Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.

Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometre-scale resolution. With high-resolution images of predation in a native (hydrated, unstained) state, we discover several surprising features of the process, including macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following growth and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid.

Structure and Function of the Dot/Icm T4SS.

The Legionella pneumophila Dot/Icm type IV secretion system (T4SS) delivers effector proteins into host cells during infection. Despite its significance as a potential drug target, our current understanding of its atomic structure is limited to isolated subcomplexes. In this study, we used subtomogram averaging and integrative modeling to construct a nearly-complete model of the Dot/Icm T4SS accounting for seventeen protein components. We locate and provide insights into the structure and function of six new components including DotI, DotJ, DotU, IcmF, IcmT, and IcmX. We find that the cytosolic N-terminal domain of IcmF, a key protein forming a central hollow cylinder, interacts with DotU, providing insight into previously uncharacterized density. Furthermore, our model, in combination with analyses of compositional heterogeneity, explains how the cytoplasmic ATPase DotO is connected to the periplasmic complex via interactions with membrane-bound DotI/DotJ proteins. Coupled with in situ infection data, our model offers new insights into the T4SS-mediated secretion mechanism.

Functional characterization and transcriptional repression by Lacticaseibacillus paracasei DinJ-YafQ.

DinJ-YafQ is a bacterial type II TA system formed by the toxin RNase YafQ and the antitoxin protein DinJ. The activity of YafQ and DinJ has been rigorously studied in Escherichia coli, but little has been reported about orthologous systems identified in different microorganisms. In this work, we report an in vitro and in vivo functional characterization of YafQ and DinJ identified in two different strains of Lacticaseibacillus paracasei and isolated as recombinant proteins. While DinJ is identical in both strains, the two YafQ orthologs differ only for the D72G substitution in the catalytic site. Both YafQ orthologs digest ribosomal RNA, albeit with different catalytic efficiencies, and their RNase activity is neutralized by DinJ. We further show that DinJ alone or in complex with YafQ can bind cooperatively to a 28-nt inverted repeat overlapping the -35 element of the TA operon promoter. Atomic force microscopy imaging of DinJ-YafQ in complex with DNA harboring the cognate site reveals the formation of different oligomeric states that prevent the binding of RNA polymerase to the promoter. A single amino acid substitution (R13A) within the RHH DNA-binding motif of DinJ is sufficient to abolish DinJ and DinJ-YafQ DNA binding in vitro. In vivo experiments confirm the negative regulation of the TA promoter by DinJ and DinJ-YafQ and unveil an unexpected high expression-related toxicity of the gfp reporter gene. A model for the binding of two YafQ-(DinJ)2-YafQ tetramers to the promoter inverted repeat showing the absence of protein-protein steric clash is also presented. KEY POINTS: • The RNase activity of L. paracasei YafQ toxin is neutralized by DinJ antitoxin. • DinJ and DinJ-YafQ bind to an inverted repeat to repress their own promoter. • The R13A mutation of DinJ abolishes DNA binding of both DinJ and DinJ-YafQ.

Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines.

Polycationic resurfaced proteins hold great promise as cell-penetrating bioreagents but their use as carriers for the intracellular delivery of peptide immuno-epitopes has not thus far been explored. Here, we report on the construction and functional characterization of a positively supercharged derivative of Pyrococcus furiosus thioredoxin (PfTrx), a thermally hyperstable protein we have previously validated as a peptide epitope display and immunogenicity enhancing scaffold. Genetic conversion of 13 selected amino acids to lysine residues conferred to PfTrx a net charge of +21 (starting from the -1 charge of the wild-type protein), along with the ability to bind nucleic acids. In its unfused form, +21 PfTrx was readily internalized by HeLa cells and displayed a predominantly cytosolic localization. A different intracellular distribution was observed for a +21 PfTrx-eGFP fusion protein, which although still capable of cell penetration was predominantly localized within endosomes. A mixed cytosolic/endosomal partitioning was observed for a +21 PfTrx derivative harboring three tandemly repeated copies of a previously validated HPV16-L2 (aa 20-38) B-cell epitope grafted to the display site of thioredoxin. Compared to its wild-type counterpart, the positively supercharged antigen induced a faster immune response and displayed an overall superior immunogenicity, including a substantial degree of self-adjuvancy. Altogether, the present data point to +21 PfTrx as a promising novel carrier for intracellular antigen delivery and the construction of potentiated recombinant subunit vaccines.

Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System.

The bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS. Mutant analysis suggests a relationship between this novel structure and the fT3SS, but not the NF-T3SS. While the function of this novel structure remains unknown, we hypothesize that either some of the fT3SS proteins assemble within the hat-like structure, perhaps including the fT3SS core complex, or that fT3SS components regulate other proteins that form part of this novel structure. IMPORTANCE The type III secretion system (T3SS) is a fascinating suite of proteins involved in building diverse macromolecular systems, including the bacterial flagellar motility machine, the injectisome machinery that bacteria use to inject effector proteins into host cells, and probably membrane nanotubes which connect bacterial cells. Here, we accidentally discovered a novel inner membrane-associated complex related to the flagellar T3SS. Examining our lab database, which is comprised of more than 40,000 cryo-tomograms of dozens of species, we discovered that this novel structure is both ubiquitous and ancient, being present in highly divergent classes of bacteria. Discovering a novel, widespread structure related to what are among the best-studied molecular machines in bacteria will open new venues for research aiming at understanding the function and evolution of T3SS proteins.

Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin-Antitoxin Systems.

A large number of bacterial toxin-antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cell transformation, culturing, and stability of the transformants, due to the expression of highly toxic proteins. Recently, in dealing with the type I Lpt/RNAII and the type II YafQ/DinJ TA systems, we encountered several of these problems that urged us to optimize methodological strategies to study the phenotype of recombinant Escherichia coli host cells. In particular, we have found conditions to tightly repress toxin expression by combining the pET expression system with the E. coli C41(DE3) pLysS strain. To monitor the RNase activity of the YafQ toxin, we developed a fluorescence approach based on Thioflavin-T which fluoresces brightly when complexed with bacterial RNA. Fluorescence microscopy was also applied to reveal loss of membrane integrity associated with the activity of the type I toxin Lpt, by using DAPI and ethidium bromide to selectively stain cells with impaired membrane permeability. We further found that atomic force microscopy can readily be employed to characterize toxin-induced membrane damages.

Beyond BRCA1 and BRCA2: Deleterious Variants in DNA Repair Pathway Genes in Italian Families with Breast/Ovarian and Pancreatic Cancers.

The 5-10% of breast/ovarian cancers (BC and OC) are inherited, and germline pathogenic (P) variants in DNA damage repair (DDR) genes BRCA1 and BRCA2 explain only 10-20% of these cases. Currently, new DDR genes have been related to BC/OC and to pancreatic (PC) cancers, but the prevalence of P variants remains to be explored. The purpose of this study was to investigate the spectrum and the prevalence of pathogenic variants in DDR pathway genes other than BRCA1/2 and to correlate the genotype with the clinical phenotype. A cohort of 113 non-BRCA patients was analyzed by next-generation sequencing using a multigene panel of the 25 DDR pathways genes related to BC, OC, and PC. We found 43 unique variants in 18 of 25 analyzed genes, 14 classified as P/likely pathogenic (LP) and 28 as variants of uncertain significance (VUS). Deleterious variants were identified in 14% of index cases, whereas a VUS was identified in 20% of the probands. We observed a high incidence of deleterious variants in the CHEK2 gene, and a new pathogenic variant was detected in the RECQL gene. These results supported the clinical utility of multigene panel to increase the detection of P/LP carriers and to identify new actionable pathogenic gene variants useful for preventive and therapeutic approaches.

Functional characterization of the type I toxin Lpt from Lactobacillus rhamnosus by fluorescence and atomic force microscopy.

Lpt is a 29 amino acid long type I toxin identified in the plasmid DNA of wild Lactobacillus rhamnosus strains isolated from food. We previously reported that transcription of the encoding gene was upregulated under nutritional starvation conditions mimicking cheese ripening environment. The heterologous expression of the Lpt peptide in E. coli resulted in cell growth inhibition, nucleoid condensation and compromised integrity of the cell membrane. Fusion of the Lpt peptide with the fluorescent protein mCherry allowed to visualize the accumulation of the peptide into the membrane, while mutagenesis experiments showed that either the insertion of a negatively charged amino acid into the hydrophobic α-helix or deletion of the hydrophilic C-terminal region, leads to a non-toxic peptide. AFM imaging of Lpt expressing E. coli cells has revealed the presence of surface defects that are compatible with the loss of portions of the outer membrane bilayer. This observation provides support for the so-called "carpet" model, by which the Lpt peptide is supposed to destabilize the phospholipid packing through a detergent-like mechanism leading to the removal of small patches of bilayer through micellization.

