Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Clinical and histopathological features of lentigo maligna and lentigo maligna melanoma: a retrospective analysis in Korea.

Introduction: Lentigo maligna (LM) and lentigo maligna melanoma (LMM) are rare in Asian countries. The histopathological diagnosis of LM is often challenging, and misdiagnosis is common. Although histopathologic features of LM/LMM are known, statistical analysis of them were scarcely reported. In this study, we aimed to investigate the histopathological characteristics of LM/LMM in Korean patients and identify key histopathological clues distinguishing LM from benign lentigo. Methods: We performed a retrospective study of the clinical and histopathological features of patients diagnosed with LM/LMM at our center between 2011 and 2022. We assessed the histopathological features in each case based on 16 pathological criteria according to previous literature. Pathologically confirmed cases of benign lentigo were analyzed for comparison. Results: Twenty-one patients (10 with LM and 11 with LMM) were analyzed. Several statistically significant difference existed between the features of LM and benign lentigo (N = 10), including asymmetry of overall structure (p < 0.001), cytologic atypia (p < 0.001), predominant single-cell proliferation (p < 0.001), melanocytic nests (p = 0.033), melanocytes forming rows (p = 0.003), pagetoid spread of melanocytes (p < 0.001), and hair follicle invasion by atypical melanocytes (p < 0.001). Degree of solar elastosis was more severe in group "Age ≥ 60" (p = 0.015), and group "Diameter ≥ 20 mm" (p = 0.043). Presence of elongated rete ridges were less common in the older than 60 age group (p = 0.015) and group "Diameter ≥ 20 mm." Invasion was associated with mitosis (p = 0.001, OR 49.285), multinucleated cells (p = 0.035, OR 17.769), and degree of lymphocyte infiltration (p = 0.004). Conclusion: This study investigated the clinical and histopathologic characteristics of LM and LMM in Koreans. Although histopathological diagnosis is challenging, especially in the early stages of LM, our data showed essential histopathological changes in architectural, cytological, and dermal patterns. Considering the potential aggressiveness of LM/LMM, it is essential to recognize its histopathological features and provide timely management.

Enhanced Luminescent Detection of Circulating Tumor Cells by a 3D Printed Immunomagnetic Concentrator.

Circulating tumor cells (CTCs) are an indicator of metastatic progression and relapse. Since non-CTC cells such as red blood cells outnumber CTCs in the blood, the separation and enrichment of CTCs is key to improving their detection sensitivity. The ATP luminescence assay can measure intracellular ATP to detect cells quickly but has not yet been used for CTC detection in the blood because extracellular ATP in the blood, derived from non-CTCs, interferes with the measurement. Herein, we report on the improvement of the ATP luminescence assay for the detection of CTCs by separating and concentrating CTCs in the blood using a 3D printed immunomagnetic concentrator (3DPIC). Because of its high-aspect-ratio structure and resistance to high flow rates, 3DPIC allows cancer cells in 10 mL to be concentrated 100 times within minutes. This enables the ATP luminescence assay to detect as low as 10 cells in blood, thereby being about 10 times more sensitive than when commercial kits are used for CTC concentration. This is the first time that the ATP luminescence assay was used for the detection of cancer cells in blood. These results demonstrate the feasibility of 3DPIC as a concentrator to improve the detection limit of the ATP luminescence assay for the detection of CTCs.

Probing mechanobiological role of filamin A in migration and invasion of human U87 glioblastoma cells using submicron soft pillars.

Filamin A (FLNa) belongs to an actin-binding protein family in binding and cross-linking actin filaments into a three-dimensional structure. However, little attention has been given to its mechanobiological role in cancer cells. Here, we quantitatively investigated the role of FLNa by analyzing the following parameters in negative control (NC) and FLNa-knockdown (KD) U87 glioma cells using submicron pillars (900 nm diameter and 2 µm height): traction force (TF), rigidity sensing ability, cell aspect ratio, migration speed, and invasiveness. During the initial phase of cell adhesion (< 1 h), FLNa-KD cells polarized more slowly than did NC cells, which can be explained by the loss of rigidity sensing in FLNa-KD cells. The higher motility of FLNa-KD cells relative to NC cells can be explained by the high TF exerted by FLNa-KD cells when compared to NC cells, while the higher invasiveness of FLNa-KD cells relative to NC cells can be explained by a greater number of filopodia in FLNa-KD cells than in NC cells. Our results suggest that FLNa plays important roles in suppressing motility and invasiveness of U87 cells.

