Searching for the "Holy Grail" of breast cancer recurrence risk: a narrative review of the hunt for a better biomarker and the promise of circulating tumor DNA (ctDNA).

BACKGROUND: This paper is a narrative review of a major clinical challenge at the heart of breast cancer care: determining which patients are at risk of recurrence, which require systemic therapy, and which remain at risk in the survivorship phase of care despite initial therapy. METHODS: We review the literature on prognostic and predictive biomarkers in breast cancer with a focus on detection of minimal residual disease. RESULTS: While we have many tools to estimate and refine risk that are used to individualize local and systemic therapy, we know that we continue to over treat many patients and undertreat others. Many patients also experience what is, at least in hindsight, needless fear of recurrence. In this review, we frame this dilemma for the practicing breast oncologist and discuss the search for what we term the "holy grail" of breast cancer evaluation: the ideal biomarker of residual distant disease. We review the history of attempts to address this problem and the up-to-date science on biomarkers, circulating tumor cells and circulating tumor DNA (ctDNA). CONCLUSION: This review suggests that the emerging promise of ctDNA may help resolve a crticical dilemma at the heart of breast cancer care, and improve prognostication, treatment selection, and outcomes for patients with breast cancer.

[NOVEL TECHNOLOGIES AND FUTURE TRENDS IN TRANSLATIONAL RESEARCH IN ONCOLOGY].

INTRODUCTION: Translational research in medicine has undergone significant changes in the last decade, primarily due to the remarkable technological advancements made during this period. Oncology research is at the forefront of translational research in medicine and is heavily influenced by these changes. In this article, we briefly review the technologies that form the basis for the "next generation of translational research" in oncology in the coming decades, as well as the emerging trends in translational research in oncology through the implementation of these technologies.

The Evolving Role of Neoadjuvant Therapy for Operable Breast Cancer.

The role of neoadjuvant therapy (NAT) for localized breast cancer has evolved tremendously over the past several years. Currently, NAT is the preferred option for high-risk early triple-negative (TN) and HER2-positive (HER2+) breast cancers and is indicated for some estrogen receptor-positive (ER+) breast cancers. In addition to traditional absolute indications for NAT, relative indications such as the assessment of outcomes at the time of surgery and guidance of treatment escalation and de-escalation have greatly evolved in recent years. Pathologic complete response (pCR) and the Residual Cancer Burden (RCB) index are highly prognostic for disease recurrence and survival, mainly in patients with TN or HER2+ disease. Furthermore, post-NAT escalation strategies have been shown to improve long-term outcomes of patients who do not achieve pCR. Additionally, by allowing the direct assessment of drug effect on the tumor, the neoadjuvant setting has become an attractive setting for the exploration of novel agents and the identification of predictive biomarkers. Neoadjuvant trial design has also evolved, using adaptive treatment approaches that enable treatment de-escalation or escalation based on response. However, despite multiple practice-changing neoadjuvant trials and the addition of various new agents to the neoadjuvant setting for early breast cancer, many key questions remain. For example, patient selection for neoadjuvant immunotherapy in TN breast cancer, de-escalation methods in HER2+ breast cancer, and the use of gene expression profiles to guide NAT recommendations in ER+ breast cancer. This article reviews the current approach for NAT in localized breast cancer as well as evolving NAT strategies, the key remaining challenges, and the ongoing work in the field.

An occupation-based strategy training approach to managing age-related executive changes: a pilot randomized controlled trial.

OBJECTIVE: To determine the feasibility of recruitment and retention of healthy older adults and the effectiveness of an intervention designed to manage age-related executive changes. DESIGN: A pilot randomized controlled trial. SETTING: Research centre and participants' homes. PARTICIPANTS: Nineteen healthy, community dwelling older adults with complaints of cognitive difficulties and everyday problems, but no evidence of mild cognitive impairment, dementia or depression on objective testing. INTERVENTIONS: Seventeen hours of group and individual training. Participants in the experimental arm received education about self-management, successful aging and an occupation-based meta-cognitive strategy-training program. Participants in the control arm received education about brain health and participated in cognitively stimulating exercises. MAIN MEASURES: Changes on untrained, everyday life goals were identified using the Canadian Occupational Performance Measure. Generalization of benefits was measured using the Stanford Chronic Disease Questionnaire, general self-efficacy and changes in executive function (Delis-Kaplan Executive Function System Tower Test, Word Fluency and Trail-Making Test). RESULTS: 20% (19/96) of healthy older adults approached were eligible, consented and were enrolled in the study, 90% (17/19) were retained to three-month follow-up. Participants in the experimental arm reported significantly more improvement on untrained goals (11/22 compared with 9/46, χ(2)=4.92, p<0.05), maintenance of physical activity (p<0.05) and better preparation for doctors' visits (p<0.05) relative to the control group. There were no significant between group differences on objective measures of executive function. CONCLUSIONS: These data support the feasibility of a larger trial where a sample of 72 (36 participants in each arm) would be required to confirm or refute these findings.