Positive margins (R1) risk factors in breast cancer conservative surgery.

BACKGROUND: The primary goal in conservative breast cancer surgery is the complete excision of the tumor, but at the same time attempting to obtain a satisfactory postoperative esthetic result. The notion of "No Ink on Tumor" that indicates exclusively the presence of tumor cells on the inked surface of the surgical specimen is now the gold standard; however, the problem of the free margin is still a fundamental topic of debate that has not yet found a definitive solution. METHODS: Our retrospective analysis takes into account 1440 patients undergoing breast conservative surgery, from October 2004 to November 2018, all treated at the breast unit of our institution. RESULTS: Positive margins (R1) rate was 10.2% (147 cases out of 1440). Overall survival was 95% at 5 years and 89% at 10 years. No differences in mortality and local recurrence rate between R0 and R1 patients were found. Half of the R1 patients underwent secondary surgery with enlargement of margins, while in the other half we performed direct mastectomy. Among the analyzed variables, age, histological size, histological type, grading, multifocality, lympho-vascular invasion and lymph node status were significantly correlated with the R1 status. The multivariate analysis shows  the association of age and surgical technique (oncoplastic) with R1 status. CONCLUSION: Further studies will allow the creation of a statistical model, for better pre-operative prediction of patients with higher risk of R1 and better selection of patients to be candidates for conservative surgery.

Identification and first characterization of DinJ-YafQ toxin-antitoxin systems in Lactobacillus species of biotechnological interest.

DinJ-YafQ is a type II TA system comprising the ribosome-dependent RNase YafQ toxin and the DinJ antitoxin protein. Although the module has been extensively characterized in Escherichia coli, little information is available for homologous systems in lactic acid bacteria. In this study, we employed bioinformatics tools to identify DinJ-YafQ systems in Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus species, commonly used in biotechnological processes. Among a total of nineteen systems found, two TA modules from Lactobacillus paracasei and two modules from Lactobacillus rhamnosus wild strains were isolated and their activity was verified by growth assays in Escherichia coli either in liquid and solid media. The RNase activity of the YafQ toxins was verified in vivo by probing mRNA dynamics and metabolism with single-cell Thioflavin T fluorescence. Our findings demonstrate that, albeit DinJ-YafQ TA systems are widely distributed in lactic acid bacteria, only few are fully functional, while others have lost toxicity even though they maintain high sequence identity with wild type YafQ and a likely functional antitoxin protein.

Rapid detection of copy number variations and point mutations in BRCA1/2 genes using a single workflow by ion semiconductor sequencing pipeline.

Molecular analysis of BRCA1 (MIM# 604370) and BRCA2 (MIM #600185) genes is essential for familial breast and ovarian cancer prevention and treatment. An efficient, rapid, cost-effective accurate strategy for the detection of pathogenic variants is crucial. Mutations detection of BRCA1/2 genes includes screening for single nucleotide variants (SNVs), small insertions or deletions (indels), and Copy Number Variations (CNVs). Sanger sequencing is unable to identify CNVs and therefore Multiplex Ligation Probe amplification (MLPA) or Multiplex Amplicon Quantification (MAQ) is used to complete the BRCA1/2 genes analysis. The rapid evolution of Next Generation Sequencing (NGS) technologies allows the search for point mutations and CNVs with a single platform and workflow. In this study we test the possibilities of NGS technology to simultaneously detect point mutations and CNVs in BRCA1/2 genes, using the OncomineTM BRCA Research Assay on Personal Genome Machine (PGM) Platform with Ion Reporter Software for sequencing data analysis (Thermo Fisher Scientific). Comparison between the NGS-CNVs, MLPA and MAQ results shows how the NGS approach is the most complete and fast method for the simultaneous detection of all BRCA mutations, avoiding the usual time consuming multistep approach in the routine diagnostic testing of hereditary breast and ovarian cancers.