Conformally Gated Surface Conducting Behaviors of Single-Walled Carbon Nanotube Thin-Film-Transistors.

Semiconducting single-walled carbon nanotubes (s-SWCNTs) have gathered significant interest in various emerging electronics due to their outstanding electrical and mechanical properties. Although large-area and low-cost fabrication of s-SWCNT field effect transistors (FETs) can be easily achieved via solution processing, the electrical performance of the solution-based s-SWCNT FETs is often limited by the charge transport in the s-SWCNT networks and interface between the s-SWCNT and the dielectrics depending on both s-SWCNT solution synthesis and device architecture. Here, we investigate the surface and interfacial electro-chemical behaviors of s-SWCNTs. In addition, we propose a cost-effective and straightforward process capable of minimizing polymers bound to s-SWCNT surfaces acting as an interfering element for the charge carrier transport via a heat-assisted purification (HAP). With the HAP treated s-SWCNTs, we introduced conformal dielectric configuration for s-SWCNT FETs, which are explored by a carefully designed wide array of electrical and chemical characterizations with finite-element analysis (FEA) computer simulation. For more favorable gate-field-induced surface and interfacial behaviors of s-SWCNT, we implemented conformally gated highly capacitive s-SWCNT FETs with ion-gel dielectrics, demonstrating field-effect mobility of ~8.19 cm2/V⋅s and on/off current ratio of ~105 along with negligible hysteresis.

Enhanced Sensing Behavior of Three-Dimensional Microfluidic Paper-Based Analytical Devices (3D-µPADs) with Evaporation-Free Enclosed Channels for Point-of-Care Testing.

Despite the potential in fabrication of microfluidic paper-based analytical devices (µPADs) for point-of-care testing (POCT) kits, the development of simple, accurate, and rapid devices with higher sensitivity remains challenging. Here, we report a novel method for 3D-µPAD fabrication with enclosed channels using vat photopolymerization to avoid fluid evaporation. In detail, height of the enclosed channels was adjusted from 0.3 to 0.17 mm by varying the UV exposure time from 1 to 4 s for the top barrier, whereas the exposure time for the bottom and side barriers was fixed. As a result, sample flow in the enclosed channels of 3D-µPADs showed lesser wicking speed with very scant evaporation compared to that in the hemi channels in the 3D-µPADs. The stoppage of evaporation in the enclosed channels significantly improved the gray intensity and uniformity in the detection zone of the 3D-µPADs, resulting in as low as 0.3 mM glucose detection. Thus 3D-µPADs with enclosed channels showed enhanced sensitivity compared to the 3D-µPADs with hemi channels when dealing with a small volume sample. Our work provides a new insight into 3D-µPAD design with enclosed channels, which redefines the methodology in 3D printing.

A multicentre, randomised, open-label, parallel-group Phase 2b study of belotecan versus topotecan for recurrent ovarian cancer.

BACKGROUND: This Phase 2b study compared the efficacy and toxicity of belotecan and topotecan in recurrent ovarian cancer. METHODS: Patients with platinum-sensitive recurrent or platinum-resistant recurrent ovarian cancer (PRROC) were randomised 1:1 to receive belotecan 0.5 mg/m2 or topotecan 1.5 mg/m2 for five consecutive days every 3 weeks. The primary endpoint was overall response rate (ORR); secondary endpoints were progression-free survival (PFS), overall survival (OS), and toxicity. RESULTS: A total of 140 (belotecan, n = 71; topotecan, n = 69) and 130 patients (belotecan, n = 66; topotecan, n = 64) were included in the intention-to-treat (ITT) and per-protocol (PP) populations. ORR did not differ significantly between the belotecan and topotecan groups (ITT, 29.6% versus 26.1%; PP, 30.3% versus 25%). Although PFS did not differ between the groups, belotecan was associated with improved OS compared with topotecan in the PP population (39.7 versus 26.6 months; P = 0.034). In particular, belotecan showed longer OS in PRROC and non-high-grade serous carcinoma (non-HGSC; PP, adjusted hazard ratios, 0.499 and 0.187; 95% confidence intervals 0.255-0.977 and 0.039-0.895). Furthermore, there were no differences in toxicities between the two groups. CONCLUSIONS: Belotecan was not inferior to topotecan in terms of overall response for recurrent ovarian cancer. CLINICAL TRIAL REGISTRATION: NCT01630018.