Genomic spectrum of actionable alterations in serial cell free DNA (cfDNA) analysis of patients with metastatic breast cancer.

We aimed to study the incidence and genomic spectrum of actionable alterations (AA) detected in serial cfDNA collections from patients with metastatic breast cancer (MBC). Patients with MBC who underwent plasma-based cfDNA testing (Guardant360®) between 2015 and 2021 at an academic institution were included. For patients with serial draws, new pathogenic alterations in each draw were classified as actionable alterations (AA) if they met ESCAT I or II criteria of the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). A total of 344 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) MBC, 95 patients with triple-negative (TN) MBC and 42 patients with HER2-positive (HER2 + ) MBC had a baseline (BL) cfDNA draw. Of these, 139 HR+/HER2-, 33 TN and 13 HER2+ patients underwent subsequent cfDNA draws. In the HR+/HER2- cohort, the proportion of patients with new AA decreased from 63% at BL to 27-33% in the 2nd-4th draws (p < 0.0001). While some of the new AA in subsequent draws from patients with HR+/HER2- MBC were new actionable variants in the same genes that were known to be altered in previous draws, 10-24% of patients had new AA in previously unaltered genes. The incidence of new AA also decreased with subsequent draws in the TN and HER2+ cohorts (TN: 25% to 0-9%, HER2 + : 38% to 14-15%). While the incidence of new AA in serial cfDNA decreased with subsequent draws across all MBC subtypes, new alterations with a potential impact on treatment selection continued to emerge, particularly for patients with HR+/HER2- MBC.

Redifferentiation of expanded human pancreatic ß-cell-derived cells by inhibition of the NOTCH pathway.

In vitro expansion of ß-cells from adult human pancreatic islets would overcome donor ß-cell shortage for cell replacement therapy for diabetes. Using a ß-cell-specific labeling system we have shown that ß-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key ß-cell genes maintain open chromatin structure in expanded ß-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator. We have reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced ß-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace ß-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells.

"It's better than nothing, but I do not find it to be ideal": Older adults' experience of TeleRehab during the first COVID-19 lockdown.

This qualitative study used descriptive thematic analysis to explore the experiences of 16 older adults (age: 71 ± 6.4) who transitioned from an in-person to telerehabilitation (TeleRehab) group intervention in March 2020. We found the following themes: (1A) Technology Use, describing challenges and need for support; and (1B) Technology Self-Efficacy, describing how technological ability was attributed to past-experience and/or age. Four themes described the intervention experience. First, "Not The Same, But Better Than Nothing" (2A), reflected a preference for in-person intervention. Specifically, in-person training provided a better social experience (theme 2B), and stronger accountability, although the content was well delivered in both modalities (theme 2C). Contextual factors (theme 2D) that played a role were ease of commute, especially important during the winter, and the context of the lockdown, that positioned the TeleRehab intervention as a meaningful social activity. However, sensory impairments, and/or distractions in the home diminished the TeleRehab experience.

Adjuvant trastuzumab and vinorelbine for early-stage HER2+ breast cancer.

