Essential Guidelines for Manufacturing and Application of Organoids.

An organoid is a self-organized three-dimensional structure derived from stem cells that mimics the structure, cell composition, and functional characteristics of specific organs and tissues and is used for evaluating the safety and effectiveness of drugs and the toxicity of industrial chemicals. Organoid technology is a new methodology that could replace testing on animals testing and accelerate development of precision and regenerative medicine. However, large variations in production can occur between laboratories with low reproducibility of the production process and no internationally agreed standards for quality evaluation factors at endpoints. To overcome these barriers that hinder the regulatory acceptance and commercialization of organoids, Korea established the Organoid Standards Initiative in September 2023 with various stakeholders, including industry, academia, regulatory agencies, and standard development experts, through public and private partnerships. This developed general guidelines for organoid manufacturing and quality evaluation and for quality evaluation guidelines for organoid-specific manufacturing for the liver, intestines, and heart through extensive evidence analysis and consensus among experts. This report is based on the common standard guideline v1.0, which is a general organoid manufacturing and quality evaluation to promote the practical use of organoids. This guideline does not focus on specific organoids or specific contexts of use but provides guidance to organoid makers and users on materials, procedures, and essential quality assessment methods at end points that are essential for organoid production applicable at the current technology level.

Diagnostic Usefulness of Diffusion-Weighted MRI for Axillary Lymph Node Evaluation in Patients with Breast Cancer.

This study aimed to determine whether apparent diffusion coefficient (ADC) and morphological features on diffusion-weighted MRI (DW-MRI) can discriminate metastatic axillary lymph nodes (ALNs) from benign in patients with breast cancer. Two radiologists measured ADC, long and short diameters, long-to-short diameter ratio, and cortical thickness and assessed eccentric cortical thickening, loss of fatty hilum, irregular margin, asymmetry in shape or number, and rim sign of ALNs on DW-MRI and categorized them into benign or suspicious ALNs. Pathologic reports were used as a reference standard. Statistical analysis was performed using the Mann-Whitney U test and chi-square test. Overall sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of DW-MRI were calculated. The ADC of metastatic ALNs was 0.905 × 10-3 mm2/s, and that of benign ALNs was 0.991 × 10-3 mm2/s (p = 0.243). All morphologic features showed significant difference between the two groups. The sensitivity, specificity, PPV, NPV, and diagnostic accuracy of the final categorization on DW-MRI were 77.1%, 93.3%, 79.4%, 92.5%, and 86.2%, respectively. Our results suggest that morphologic evaluation of ALNs on DWI can discriminate metastatic ALNs from benign. The ADC value of metastatic ALNs was lower than that of benign nodes, but the difference was not statistically significant.

Computer-Aided Diagnosis Parameters of Invasive Carcinoma of No Special Type on 3T MRI: Correlation with Pathologic Immunohistochemical Markers.

Purpose: To investigate the correlation between computer-aided diagnosis (CAD) parameters in 3-tesla (T) MRI and pathologic immunohistochemical (IHC) markers in invasive carcinoma of no special type (NST). Materials and Methods: A total of 94 female who were diagnosed with NST carcinoma and underwent 3T MRI using CAD, from January 2018 to April 2019, were included. The relationship between angiovolume, curve peak, and early and late profiles of dynamic enhancement from CAD with pathologic IHC markers and molecular subtypes were retrospectively investigated using Dwass, Steel, Critchlow-Fligner multiple comparison analysis, and univariate binary logistic regression analysis. Results: In NST carcinoma, a higher angiovolume was observed in tumors of higher nuclear and histologic grades and in lymph node (LN) (+), estrogen receptor (ER) (-), progesterone receptor (PR) (-), human epidermal growth factor 2 (HER2) (+), and Ki-67 (+) tumors. A high rate of delayed washout and a low rate of delayed persistence were observed in Ki-67 (+) tumors. In the binary logistic regression analysis of NST carcinoma, a high angiovolume was significantly associated with a high nuclear and histologic grade, LN (+), ER (-), PR (-), HER2 (+) status, and non-luminal subtypes. A high rate of washout and a low rate of persistence were also significantly correlated with the Ki-67 (+) status. Conclusion: Angiovolume and delayed washout/persistent rate from CAD parameters in contrast enhanced breast MRI correlated with predictive IHC markers. These results suggest that CAD parameters could be used as clinical prognostic, predictive factors.