Three-Dimensional Paper-Based Microfluidic Analysis Device for Simultaneous Detection of Multiple Biomarkers with a Smartphone.

Paper-based microfluidic analysis devices (µPADs) have attracted attention as a cost-effective platform for point-of-care testing (POCT), food safety, and environmental monitoring. Recently, three-dimensional (3D)-µPADs have been developed to improve the performance of µPADs. For accurate diagnosis of diseases, however, 3D-µPADs need to be developed to simultaneously detect multiple biomarkers. Here, we report a 3D-µPADs platform for the detection of multiple biomarkers that can be analyzed and diagnosed with a smartphone. The 3D-µPADs were fabricated using a 3D digital light processing printer and consisted of a sample reservoir (300 µL) connected to 24 detection zones (of 4 mm in diameter) through eight microchannels (of 2 mm in width). With the smartphone application, eight different biomarkers related to various diseases were detectable in concentrations ranging from normal to abnormal conditions: glucose (0-20 mmol/L), cholesterol (0-10 mmol/L), albumin (0-7 g/dL), alkaline phosphatase (0-800 U/L), creatinine (0-500 µmol/L), aspartate aminotransferase (0-800 U/L), alanine aminotransferase (0-1000 U/L), and urea nitrogen (0-7.2 mmol/L). These results suggest that 3D-µPADs can be used as a POCT platform for simultaneous detection of multiple biomarkers.

Highly Scalable and Robust Mesa-Island-Structure Metal-Oxide Thin-Film Transistors and Integrated Circuits Enabled by Stress-Diffusive Manipulation.

The increasing interest in flexible and wearable electronics has demanded a dramatic improvement of mechanical robustness in electronic devices along with high-resolution implemented architectures. In this study, a site-specific stress-diffusive manipulation is demonstrated to fulfill highly robust and ultraflexible amorphous indium-gallium-zinc oxide (a-IGZO) thin-film transistors (TFTs) and integrated circuits. The photochemically activated combustion sol-gel a-IGZO TFTs on a mesa-structured polyimide show an average saturation mobility of 6.06 cm2 V-1 s-1 and a threshold voltage of -0.99 V with less than 9% variation, followed by 10 000 bending cycles with a radius of 125 µm. More importantly, the site-specific monolithic formation of mesa pillar-structured devices can provide fully integrated logic circuits such as seven-stage ring-oscillators, meeting the industrially needed device density and scalability. To exploit the underlying stress-diffusive mechanism, a physical model is provided by using a variety of chemical, structural, and electrical characterizations along with multidomain finite-element analysis simulation. The physical models reveal that a highly scalable and robust device can be achieved via the site-specific mesa architecture, by enabling generation of multineutral layers and fine-tuning the accumulated stresses on specific element of devices with their diffusion out into the boundary of the mesa regions.

Electron tunneling of hierarchically structured silver nanosatellite particles for highly conductive healable nanocomposites.

Healable conductive materials have received considerable attention. However, their practical applications are impeded by low electrical conductivity and irreversible degradation after breaking/healing cycles. Here we report a highly conductive completely reversible electron tunneling-assisted percolation network of silver nanosatellite particles for putty-like moldable and healable nanocomposites. The densely and uniformly distributed silver nanosatellite particles with a bimodal size distribution are generated by the radical and reactive oxygen species-mediated vigorous etching and reduction reaction of silver flakes using tetrahydrofuran peroxide in a silicone rubber matrix. The close work function match between silicone and silver enables electron tunneling between nanosatellite particles, increasing electrical conductivity by ~5 orders of magnitude (1.02×103 Scm-1) without coalescence of fillers. This results in ~100% electrical healing efficiency after 1000 breaking/healing cycles and stability under water immersion and 6-month exposure to ambient air. The highly conductive moldable nanocomposite may find applications in improvising and healing electrical parts.