Background: The single-arm phase II APT trial established trastuzumab and paclitaxel (TH) as the standard adjuvant regimen for small human epidermal growth factor receptor 2 (HER2+) tumors. However, paclitaxel causes alopecia and has high rates of neuropathy and hypersensitivity reactions. In patients with metastatic HER2+ breast cancer (BC), the combination of trastuzumab and vinorelbine (TV) is effective and well tolerated. There is a need for alternative non-anthracycline/taxane-based regimens for patients with HER2+ early-stage BC, especially for those with contraindications or who wish to avoid side effects of taxane-based regimens. Here we describe our institutional experience with adjuvant TV for patients with early-stage HER2+ BC. Methods: Clinicopathological characteristics, treatment details, and outcomes of patients with localized HER2+ BC treated with adjuvant TV from 2007 to 2021 at a large academic medical institution were collected. Study endpoints included invasive disease-free survival (IDFS), overall survival (OS), and safety/tolerability. IDFS and OS were measured from start date of TV treatment to date of event/last follow-up and date of death/last follow-up, respectively. Results: A total of 30 patients were treated with TV. All patients received trastuzumab at standard dosing and vinorelbine at a starting dose of 25 mg/m2 either on days 1/8 or on days 1/8/21 (weekly) of a 21-day cycle with four planned cycles. Median age at diagnosis was 59 years (range: 36-81). 90.3% of patients had anatomic pathologic stage IA BC and 9.7% stage IIA BC. Of the 30 patients, 24 of them opted to pursue TV due to concerns related to alopecia, neuropathy, and other toxicities, and 6 switched from treatment with TH to TV due to toxicities. Eight patients experienced neutropenia with no cases of febrile neutropenia. No patients experienced alopecia or long-term neuropathy. With a median follow-up of 68 months (5.7 years), the 5-year IDFS rate was 90.9%, with one local and one distant recurrence. The 5-year OS was 100%. Conclusions: Trastuzumab in combination with vinorelbine in the adjuvant, early-stage setting for low-risk HER2+ BC demonstrated clinical efficacy and appeared to be well tolerated. TV warrants further evaluation as an alternative regimen to TH for patients with early-stage HER2+ BC.

Combining elements of the CO-OP Approach™ with education to promote healthy eating among older adults: A pilot study.

This paper describes an exploratory study developing the Baycrest Brain-healthy Eating Approach (BBEA). Poor diet is a modifiable risk factor for many health problems including dementia. Mediterranean type diets, high in plant-based foods, rich in poly- and mono- unsaturated fatty acids with minimal consumption of saturated fat, red meat, and processed foods, are considered brain healthful. While several dementia prevention trials randomized controlled trials have included nutritional counselling in favor of these diets as one component of their interventions, the extent to which dietary change occurred is not known. Based on observations that a strategy training approach, the Cognitive Orientation to daily Occupational Performance (CO-OP) Approach, was beneficial for promoting lifestyle changes in older adults with complaints of cognitive changes, we undertook to develop the BBEA combining elements of CO-OP with didactic nutrition education. This exploratory, descriptive study assesses the feasibility and acceptability of the BBEA. Healthy community dwelling older adults (n = 5) were recruited using convenience sampling. Participants received five, 2 h, group sessions. During these sessions participants were supported in adopting dietary practices consistent with brain healthy eating. Each participant set specific dietary goals important to them. Feasibility of the intervention was demonstrated through high levels of attendance and by the findings that at each session, all participants set personally meaningful goals and received education on selected brain healthy eating topics. Acceptability was demonstrated through participants' positive reports of their experiences and perspectives obtained via semi-structured interviews. Thus, the BBEA appears to be feasible and acceptable.

Strategy-training post-stroke via tele-rehabilitation: a pilot randomized controlled trial.

PURPOSE: Long-term limitations in social participation are common after stroke. Whether these can be attenuated through a tele-rehabilitation approach is unknown. We were particularly interested in examining transfer of learning effects which could result in broader improvements in social participation. METHODS: We adapted a strategy training rehabilitation approach (tele-CO-OP) for remote delivery. Participants with chronic stroke were randomized to receive the intervention (EXPT) or to a wait list (Control). Feasibility and acceptability were measured via attendance scores, satisfaction with the training and therapist evaluation of engagement with the training. The primary outcome measure was the Canadian Occupational Performance Measure (COPM), a standardized semi-structured interview which elicits difficulties in day-to-day life. RESULTS: Seventeen participants were randomized. Tele-CO-OP was found to be feasible and acceptable: participants reported high satisfaction and engagement, and missed few sessions. Large effect sizes for transfer of learning effects were observed in favor of receiving tele-CO-OP vs being waitlisted. Significant benefits were also conferred to the Control group following receipt of tele-CO-OP. The intervention also appeared to improve mood. CONCLUSIONS: This exploratory study demonstrates the feasibility and acceptability of tele-CO-OP and provides preliminary evidence for transfer of learning effects to untrained everyday social participation activities. Trial registration number: NCT02724813.