In vivo imaging of invasive aspergillosis with 18F-fluorodeoxysorbitol positron emission tomography.

Invasive aspergillosis is a critical complication in immunocompromised patients with hematologic malignancies or with viral pneumonia caused by influenza virus or SARS­CoV­2. Although early and accurate diagnosis of invasive aspergillosis can maximize clinical outcomes, current diagnostic methods are time-consuming and poorly sensitive. Here, we assess the ability of 2-deoxy-2-18F-fluorosorbitol (18F-FDS) positron emission tomography (PET) to specifically and noninvasively detect Aspergillus infections. We show that 18F-FDS PET can be used to visualize Aspergillus fumigatus infection of the lungs, brain, and muscles in mouse models. In particular, 18F-FDS can distinguish pulmonary aspergillosis from Staphylococcus aureus infection, both of which induce pulmonary infiltrates in immunocompromised patients. Thus, our results indicate that the combination of 18F-FDS PET and appropriate clinical information may be useful in the differential diagnosis and localization of invasive aspergillosis.

Caveolin-1 enhances brain metastasis of non-small cell lung cancer, potentially in association with the epithelial-mesenchymal transition marker SNAIL.

BACKGROUND: Caveolin-1 (Cav-1) plays an important role in the development of various human cancers. We investigated the relationship between Cav-1 expression and non-small cell lung cancer (NSCLC) progression in the context of brain metastasis (BM). METHODS: Cav-1 expression was investigated in a series of 102 BM samples and 49 paired primary NSCLC samples, as well as 162 unpaired primary NSCLC samples with (63 cases) or without (99 cases) metastasis to distant organs. Human lung cancer cell lines were used for in vitro functional analysis. RESULTS: High Cav-1 expression in tumor cells was observed in 52% (38/73) of squamous cell carcinomas (SQCs) and 33% (45/138) of non-SQCs. In SQC, high Cav-1 expression was increased after BM in both paired and unpaired samples of lung primary tumors and BM (53% vs. 84% in paired samples, P = 0.034; 52% vs. 78% in unpaired samples, P = 0.020). Although the difference in median overall survival in patients NSCLC was not statistically significant, high Cav-1 expression in tumor cells (P = 0.005, hazard ratio 1.715, 95% confidence index 1.175-2.502) was independent prognostic factors of overall survival on multivariate Cox regression analyses, in addition to the presence of BM and non-SQC type. In vitro assays revealed that Cav-1 knockdown inhibited the invasion and migration of lung cancer cells. Genetic modulation of Cav-1 was consistently associated with SNAIL up- and down-regulation. These findings were supported by increased SNAIL and Cav-1 expression in BM samples of SQC. CONCLUSIONS: Cav-1 plays an important role in the BM of NSCLC, especially in SQC. The mechanism may be linked to SNAIL regulation.

The Role Played by SLUG, an Epithelial-Mesenchymal Transition Factor, in Invasion and Therapeutic Resistance of Malignant Glioma.

In malignant gliomas, invasive phenotype and cancer stemness promoting resurgence of residual tumor cells render treatment very difficult. Hence, identification of epithelial-mesenchymal transition (EMT) factors associated with invasion and stemness of glioma cells is critical. To address the issue, we investigated several EMT factors in hypermotile U87MG and U251 cells, orthotopic mouse glioma model, and human glioma samples. Of several EMT markers, SLUG expression was notably increased at the invasive fronts of gliomas, both in mouse tumor grafts and human glioma samples. The biological role played by SLUG was investigated using a colony-forming assay after chemotherapy and irradiation, and by employing a neurosphere culture assay. The effect of SLUG on glioma progression was examined in our patient cohort and samples, and compared to large public data from the REMBRANDT and TCGA. Genetic upregulation of SLUG was associated with increased levels of stemness factors and enhanced resistance to radiation and temozolomide. In our cohort, patients exhibiting lower-level SLUG expression evidenced longer progression-free survival (P = 0.042). Also, in the REMBRANDT dataset, a group in which SLUG was downregulated exhibited a significant survival benefit (P < 0.001). Although paired glioblastoma samples from our patients did not show a significant increase of SLUG expression, increased mRNA levels of SLUG were found in recurrent glioblastoma from TCGA (P = 0.052), and in temozolomide-treated glioma cells and mouse tumor grafts. SLUG may contribute to glioma progression by controlling invasion at infiltrating margins, associated with increased stemness and therapeutic resistance.