Three-Dimensional Paper-Based Microfluidic Analytical Devices Integrated with a Plasma Separation Membrane for the Detection of Biomarkers in Whole Blood.

Paper-based microfluidic analytical devices (µPADs) have recently attracted attention as a point-of-care test kit because of their low cost and nonrequirement for external forces. To directly detect biomarkers in whole blood, however, they need to be assembled with a filter such as a plasma separation membrane (PSM) because the color of the blood cells interferes with the colorimetric assay. However, this assembly process is rather complicated and cumbersome, and the fluid does not uniformly move to the detection zone when the adhesion between the paper and PSM is not perfect. In this study, we report a simple three-dimensional (3D) printing method for fabricating PSM-integrated 3D-µPADs made of plastics without the need for additional assembly. In detail, PSM was coated with parylene C to prevent its dissolution from organic solvent during 3D printing. Then, the coated PSM was superimposed on the paper. Detection zones and a reservoir were printed on the paper and PSM via liquid photopolymerization, using a digital light processing printer. The limit of detection of the PSM-integrated 3D-µPADs for glucose in whole blood was 0.3 mM, and these devices demonstrated clinically relevant performance on diabetes patient blood samples. Our 3D-µPADs can also simultaneously detect multiple metabolic disease markers including glucose, cholesterol, and triglycerides in whole blood. Our results suggest that our printing method is useful for fabricating 3D-µPADs integrated with PSM for the direct detection of biomarkers in whole blood.

Dual anode single-photon avalanche diode for high-speed and low-noise Geiger-mode operation.

The after-pulsing effect is a common problem in high-speed and low-noise single-photon detection based on single-photon avalanche diodes (SPADs). This article presents a dual anode InGaAs/InP SPAD (DA-SPAD) with two separate anode output ports that can be utilized for discriminating relatively weak avalanche signals, providing a simple and robust configuration of the SPAD-based single-photon detection system. Weak avalanche signals with amplitudes below the amplitude of the parasitic capacitive response of the SPAD were easily detected by the DA-SPAD and a simple subtraction circuit. The gated Geiger-mode performance of the DA-SPAD was also investigated. At a gating frequency of 1 GHz, the detection efficiency was 20.4% with an after-pulse probability of 3.5% at a temperature of -20 °C.

Double-sided 3D printing on paper towards mass production of three-dimensional paper-based microfluidic analytical devices (3D-µPADs).

Recently, much effort has been focused on developing three-dimensional, paper-based microfluidic analytical devices (3D-µPADs) targeting in vitro diagnostics. However, 3D-µPAD fabrication typically requires tedious assembly that hinders mass production. Here, we report on a fabrication method for 3D-µPADs made of plastics without the need for additional assembly. Both sides of the paper were printed via liquid resin photopolymerization using a digital light processing (DLP) printer. The sample reservoir and detection zones are located on the top of the 3D-µPADs, and three microchannels are located on the bottom. The detection limits for glucose, cholesterol, and triglyceride (TG) in phosphate-buffered saline (PBS) were 0.3 mM, 0.2 mM, and 0.3 mM, respectively. The detectable ranges of glucose, cholesterol, and TG in human serum were 5-11 mM, 2.6-6.7 mM, and 1-2.3 mM. These results suggest that our fabrication method is suitable to mass produce 3D-µPADs with relative ease using simple fabrication processes.

Dual Mechanism of Action of 5-Nitro-1,10-Phenanthroline against Mycobacterium tuberculosis.