Role of frozen section in sentinel lymph node biopsy for breast cancer in the era of the ACOSOG Z0011 and IBCSG 23-10 trials.

BACKGROUND: Intraoperative frozen sections (FS) of sentinel lymph nodes (SLN) were evaluated to avoid the need for deferred axillary lymph node dissection (ALND) in patients with early breast cancer (EBC). However, FS has low sensitivity for detecting micro-metastases (<2 mm), resulting in patients who later undergo deferred ALND. The aim of the study was to determine the best clinical approach for selecting patients who would derive real benefit from ALND, as well as to minimize the functional and psychological damage caused by delayed surgery, and the risk of undertreating EBC patients. METHODS: This study evaluated 1453 patients with early breast cancer (EBC) who underwent SLN biopsy, FS and definitive evaluation. Causes of discrepancies between SLN biopsy and FS results and the need for further surgery were evaluated. RESULTS: A total of 1226 (86%) patients underwent FS; of these patients, 146 (11.9%) were false negatives. The global sensitivity of FS in detecting both macro and micrometastases was 53.7%. Although ACOSOG Z0011 criteria found that ALND could be avoided in 236 patients, 40 (17%) of these had >3 positive axillary lymph nodes. In contrast, application of the IBCSG 23-10 trial criteria, found that only three patients (3.1%) had >3 positive axillary lymph nodes. CONCLUSIONS: FS has a low sensitivity in detecting micrometastases (19%), but a reasonable sensitivity for macrometastases (75%). Most false negatives were smaller metastases (mean 2.1 mm) and more likely in patients with infiltrating lobular carcinoma. Retrospective modelling of the IBCSG 23-10 criteria reduced the percentage of patients requiring deferred surgery from 12% to 4%. Guidelines recommend irradiation of lymph node drainage stations in patients with ≥4 axillary metastatic lymph nodes. Omission of ALND from 40% of patients who met Z0011 criteria would have resulted in their undertreatment. This risk decreases to 3% by omitting axillary clearing only in patients with micrometastases.

Broadly neutralizing antiviral responses induced by a single-molecule HPV vaccine based on thermostable thioredoxin-L2 multiepitope nanoparticles.

Vaccines targeting the human papillomavirus (HPV) minor capsid protein L2 are emerging as chemico-physically robust and broadly protective alternatives to the current HPV (L1-VLP) vaccines. We have previously developed a trivalent L2 vaccine prototype exploiting Pyrococcus furiosus thioredoxin (PfTrx) as a thermostable scaffold for the separate presentation of three distinct HPV L2(20-38) epitopes. With the aim of achieving a highly immunogenic, yet simpler and more GMP-production affordable formulation, we report here on a novel thermostable nanoparticle vaccine relying on genetic fusion of PfTrx-L2 with the heptamerizing coiled-coil polypeptide OVX313. A prototype HPV16 monoepitope version of this nanoparticle vaccine (PfTrx-L2-OVX313; median radius: 8.6 ± 1.0 nm) proved to be approximately 10-fold more immunogenic and with a strikingly enhanced cross-neutralization capacity compared to its monomeric counterpart. Vaccine-induced (cross-)neutralizing responses were further potentiated in a multiepitope derivative displaying eight different L2(20-38) epitopes, which elicited neutralizing antibodies against 10 different HPVs including three viral types not represented in the vaccine. Considering the prospective safety of the PfTrx scaffold and of the OVX313 heptamerization module, PfTrx-OVX313 nanoparticles lend themselves as robust L2-based immunogens with a high translational potential as a 3rd generation HPV vaccine, but also as a novel and extremely versatile peptide-antigen presentation platform.

Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor.

