Diversity and complexity of mouse allergens in urine, house dust, and allergen extracts assessed with an immuno-allergomic approach.

BACKGROUND: Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone-binding lipocalins called major urinary proteins (MUPs). METHODS: We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two-dimensional electrophoresis and immunoblotting; liquid chromatography-high-resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N-66 residue of MUP3. RESULTS: Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice. CONCLUSIONS: Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content.

Prognostic values of cancer associated macrophage-like cells (CAML) enumeration in metastatic breast cancer.

PURPOSE: Circulating cancer associated macrophage-like cells (CAMLs) have been detected in the peripheral blood of patients with solid tumors including breast cancer. However, the prognostic relevance of CAMLs in metastatic breast cancer (MBC) has not been evaluated. In the present study, we aimed to measure CAMLs and circulating tumor cells (CTCs) at baseline and examine their prognostic value in patients with MBC. METHODS: Peripheral blood samples from 127 MBC patients were collected at baseline before starting a new treatment. The detection and enumeration of CAMLs and CTCs in 7.5 ml whole blood were performed using the CellSearch™ system. The associations of CAMLs and CTCs with the progression-free survival (PFS) and overall survival (OS) in the patients were evaluated using Kaplan-Meier curves and Cox proportional hazards modeling. RESULTS: Among 127 MBC patients, 21 (16.5%) were detected with CAMLs and 38 (29.9%) had elevated CTCs (≥5 CTCs/7.5 ml). Patients with CAMLs at baseline had worse PFS and OS with an adjusted hazard ratio (HR) of 1.75 (95% CI 1.03-2.98, P = 0.0374) and 3.75 (95% CI 1.52-9.26, P = 0.0042), compared to patients without CAMLs. Compared with patients with <5 CTCs and without CAMLs, patients with <5 CTCs and with CAMLs, with ≥5 CTCs but without CAMLs, or with ≥5 CTCs and with CAMLs, had an increasing trend of risk of disease progression (HR = 0.84, 3.42 and 4.04 respectively, P for trend <0.0001) and death (HR = 2.66, 6.14, and 9.13, respectively, P for trend <0.0001). CONCLUSION: Baseline enumeration of individual CAMLs is an independent indicator for MBC patients' survival. Evaluation of CAMLs in peripheral blood might provide a potential biomarker with additional prognostic values over CTC enumeration alone in MBC patients.

Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer.

PURPOSE: Circulating tumor cell (CTC) is a well-established prognosis predictor for metastatic breast cancer (MBC), and CTC-cluster exhibits significantly higher metastasis-promoting capability than individual CTCs. Because measurement of CTCs and CTC-clusters at a single time point may underestimate their prognostic values, we aimed to analyze longitudinally collected CTCs and CTC-clusters in MBC prognostication. METHODS: CTCs and CTC-clusters were enumerated in 370 longitudinally collected blood samples from 128 MBC patients. The associations between baseline, first follow-up, and longitudinal enumerations of CTCs and CTC-clusters with patient progression-free survival (PFS) and overall survival (OS) were analyzed using Cox proportional hazards models. RESULTS: CTC and CTC-cluster counts at both baseline and first follow-up were significantly associated with patient PFS and OS. Time-dependent analysis of longitudinally collected samples confirmed the significantly unfavorable PFS and OS in patients with ≥5 CTCs, and further demonstrated the independent prognostic values by CTC-clusters compared to CTC-enumeration alone. Longitudinal analyses also identified a link between the size of CTC-clusters and patient OS: compared to the patients without any CTC, those with 2-cell CTC-clusters and ≥3-cell CTC-clusters had a hazard ratio (HR) of 7.96 [95 % confidence level (CI) 2.00-31.61, P = 0.003] and 14.50 (3.98-52.80, P < 0.001), respectively. CONCLUSIONS: In this novel time-dependent analysis of longitudinally collected CTCs and CTC-clusters, we showed that CTC-clusters added additional prognostic values to CTC enumeration alone, and a larger-size CTC-cluster conferred a higher risk of death in MBC patients.