New chemotherapeutic agents with novel mechanisms of action are urgently required to combat the challenge imposed by the emergence of drug-resistant mycobacteria. In this study, a phenotypic whole-cell screen identified 5-nitro-1,10-phenanthroline (5NP) as a lead compound. 5NP-resistant isolates harbored mutations that were mapped to fbiB and were also resistant to the bicyclic nitroimidazole PA-824. Mechanistic studies confirmed that 5NP is activated in an F420-dependent manner, resulting in the formation of 1,10-phenanthroline and 1,10-phenanthrolin-5-amine as major metabolites in bacteria. Interestingly, 5NP also killed naturally resistant intracellular bacteria by inducing autophagy in macrophages. Structure-activity relationship studies revealed the essentiality of the nitro group for in vitro activity, and an analog, 3-methyl-6-nitro-1,10-phenanthroline, that had improved in vitro activity and in vivo efficacy in mice compared with that of 5NP was designed. These findings demonstrate that, in addition to a direct mechanism of action against Mycobacterium tuberculosis, 5NP also modulates the host machinery to kill intracellular pathogens.

Intersubband transition in lattice-matched BGaN/AlN quantum well structures with high absorption coefficients.

Intersubband absorption properties of lattice-matched BGaN/AlN quantum well (QW) structures grown on AlN substrate are theoretically investigated using an effective mass theory considering the nonparabolicity of the conduction band. These results are compared with those of GaN/AlN QW structures. The intersubband absorption coefficient of the BGaN/AlN QW structure is shown to be enhanced significantly, compared to that of the conventional GaN/AlN QW structure. This can be explained by the fact that the BGaN/AlN QW structure exhibits larger intersuband dipole moment and quasi-Fermi-level separation than the GaN/AlN QW structure, due to the increase in the carrier confinement by a larger internal field. We expect that the BGaN/AlN QW structure with a high absorption coefficient can be used for telecommunication applications at 1.55 µm under the lattice-matched condition, instead of the conventional GaN/AlN QW structure with the large strain.

Design of Rehabilitation Treatment Coach Robot.

In the rehabilitation treatment for at-home patients, it is not only costly for a patient to hire a professional therapist or personal trainer, but also time consuming for a therapist or trainer to visit all the patients. To improve the situation, we propose the rehabilitation treatment coach robot which helps the patient to do rehabilitation exercises alone. Designed economically with cheap parts, our robot provides multiple functions: rehabilitation program suggestion, rehabilitation posture correction, and emergency detection. A brief plan is presented for data collection and performance evaluation.

3D-printed microfluidic magnetic preconcentrator for the detection of bacterial pathogen using an ATP luminometer and antibody-conjugated magnetic nanoparticles.

Various types of microfluidic systems have been developed to detect bacterial pathogens. However, most of these require enrichment steps that take at least several hours when detecting bacteria that are present with a low number of cells and, in addition, fabrication requires complicated assembly steps. In this study, we report the development of 3D microfluidic magnetic preconcentrator (3DµFMP) made of plastic via 3D printing without the need for any assembly. 3DµFMP could selectively preconcentrate enterohemorrhagic Escherichia coli O157:H7 in 100mL by a factor of 700 within 1h using antibody-conjugated magnetic nanoparticles (Ab-MNPs). With the combined use of an ATP luminometer, as low as 10 E. coli O157:H7 CFU (colony forming unit)/mL could be detected in blood. These results demonstrate the feasibility of 3DµFMP as a preconcentrator to improve the detection limit of existing bacterial detection systems.

Effect of number of retrieved lymph nodes on prognosis in FIGO stage IB-IIA cervical cancer patients treated with primary radical surgery.