BACKGROUND: GabR is a transcriptional regulator belonging to the MocR/GabR family, characterized by a N-terminal wHTH DNA-binding domain and a C-terminal effector binding and/or oligomerization domain, structurally homologous to aminotransferases (ATs). In the presence of γ-aminobutyrate (GABA) and pyridoxal 5'-phosphate (PLP), GabR activates the transcription of gabT and gabD genes involved in GABA metabolism. METHODS: Here we report a biochemical and atomic force microscopy characterization of Bacillus subtilis GabR in complex with DNA. Complexes were assembled in vitro to study their stoichiometry, stability and conformation. RESULTS: The fractional occupancy of the GabR cognate site suggests that GabR binds as a dimer with Kd of 10nM. Upon binding GabR bends the DNA by 80° as measured by anomalous electrophoretic mobility. With GABA we observed a decrease in affinity and conformational rearrangements compatible with a less compact nucleo-protein complex but no changes of the DNA bending angle. By employing promoter and GabR mutants we found that basic residues of the positively charged groove on the surface of the AT domain affect DNA affinity. CONCLUSIONS: The present data extend current understanding of the GabR-DNA interaction and the effect of GABA and PLP. A model for the GabR-DNA complex, corroborated by a docking simulation, is proposed. GENERAL SIGNIFICANCE: Characterization of the GabR DNA binding mode highlights the key role of DNA bending and interactions with bases outside the canonical direct repeats, and might be of general relevance for the action mechanism of MocR transcription factors.

Metal-responsive promoter DNA compaction by the ferric uptake regulator.

Short-range DNA looping has been proposed to affect promoter activity in many bacterial species and operator configurations, but only few examples have been experimentally investigated in molecular detail. Here we present evidence for a metal-responsive DNA condensation mechanism controlled by the Helicobacter pylori ferric uptake regulator (Fur), an orthologue of the widespread Fur family of prokaryotic metal-dependent regulators. H. pylori Fur represses the transcription of the essential arsRS acid acclimation operon through iron-responsive oligomerization and DNA compaction, encasing the arsR transcriptional start site in a repressive macromolecular complex. A second metal-dependent regulator NikR functions as nickel-dependent anti-repressor at this promoter, antagonizing the binding of Fur to the operator elements responsible for the DNA condensation. The results allow unifying H. pylori metal ion homeostasis and acid acclimation in a mechanistically coherent model, and demonstrate, for the first time, the existence of a selective metal-responsive DNA compaction mechanism controlling bacterial transcriptional regulation.

Importance of perforating vessels in nipple-sparing mastectomy: an anatomical description.

BACKGROUND: Nipple-sparing mastectomy (NSM), understood as an oncologically valid procedure, is relatively new, and is an evolution of traditional mastectomy, particularly in relation to breast-conserving surgery. The anterior perforating branches are responsible for the cutaneous vascularization of the breast skin, and their preservation is a fundamental step to avoid possible postoperative necrosis. Therefore, evaluating the potential complications of cancer-related reconstructive surgical procedures such as NSM, both the distance of the tumoral lesion from the skin and the surgical incision site should be carefully considered. The preferred site of incision corresponds to the inframammary fold or possibly the periareolar area. METHODS: We retrospectively reviewed 113 patients who underwent NSM from January 2005 to October 2012 to evaluate skin complications. The anatomical study was performed by magnetic resonance imaging of the breast. RESULTS: Only one of the 113 women who had undergone a NSM procedure had total necrosis (0.9%) and six patients had partial necrosis (5.8%) of the nipple-areola complex.

High-resolution, handheld camera use for occult breast lesion localization plus sentinel node biopsy (SNOLL): a single-institution experience with 186 patients.

BACKGROUND: Sentinel node and occult lesion localization (SNOLL) calls for a combination of two specific procedures: intraoperative detection of sentinel lymph node (SLN) via gamma probe and radioguided occult lesion localization (ROLL). This applies to nonpalpable invasive breast cancer or high-grade in situ carcinoma. As opposed to standard techniques, today's handheld gamma cameras enable intraoperative scintigraphic images. METHODS: A cohort (N = 186) of consecutive patients with breast cancer was subjected to radioguided conservative surgery (quadrantectomy and SLN biopsy), using a standard gamma probe and a high-resolution handheld camera. Intraoperative SLN frozen section was also performed. RESULTS: Neoplastic lesions were removed in 99.4% of all patients, and SLN biopsy was achieved in 99%. Of the 137 patients with invasive cancer, SLN metastasis was confirmed in 21. In 12% of patients, a second operation was required for close or tumor-positive surgical margins. DISCUSSION: This combination of procedures represents an improvement in the surgical management of occult breast carcinomas and is the method of choice for accurate tumor localization and SLN biopsy. Handheld cameras have the potential to become highly useful intraoperative aids.