Prognostic significance of E-cadherin and N-cadherin expression in Gliomas.

BACKGROUND: Epithelial-mesenchymal transition (EMT), principally involving an E-cadherin to N-cadherin shift, linked to tumor invasion or metastasis, and therapeutic resistance in various human cancer. A growing body of recent evidence has supported the hypothesis that EMT play a crucial role in the invasive phenotype of gliomas. To evaluate the prognostic connotation of EMT traits in glioma, expression of E-cadherin and N-cadherin was explored in a large series of glioma patients in relation to patient survival rate. METHODS: Expressions of E- and N-cadherin were examined using immunohistochemical analysis in 92 glioma cases diagnosed at our hospital. These markers expressions were also explored in 21 cases of fresh frozen glioma samples and in glioma cell lines by Western blot analysis. RESULTS: Expression of E-cadherin was observed in eight cases (8.7%) with weak staining intensity in the majority of the immunoreactive cases (7/8). Expression of N-cadherin was identified in 81 cases (88.0%) with high expression in 64 cases (69.5%). Fresh frozen tissue samples and glioma cell lines showed similar results by Western blot analysis. There was no significant difference in either overall survival (OS) or progression-free survival (PFS) according to E-cadherin expression (P > 0.05). Although the OS rates were not affected by N-cadherin expression levels (P = 0.138), PFS increased in the low N-cadherin expression group with marginal significance (P = 0.058). The survival gains based on N-cadherin expression levels were significantly augmented in a larger series of publicly available REMBRANDT data (P < 0.001). CONCLUSIONS: E- and N-cadherin, as representative EMT markers, have limited prognostic value in glioma. Nonetheless, the EMT process in gliomas may be compounded by enhanced N-cadherin expression supported by unfavorable prognostic outcomes.

Human U87 glioblastoma cells with stemness features display enhanced sensitivity to natural killer cell cytotoxicity through altered expression of NKG2D ligand.

BACKGROUND: Glioblastoma (GBM) is one of the most lethal tumors with a poor prognosis. Its inevitable recurrence is frequently explained by the presence of cancer stem cells. We aimed to show that human GBM cells with stemness features are more sensitive to natural killer (NK) cells than GBM cells without stemness characteristics. METHODS: Natural killer cell cytotoxicity was measured using flow cytometry in neurosphere-forming U87 GBM cells cultured with neurobasal media (NBE condition) and compared with that in serum-cultured U87 GBM cells (serum condition). Cytotoxicity was examined after addition of blocking NKG2D monoclonal antibodies. The expression profile of NK ligands of NK cells were investigated by reverse transcription polymerase chain reaction and western blot analysis in the U87 GBM cells in both conditions. RESULTS: NBE U87 cells showed higher cytotoxicity to NK cells than serum U87 cells did (55 vs 35% at an effector to target cell ratio of 5:1). The increased cytotoxicity was diminished in NBE U87 cells by a larger gap than in serum U87 cells by adding NKG2D blocking antibodies. Of the NKG2D ligands, the expression of ULBP1 and ULBP3 was relatively increased in NBE U87 cells compared to serum U87 cells. CONCLUSIONS: U87 GBM cells with stemness features demonstrate increased cytotoxicity to NK cells in association with altered NKG2D ligand expression of NK cell activating receptor. Applying immune modulation to GBM treatment may be a promising adjuvant therapy in patients with intractable GBM.

Sonoelastographic strain ratio: how does the position of reference fat influence it?