Stroke results in long-term limitations in social participation.The Cognitive Orientation to daily Occupational Performance (CO-OP) Approach provides a potential avenue for ameliorating these limitations.This pilot randomized controlled trial demonstrated that it is feasible to deliver tele-CO-OP and that positive benefits may accrue to those receiving the intervention for both trained and untrained activities.Tele-CO-OP is a promising intervention for addressing long-term participation limitations in individuals with chronic stroke.

Next-generation sequencing in thyroid cancers: do targetable alterations lead to a therapeutic advantage?: A multicenter experience.

ABSTRACT: Radioiodine-refractory thyroid cancers (IRTCs) are uncommon and have a poor prognosis. Treatment options for radioiodine-refractory and anaplastic tumors (ATCs) are limited. Although the genomic landscape of thyroid cancer has been studied, there is little evidence on whether next-generation sequencing (NGS) findings translate to tumor control.We analyzed all patients with IRTC and ATC who underwent commercially available NGS in 3 cancer centers.Twenty-two patients were identified, 16 patients with IRTCs and 6 patients with ATCs. Eighteen (82%) had targetable findings in NGS, nine patients were treated accordingly. Median progression-free survival for targeted treatment was 50 months [95% confidence interval (CI95%) 9.8-66.6] and2 months (CI95% 0.2-16.5) for IRTC and ATC, respectively. Of 4 patients who achieved durable responses of 7 to 50 months, 2 are ongoing. The estimated median OS of IRTC receiving targeted treatment was not reached (CI95% 89.7-111.4 months) and was 77.8 months (CI95% 52.5-114.6) for patients treated conventionally (P = .3).NGS may detect clinically significant genetic alterations and benefit patients with advanced thyroid cancers.

The impact of tumor detection method on genomic and clinical risk and chemotherapy recommendation in early hormone receptor positive breast cancer.

BACKGROUND: Symptomatic breast cancers share aggressive clinico-pathological characteristics compared to screen-detected breast cancers. We assessed the association between the method of cancer detection and genomic and clinical risk, and its effect on adjuvant chemotherapy recommendations. PATIENTS AND METHODS: Patients with early hormone receptor positive (HR+) HER2neu-negative (HER2-) breast cancer, and known OncotypeDX Breast Recurrence Score test were included. A natural language processing (NLP) algorithm was used to identify the method of cancer detection. The clinical and genomic risks of symptomatic and screen-detected tumors were compared. RESULTS: The NLP algorithm identified the method of detection of 401 patients, with 216 (54%) diagnosed by routine screening, and the remainder secondary to symptoms. The distribution of OncotypeDX recurrence score (RS) varied between the groups. In the symptomatic group there were lower proportions of low RS (13% vs 23%) and higher proportions of high RS (24% vs. 13%) compared to the screen-detected group. Symptomatic tumors were significantly more likely to have a high clinical risk (59% vs 40%). Based on genomic and clinical risk and current guidelines, we found that women aged 50 and under, with a symptomatic cancer, had an increased probability of receiving adjuvant chemotherapy recommendation compared to women with screen-detected cancers (60% vs. 37%). CONCLUSIONS: We demonstrated an association between the method of cancer detection and both genomic and clinical risk. Symptomatic breast cancer, especially in young women, remains a poor prognostic factor that should be taken into account when evaluating patient prognosis and determining adjuvant treatment plans.

Next-generation sequencing in salivary gland carcinoma: Targetable alterations lead to a therapeutic advantage-Multicenter experience.

BACKGROUND: Salivary gland cancers (SGCs) are rare. The approach to metastatic patients is histology-dependent. There is little evidence on whether next-generation sequencing (NGS) findings translate to tumor control in SGCs. METHODS: We analyzed all patients with histologically confirmed SGC who underwent NGS. RESULTS: Twenty-seven patients were identified, 14 (51.8%) had targetable findings in NGS: 5 ERBB2 amplifications, 3 PIK3CA mutations, 2 RUNX1 mutations, 1 TRIM33-RET fusion, 1 FGFR3-TACC3 fusion, 1 microsatellite instability-high, and 2 high mutational burden. Ten patients were treated accordingly. Median progression-free survival for targeted treatment was 8.4 months. Of five patients who achieved durable responses of 8.4 to 31.3 months, two are ongoing. The overall median survival was not reached for patients receiving targeted treatment and was 40.4 months for patients treated conventionally (P = .18). CONCLUSIONS: In the absence of a well-established therapeutic approach, NGS may detect clinically significant genetic alterations and benefit patients with advanced SGC.