Exclusive electron intraoperative radiotherapy in early-stage breast cancer: a monoinstitutional experience.

AIM: To evaluate the effectiveness of intra-operative radiotherapy (IORT) in breast cancer in terms of local control, esthetic results and disease-free survival. PATIENTS AND METHODS: From June 2007 to October 2011, 110 patients with early-stage breast cancer were submitted to quadrantectomy and IORT. A total dose of 21 Gy prescribed at 90-100% isodose was delivered in all cases. Patients were evaluated after surgery for early and late complications. RESULTS: Median follow-up was 27 (range: 2-54) months. In 10 patients (9.1%), breast ultrasound showed liponecrosis. Six patients (5.5%) developed grade 2 fibrosis. Disease-free survival rates at 2 and 3 years were 96.8% and 92.9 %. Three patients (2.7%) developed local recurrence, two patients (1.8%) distant metastasis. Two patients died. The 2- and 3-year overall survival rates were 100% and 97.3%, respectively. CONCLUSION: IORT could be an appropriate therapeutic alternative in selected patients although it remains investigational; longer follow-up to confirm these results is required.

Non-sentinel lymph node metastases in breast cancer patients with a positive sentinel lymph node: validation of five nomograms and development of a new predictive model.

AIMS AND BACKGROUND: Discordance of intraoperative analysis with definitive histology of the sentinel lymph node in breast cancer leads to completion axillary lymph node dissection, which only in 35-50% shows additional nodal metastases. The aim of the study was to identify individual patient risk for non-sentinel lymph node metastases by validating several statistical methods present in the recent literature and by developing a new tool with the final goal of avoiding unnecessary completion axillary lymph node dissection. METHODS: We retrospectively evaluated 593 primary breast cancer patients. Completion axillary lymph node dissection was performed in 139 with a positive sentinel lymph node. The predictive accuracy of five published nomograms (MSKCC, Tenon, Cambridge, Stanford and Gur) was measured by the area under the receiver operating characteristic curve. We then developed a new logistic regression model to compare performance. Our model was validated by the leave-one-out cross-validation method. RESULTS: In 53 cases (38%), we found at least one metastatic non-sentinel lymph node. All the selected nomograms showed values greater than the 0.70 threshold, and our model reported a value of 0.77 (confidence interval = 0.69-0.86 and error rate = 0.28) and 0.72 (confidence interval = 0.63-0.81, error rate = 0.28) after the validation. With a 5% cutoff value, sensitivity was 98% and specificity 9%, for a cutoff of 10%, 96% and 2%, respectively. CONCLUSIONS: All the nomograms were good discriminators, but the alternative developed model showed the best predictive accuracy in this Italian breast cancer sample. We still confirm that these models, very accurate in the institution of origin, require a new validation if used on other populations of patients.

Is complete axillary dissection necessary for all patients with positive findings on sentinel lymph node biopsy? Validation of a breast cancer nomogram for predicting the likelihood of a non-sentinel lymph node.

AIM AND BACKGROUND: Axillary dissection in patients positive for sentinel lymph nodes is currently under discussion in the literature, since approximately only 50% of such patients has metastases in the remaining lymph nodes. To identify patients at risk for non-sentinel lymph nodes metastases, a nomogram was developed by the Breast Service of the Memorial Sloan-Kettering Cancer Center. The aim of this study was to assess the nomogram's predictive accuracy in a population of Italian breast cancer patients in our hospital. MATERIALS AND METHODS: The system of calculation used as variables prognostic factors of breast cancer: pathologic size, tumor type and nuclear grade, lymphovascular invasion, multifocality, estrogen receptor status, method of detection of the sentinel lymph nodes metastases (frozen section, serial hematoxylin-eosin, routine hematoxylin-eosin, and immunohistochemistry), number of positive and number of negative sentinel lymph nodes. RESULTS AND CONCLUSIONS: To measure the discrimination of the nomogram, a receiver-operating characteristic curve was construed, and the area under the curve was calculated. However, the area under the curve was 0.72, a very high value considering that the limit of acceptability is 0.70-0.80. The calculation system developed by the Memorial Sloan-Kettering Cancer Center provides a predictive value on the histopathologic state of sentinel lymph nodes.