psi CH2NH-(10-11)]substance P (600 fmol in 60 nl). The substance P antagonist blocked the angiotensin II-induced hypotension and bradycardia (-16 +/- 3 mmHg and -24 +/- 7 beats/min before versus -0.3 +/- 1 mmHg and -2 +/- 3 beats/min after; P < 0.05). The depressor and bradycardic effects of glutamate were not altered by the substance P antagonist. In vitro receptor autoradiography showed that the substance P antagonist (10 or 100 microM) did not compete for 125I-labeled angiotensin II binding in the dorsal medulla, suggesting that the substance P antagonist does not interact directly with angiotensin II receptors. We conclude that the cardiovascular effects of angiotensin II in the NTS are mediated at least in part by substance P.

Hypothalamic substance P release. Attenuated angiotensin responses in mRen2(27) transgenic rats.

Increases in arterial pressure and paraventricular nucleus vasopressin release in response to intracerebroventricular injections of angiotensin peptides are blunted in mRen2(27) renin transgenic [TG(+)] rats. Intraventricular injections of tachykinin peptides mimic several of the actions of angiotensin peptides, and angiotensin peptides evoke substance P release from hypothalamic brain slices. The present study assessed whether diminished substance P release occurs in response to angiotensin peptides in TG(+) rats. Systolic blood pressure at 8 to 12 weeks of age averaged 197 +/- 4 mm Hg (n = 20; P < .05) in TG(+) rats compared with 123 +/- 4 mm Hg in normotensive control [TG(-)] rats (n = 18). Body weight was lower in hypertensive than in normotensive rats (305 +/- 14 versus 344 +/- 13 g, respectively; P < .05). Brain slices from hypothalamus were perfused at 37 degrees C with oxygenated Krebs' bicarbonate buffer. Substance P was measured before (basal) and during perfusion with either Krebs' buffer (control) or 2 mumol/L angiotensin-(1-7) or angiotensin II. Basal substance P release was 92 +/- 10 pg/g wet tissue in TG(+) and 98 +/- 12 pg/g in TG(-) rats (P > .05). Angiotensin-(1-7) and angiotensin II significantly increased substance P release from hypothalamus of TG(-) rats (82% and 70% above control: P < .05) but not TG(+) rats. These studies further support the hypothesis that the cardiovascular effects of angiotensin peptides are mediated in part by substance P and that this relationship is blunted in a hypertensive model that results from excess tissue production of angiotensins.

Interactions of nonpeptide angiotensin II receptor antagonists at imidazoline/guanidinium receptor sites in rat forebrain.

Imidazoline/guanidinium receptive sites (IGRS) are shown to be present in the subfornical organ and hypothalamic arcuate nucleus by a derivative of cirazoline, 2-(3-amino-4-[125I]iodophenoxy)methylimidazoline ([125I]AMIPI). Because many of the nonpeptide angiotensin II (Ang II) receptor antagonists contain imidazole ring structures, they may interact with IGRS. Therefore, we studied competitive activity of Ang II and several nonpeptide Ang II receptor antagonists [DuP 753 (losartan), EXP 3174, CV11974, and PD123319] at IGRS in rat forebrain. The results showed specific binding of 944 +/- 169 fmol/mg protein in the subfornical organ (n = 11) and of 367 +/- 27 fmol/mg protein in the arcuate nucleus (n = 6) at 0.4 nM [125I]AMIPI, as defined by competition with 10 microM cirazoline. Specific [125I]AMIPI binding was competed for completely by 10 microM idazoxan or clonidine as further characterization of IGRS. Ang II and the nonpeptide AT1 and AT2 antagonists did not significantly compete for specific [125I]AMIPI binding in either brain region at concentrations of 10 microM (< 20% competition with each compound), which is 10- to 100-fold higher than the concentration necessary to compete completely for their respective Ang II receptor subtypes. Only at the highest concentration (100 microM) did losartan compete significantly for binding (56 +/- 8%). Therefore, Ang II receptor antagonists interact with IGRS in rat forebrain cardiovascular areas only at high concentrations.