Prognosis of sentinel node staged patients with primary cutaneous melanoma

Background

This study investigated survival probabilities and prognostic factors in sentinel lymph node biopsy (SLNB) staged patients with cutaneous melanoma (CM) with the aim of defining subgroups of patients who are at higher risk for recurrences and who should be considered for adjuvant clinical trials.

Methods

Patients with primary CM who underwent SLNB in the Department of Dermatology, University of Tuebingen, Germany, between 1996 and 2009 were included into this study. Survival probabilities and prognostic factors were evaluated by Kaplan-Meier and multivariate Cox proportional hazard models.

Results

1909 SLNB staged patients were evaluated. Median follow-up time was 44 months. Median tumor thickness was 1.8 mm, ulceration was present in 31.8% of cases. The 5-year Overall Survival (OS) was 90.3% in SLNB negative patients (IB 96.2%, IIA 87.0%, IIB 78.1%, IIC 72.6%). Patients with micrometastases (stage IIIA/B) had a 5-year OS rate of 70.9% which was clearly less favorable than for stages I–II. Multivariate analysis revealed tumor thickness, ulceration, body site, histopathologic subtype and SLNB status as independent significant prognostic factors.

Conclusion

Survival rates of patients with primary CM in stages I–II were shown to be much more favorable than previously reported from non sentinel node staged collectives. For future clinical trials, sample size calculations should be adapted using survival probabilities based on sentinel node staging.     

Different nitrogen sources modulate activity but not expression of glutamine synthetase in arbuscular mycorrhizal fungi

Glutamine synthetase (GS) is a central enzyme of nitrogen metabolism that allows assimilation of nitrogen and biosynthesis of glutamine. We isolated the cDNA encoding GS from two arbuscular mycorrhizal fungi, Glomus mosseae (GmGln1) and Glomus intraradices (GiGln1). The deduced protein orthologues have a high degree of similarity (92%) with each other as well as with GSs from other fungi. GmGln1 was constitutively expressed during all stages of the fungal life cycle, i.e., spore germination, intraradical and extraradical mycelium. Feeding experiments with different nitrogen sources did not induce any change in the mRNA level of both genes independent of the symbiotic status of the fungus. However, GS activity of extraradical hypahe in G. intraradices was considerably modulated in response to different nitrogen sources. Thus, in a N re-supplementation time-course experiment, GS activity responded quickly to addition of nitrate, ammonium or glutamine. Re-feeding with ammonium produced a general increase in GS activity when compared with hyphae grown in nitrate as a sole N source.     

Quantitative Measurement of Melanoma Spread in Sentinel Lymph Nodes and Survival

Background

Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.

Methods and Findings

We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3–123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61–2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02–2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.

Conclusions

Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically. Please see later in the article for the Editors'' Summary     

Plant signals and fungal perception during arbuscular mycorrhiza establishment

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that need their plant hosts to complete their life cycle. In the absence of the plant, germlings arrest growth after a few days and retract most of their cytoplasm back into the multinuclear spores. The spores can germinate again during more favorable conditions. How AM fungi recognize compatible host roots and activate their symbiotic program is not yet understood. However, research in this field in the last years has shed light into this topic. We, and others, have approached some of these aspects by studying changes in fungal gene expression observed at early stages of development, before and at the plant recognition stage in an attempt to identify genes and proteins featuring as key regulators in the switch between the asymbiotic and symbiotic style of life. The molecular bases of this recognition process are now starting to be understood and point to common signaling pathways shared with other microbe-plant associations and to arbuscular mycorrhiza specific signaling pathways.     

Diagnostic Accuracy of Computer-Aided Detection of Pulmonary Tuberculosis in Chest Radiographs: A Validation Study from Sub-Saharan Africa

Background

Chest radiography to diagnose and screen for pulmonary tuberculosis has limitations, especially due to inter-reader variability. Automating the interpretation has the potential to overcome this drawback and to deliver objective and reproducible results. The CAD4TB software is a computer-aided detection system that has shown promising preliminary findings. Evaluation studies in different settings are needed to assess diagnostic accuracy and practicability of use.

Methods

CAD4TB was evaluated on chest radiographs of patients with symptoms suggestive of pulmonary tuberculosis enrolled in two cohort studies in Tanzania. All patients were characterized by sputum smear microscopy and culture including subsequent antigen or molecular confirmation of Mycobacterium tuberculosis (M.tb) to determine the reference standard. Chest radiographs were read by the software and two human readers, one expert reader and one clinical officer. The sensitivity and specificity of CAD4TB was depicted using receiver operating characteristic (ROC) curves, the area under the curve calculated and the performance of the software compared to the results of human readers.

Results

Of 861 study participants, 194 (23%) were culture-positive for M.tb. The area under the ROC curve of CAD4TB for the detection of culture-positive pulmonary tuberculosis was 0.84 (95% CI 0.80–0.88). CAD4TB was significantly more accurate for the discrimination of smear-positive cases against non TB patients than for smear-negative cases (p-value<0.01). It differentiated better between TB cases and non TB patients among HIV-negative compared to HIV-positive individuals (p<0.01). CAD4TB significantly outperformed the clinical officer, but did not reach the accuracy of the expert reader (p = 0.02), for a tuberculosis specific reading threshold.

Conclusion

CAD4TB accurately distinguished between the chest radiographs of culture-positive TB cases and controls. Further studies on cost-effectiveness, operational and ethical aspects should determine its place in diagnostic and screening algorithms.     

Tumor Imaging and Targeting Potential of an Hsp70-Derived 14-Mer Peptide

Background

We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding ’healthy‘ tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro.

Methodology/Principal Findings

The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.

Conclusions/Significance

This study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70.     

KLUH/CYP78A5 promotes organ growth without affecting the size of the early primordium

Mobile signals play a key role in controlling the growth of organisms. In Arabidopsis, the cytochrome P450 CYP78A5/KLUH (KLU) non-cell autonomously stimulates cell proliferation in developing organs. In a recent study, we determined the range of KLU action, using a widely applicable system to predictably generate chimaeric plants. We showed that KLU acts not only within individual floral organs or flowers, but that its overall activity level is integrated across an inflorescence to determine organ size. Here, we address the question at which stage of petal development KLU acts to promote growth. We demonstrate that the size of the very young petal primordium in klu mutants is not altered, supporting the conclusion that KLU acts during later stages of organ outgrowth and a correspondingly longer range of the presumed KLU-dependent growth signal.Key words: Arabidopsis, KLUH, floral organ growth, signaling, flower, growth coordination