PURPOSE: The purpose of this study was to evaluate how the reference fat position influenced the calculation of the sonoelastographic strain ratio. METHODS: Three hundred fifty-eight breast masses (256 benign lesions and 102 malignant lesions) in 300 women (mean age 47.4 years; age range 17-82 years) who had been scheduled for a percutaneous needle biopsy or surgical excision were examined using B-mode ultrasonography and elastography. The sonoelastographic strain ratio was calculated twice per lesion; once by dividing the strain value of the fat near the mass by that of the mass (FLR 1) and once by dividing the strain value of the subcutaneous fat by that of the mass (FLR 2). RESULTS: Most (91.9 %) showed a difference of less than 0.5 between FLR 1 and FLR 2 values. Regardless of the position of reference fat, there was no statistically significant difference between the FLR 1 and FLR 2 values (p value = 0.077 and 0.0825, respectively). According to the pathology of the lesion, a difference between FLR 1 and FLR 2 less than 0.5 occurred in 95.3 % of the benign lesions and 84 % of the malignant lesions (p < 0.001). CONCLUSIONS: The sonoelastographic strain ratio was not significantly affected by the position of reference fat.

The Influence of Arm Swelling Duration on Shoulder Pathology in Breast Cancer Patients with Lymphedema.

PURPOSE: To evaluate the pathological effect of the duration of arm swelling on the shoulder pathology in patients with breast cancer-related lymphedema. METHODS: Forty seven breast cancer patients with unilateral arm lymphedema were assessed. The duration of the arm swelling and shoulder pain were recorded. Ultrasound examination of the shoulder joint was performed in all patients to detect any lesions. RESULTS: Abnormalities were detected by ultrasound in 41/47 (87.2%) study participants. Subacromial subdeltoid bursal thickening was found in 26/47 (55.3%) participants, distension of the biceps brachii tendon sheath was found in 14/47 (29.8%) and a supraspinatus tendon tear was found in 13/47 (27.7%). Patients with a supraspinatus tendon tear were found to have a significantly longer duration of lymphedema (1310 days vs. 398 days, p = 0.032). CONCLUSIONS: The duration of arm lymphedema has a progressive pathological effect on rotator cuff. Clinicians should adopt an early management approach of shoulder pain in patients with breast cancer-related lymphedema.

KITENIN promotes glioma invasiveness and progression, associated with the induction of EMT and stemness markers.

KITENIN (KAI1 COOH-terminal interacting tetraspanin) promotes tumor invasion and metastasis in various cancers. This study assessed the association between KITENIN expression and advanced glioma grade in patients. In vitro assays revealed that KITENIN knockdown inhibited the invasion and migration of glioma cells, whereas KITENIN overexpression promoted their invasion and migration. In orthotopic mouse tumor models, mice transplanted with KITENIN-transfected glioma cells had significantly shorter survival than mice transplanted with mock-transfected cells. Patients with low KITENIN expression showed a significantly longer progression-free survival than patients with high KITENIN expression. KITENIN induced the expression of the epithelial-mesenchymal transition (EMT) markers (N-cadherin, ZEB1, ZEB2, SNAIL and SLUG) as well as the glioma stemness markers (CD133, ALDH1 and EPH-B1). Taken together, these findings showed that high levels of KITENIN increased glioma invasiveness and progression, associated with the up-regulation of EMT and stemness markers.

Melanotic neuroectodermal tumor of infancy disseminated by a ventriculoperitoneal shunt and diagnosed from the inguinal sac.

Melanotic neuroectodermal tumor of infancy (MNTI) is a rare congenital neoplasm that originates from the neural crest cells, which give rise to the melanocytes of the skin and leptomeninges. We report a case of MNTI with neurocutaneous melanosis of a 28-month-old girl. She was born with hydrocephalus and several large congenital giant nevi. There were no findings except for hydrocephalus, after a ventriculoperitoneal (VP) shunt operation performed when she was 6 months old. She was operated on for a growing inguinal mass at 8 months. The specimen from the inguinal sac was positive for HMB45, vimentin, chromogranin, and neuron-specific enolase. Brain magnetic resonance imaging showed an extensive enhancing extra-axial mass with high signal intensity, along the cerebral spinal fluid space. We report a rare case of MNTI, diagnosed from an inguinal hernia sac, with a disseminated clinical manifestation.