AIM: In the treatment of cervical cancer, the extent of lymphadenectomy is a matter of debate. The goal of the current study was to examine the question of whether the number of retrieved lymph nodes (RLN) can influence survival of patients with early stage cervical cancer. METHODS: The medical records of 180 FIGO stage IB-IIA cervical cancer patients treated with primary radical surgery were reviewed. Patients were divided into two groups: those with ≤ 40 RLN and those with > 40 RLN. Patients were also assigned to either the bulky (tumor size > 4 cm) cervical cancer group or the non-bulky (tumor size ≤ 4 cm) cervical cancer group. RESULTS: The number of RLN had a statistically significant effect on both disease-free survival (P = 0.04) and overall survival (P = 0.02) of all patients. Patients with > 40 RLN had better prognoses than those with ≤ 40 RLN. In the bulky cervical cancer group, the number of RLN was an independent prognostic factor. In multivariate analysis for the bulky cervical cancer group, > 40 RLN had a significant positive effect on disease-free survival (adjusted hazard ratio, 0.36; 95% confidence interval, 0.13-0.97) and overall survival (adjusted hazard ratio, 0.23; 95% confidence interval, 0.06-0.90). However, number of RLN was not an independent prognostic factor in the non-bulky cervical cancer group. CONCLUSIONS: A more extensive lymphadenectomy increased the survival of bulky cervical cancer patients. This finding may be helpful in determining surgical extent before surgery for cervical cancer.

IinQ attenuates systemic inflammatory responses via selectively impairing the Myddosome complex formation upon TLR4 ligation.

A specific small-molecule inhibitor of the TLR4 signaling complex upstream of the IKK would likely provide therapeutic benefit for NF-κB-mediated inflammatory disease. We previously identified brazilin as a selective upstream IKK inhibitor targeting the Myddosome complex. In this study, using a cell-based ubiquitination assay for IRAK1 and a chemical library comprising a series of structural analogues of brazilin, a novel small molecule, 2-hydroxy-5,6-dihydroisoindolo[1,2-a]isoquinoline-3,8-dione (IinQ), was identified as a selective and potent inhibitor of IRAK1-dependent NF-κB activation upon TLR4 ligation. In RAW264.7 macrophages, IinQ drastically suppressed activation of upstream IKK signaling events including membrane-bound IRAK1 ubiquitination and IKK phosphorylation by the TLR4 ligand, resulting in reduced expression of proinflammatory mediators including IL-6, TNF-α, and nitric oxide. Interestingly, IinQ did not suppress NF-κB activation via the TLR3 ligand, DNA damaging agents, or a protein kinase C activator, indicating IinQ is specific for TLR4 signaling. Analysis of upstream signaling events further confirmed that IinQ disrupts the MyD88-IRAK1-TRAF6 complex formation induced by LPS treatment, without affecting TLR4 oligomerization. Moreover, intravenous administration of IinQ significantly reduced lethality and attenuated systemic inflammatory responses in an in vivo mouse model of endotoxin shock following LPS challenge. Thus, IinQ represents a novel class of brazilin analogues with improved potency and specificity toward disruption of Myddosome complex formation in TLR4 signaling, indicating that IinQ may be a promising therapeutic candidate for the treatment of systemic inflammatory diseases.

The influence of number of high risk factors on clinical outcomes in patients with early-stage cervical cancer after radical hysterectomy and adjuvant chemoradiation.

OBJECTIVE: The purpose of this study was to evaluate the prognosis according to the number of high risk factors in patients with high risk factors after radical hysterectomy and adjuvant chemoradiation therapy for early stage cervical cancer. METHODS: Clinicopathological variables and clinical outcomes of patients with FIGO (International Federation of Gynecology and Obstetrics) stage IB1 to IIA cervical cancer who had one or more high risk factors after radical hysterectomy and adjuvant chemoradiation therapy were retrospectively analyzed. Patients were divided into two groups according to the number of high risk factors (group 1, single high risk factor; group 2, two or more high risk factors). RESULTS: A total of 93 patients were enrolled in the present study. Forty nine out of 93 (52.7%) patients had a single high risk factor, and 44 (47.3%) had two or more high risk factors. Statistically significant differences in stage and stromal invasion were observed between group 1 and group 2. However, age, histology, tumor size, and lymphovascular space invasion did not differ significantly between the groups. Distant recurrence occurred more frequently in group 2, and the probability of recurrence and death was higher in group 2. CONCLUSION: Patients with two or more high risk factors had worse prognosis in early stage cervical cancer. For these patients, consideration of new strategies to improve survival may be worthwhile. Conduct of further clinical trials is warranted for development of adjuvant treatment strategies individualized to each risk group.