N-Acetylglucosaminyltransferase V triggers overexpression of MT1-MMP and reinforces the invasive/metastatic potential of cancer cells.

N-Acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes the formation of a ß1,6-N-acetylglucosamine (GlcNAc) side chain to a core mannosyl residue in N-linked glycoproteins. Besides its direct function of producing aberrant glycoproteins, it promotes cancer progression by its involvement in the stimulation of oncoproteins. Herein, we report that GnT-V guided the transcriptional activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) in cancer cells. The activated MT1-MMP expression had dual effects on cancer progression. It not only promoted proteolytic activity for cancer cells per se, but also led to the activation of MMP-2. Consequently, the activation of the two MMPs triggered by GnT-V intensified the invasive potential. A quantitative analysis using clinical tissues revealed a relatively strong correlation between GnT-V overexpression and MT1-MMP upregulation. In this study, we report for the first time that GnT-V directs cancer progression by modulating MMPs in cancer.

Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer.

A novel circulating tumor-associated autoantibody, K94, obtained from a hepatocellular carcinoma (HCC) mouse model was characterized. The target antigen of K94 autoantibody was expressed in various tumor cell lines including liver cancer, and its secretion was detectable using MCF-7 breast carcinoma cells. Proteomic analysis revealed that the protein bands reactive to K94 included cytokeratin (CK) 8 and 18, which are known to be related to tumorigenesis and form a heterotypic complex with each other. However, K94 showed no activity toward CK8 or CK18 separately. The epitope of the K94 antibody was only presented by a complex between CK8 and CK18, which was confirmed by analysis using recombinant CK8 and CK18 proteins. To formulate an assay for anti-CK8/18 complex autoantibody, a mimotope peptide reactive to K94 was selected from loop-constrained heptapeptide (-CX7C-) display phage library, of which sequence was CISPDAHSC (K94p1). A mimotope enzyme-linked immunosorbent assay (ELISA) using phage-displayed K94p1 peptide as a coating antigen was able to discriminate breast cancer (n=30) patients from normal subjects (n=30) with a sensitivity of 50% and a specificity of 82.61%. CA15.3 was detected at very low levels in the same breast cancer subjects and did not discriminate breast cancer patients from normal subjects, although it is a conventional biomarker of breast cancer. These results suggest that a mimotope ELISA composed of K94p1 peptide may be useful for the diagnosis of breast cancer.

Galectin-3 binding protein promotes cell motility in colon cancer by stimulating the shedding of protein tyrosine phosphatase kappa by proprotein convertase 5.

It has previously been reported that shedding of the PTPκ ectodomain drives enhanced motility of colon cancer cells. Herein, we provide mechanism underlying the regulation of PTPκ shedding by galectin-3 binding protein. PTPκ was inarguably scissored by the processed form of proprotein convertase 5 (subtilisin/kexin type 5), and galectin-3 binding protein which is over-produced in colon cancer cells and tissues contributed to increased cancer cell motility by acting as a negative regulator of galectin-3 at the cell surface. The high expression ratio of galectin-3 binding protein to galectin-3 was clinically correlated to lymphatic invasion. These results suggest that galectin-3 binding protein may be a potential therapeutic target for treatment of, at least, colon cancer patients with high expression of galectin-3 binding protein.

Elective laparoscopic repair after colonoscopic decompression for incarcerated morgagni hernia.

Plain radiographs of an 88-year-old woman who had experienced vomiting and abdominal distention for 3 days revealed a severely obstructed ileus, and abdominopelvic computed tomography revealed an incarcerated Morgagni hernia. The endoscope was passed through the constrictions from the diaphragmatic indentations and a thin catheter was placed for decompression. The obstructive ileus regressed markedly after the procedure; the patient underwent elective laparoscopic repair of the hernia 1 week later. This is believed to be the first case of endoscopic preoperative decompression for an incarcerated Morgagni hernia.

Lectin precipitation using phytohemagglutinin-L(4) coupled to avidin-agarose for serological biomarker discovery in colorectal cancer.

N-acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L(4) (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin-agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs.

Functional proteomics study reveals that N-Acetylglucosaminyltransferase V reinforces the invasive/metastatic potential of colon cancer through aberrant glycosylation on tissue inhibitor of metalloproteinase-1.

N-Acetylglucosaminyltransferase-V (GnT-V) has been reported to be up-regulated in invasive/metastatic cancer cells, but a comprehensive understanding of how the transferase correlates with the invasive/metastatic potential is not currently available. Through a glycomics approach, we identified 30 proteins, including tissue inhibitor of metalloproteinase-1 (TIMP-1), as a target protein for GnT-V in human colon cancer cell WiDr. TIMP-1 was aberrantly glycosylated as characterized by the addition of beta1,6-N-acetylglucosamine, polylactosaminylation, and sialylation in GnT-V-overexpressing WiDr cells. Compared with normal TIMP-1, the aberrantly glycosylated TIMP-1 showed the weaker inhibition on both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy was closely associated with cancer cell invasion and metastasis in vivo as well as in vitro. Integrated data, both of TIMP-1 expression level and aberrant glycosylation, could provide important information to aid to improve the clinical outcome of colon cancer patients.

Identification of target proteins of N-acetylglucosaminyl transferase V in human colon cancer and implications of protein tyrosine phosphatase kappa in enhanced cancer cell migration.

To gain a better understanding of the mechanism underlying colon cancer and to search for potential markers of colon cancer prognosis, a comparative proteomic analysis of colon cancer WiDr cells was conducted using 2-DE and lectin blot, followed by identification based on ESI-MS. Through these approaches 14 proteins were identified as candidate target proteins for N-acetylglucosaminyl transferase V (GnT-V) that would be expected to be implicated in the progression of colon cancer. We selected protein tyrosine phosphatase kappa (PTPkappa) as a model protein to validate this approach to the discovery of novel biomarkers in colon cancer. PTPkappa underwent an aberrant glycosylation in GnT-V-overexpressing WiDr cells, and the aberrantly glycosylated PTPkappa was vulnerable to proteolytic cleavage. The enhanced cleavage of PTPkappa in GnT-V-overexpressing cells was responsible for the mitigation of the homophilic binding capacity, resulting in an increase in cancer cell migration.

The effect of simulation on recurrence after breast-conserving surgery and radiotherapy: preliminary results.

PURPOSE: To evaluate the effect of the simulation method on recurrence among the patients who received radiotherapy after breast-conserving surgery (BCS) for early breast carcinoma. MATERIALS AND METHODS: Between 1995 and 2000, 70 patients with stage I-II breast carcinoma underwent breast-conserving surgery and adjuvant radiotherapy. Twenty nine patients (41.4%) were simulated with the 2D contour-based method (September 1995 to August 1997) and 41 patients (58.6%) were simulated with the 3D CT-based method (September 1997 to February 2000). To analyze the effect of the simulation method, the patient and treatment characteristics were compared. RESULTS: The characteristics were similar for the patients between the 2D contour-based simulation group and the 3D CT-based simulation group. During a median follow-up period of 75 months, 4 (13.8%) of 29 patients who were treated with 2D simulation and 1 (2.4%) of 41 patients who were treated with 3D simulation group developed treatment failure. The five-year survival rates were 89.2% and 95.1% between the 2D and 3D simulation groups (p=0.196). The five-year disease free survival (DFS) rates were 86.2% and 97.5% between the 2D and 3D simulation groups (p=0.0636). On univariate analysis, age > 40 (p= 0.0226) and the number of dissected axillary lymph node >or= 10 (p=0.0435) were independent predictors of improved 5-year DFS. CONCLUSIONS: Although our data showed marginal significance for the DFS between the two groups, it is insufficient, due to the small number of patients in our study, to prove whether 3D CT-based simulation might improve the DFS and reduce the risk of recurrence when compared with 2D contour-based simulation. Further study is needed with a larger group of patients.