Soft-Tissue Sarcoma following Traumatic Injury: Case Report and Review of the Literature.

Soft-tissue sarcomas (STSs) are a heterogeneous group of tumors, which accounts for 1-2% of adult cancers worldwide. Despite quite a few reports on traumatic events followed by STS formation, the link between the two events remains a point of controversy. In this paper, we present the case of a young patient who had a rhabdomyosarcoma in the lower extremity, which had developed in the same location where the patient was wounded by a gunshot 9 years earlier. X-ray and CT scans clearly showed metal fragments in the area of sarcoma formation. The patient underwent neoadjuvant chemotherapy treatment, to which the tumor was, unfortunately, unresponsive. Therefore, the patient was referred to below-knee amputation of the injured leg. There are several possible etiological factors for sarcoma development in this patient, including tissue damage and inflammation, as well as the presence of metal fragments in the tissue and the limb's exposure to radiation during multiple imaging tests. Here, we will discuss the potential influence wielded by the injury itself, as well as its complications and its medical management on the formation of the sarcoma, in light of the current literature.

Quantitative and semi-quantitative histopathological examination of renal biopsies in healthy individuals, and associations with kidney function.

This study assesed the prevalence of histopathological changes in renal biopsies from healthy individuals, and the association with age, sex and smoking. Donor biopsies from 109 subjects were obtained from living kidney donors, and blood and urine samples were collected together with medical history. All biopsies were scored according to the Banff '97 classification with some modifications. The parameters included in this study were tubular atrophy, interstitial fibrosis, glomerulosclerosis, arteriosclerosis, arteriolohyalinosis and a sclerosis score. An alternative scoring system for tubular atrophy was examined (using ≤5% rather than <1% as a cut-off for grade 0). Glomerular filtration rate was measured in most cases as chromium ethylenediaminetetra-acetic acid (Cr-EDTA) clearance. Age was a significant predictor for tubular atrophy, fibrosis and sclerosis. Pack-years of smoking increased the risk of tubular atrophy, fibrosis and arteriolohyalinosis. The alternative scoring of tubular atrophy showed a stronger association with smoking, but a weaker association with age, compared with the original one. Limited histopathological changes are common in healthy kidney donors around 50 years of age with normal kidney function. We propose that a cut-off of ≤5% yields a better definition of grade 0 tubular atrophy compared with the established cut-off of >0%.

Identifying instruments to quantify financial management skills in adults with acquired cognitive impairments.

INTRODUCTION: Financial management skills-that is, the skills needed to handle personal finances such as banking and paying bills-are essential to a person's autonomy, independence, and community living. To date, no comprehensive review of financial management skills instruments exists, making it difficult for clinicians and researchers to choose relevant instruments. The objectives of this review are to: (a) identify all available instruments containing financial management skill items that have been used with adults with acquired cognitive impairments; (b) categorize the instruments by source (i.e., observation based, self-report, proxy report); and (c) describe observation-based performance instruments by populations, overarching concepts measured, and comprehensiveness of financial management items. Objective (c) focuses on observation-based performance instruments as these measures can aid in situations where the person with cognitive impairment has poor self-awareness or where the proxy has poor knowledge of the person's current abilities. METHOD: Two reviewers completed two systematic searches of five databases. Instruments were categorized by reviewing published literature, copies of the instruments, and/or communication with instrument authors. Comprehensiveness of items was based on nine key domains of financial management skills developed by the authors. RESULTS: A total of 88 discrete instruments were identified. Of these, 44 were categorized as observation-based performance and 44 as self- and/or proxy-reports. Of the 44 observation-based performance instruments, 8 had been developed for acquired brain injury populations and 24 for aging and dementia populations. Only 7 of the observation-based performance instruments had items spanning 6 or more of the 9 financial management skills domains. CONCLUSIONS: The majority of instruments were developed for aging and dementia populations, and few were comprehensive. This review provides foundation for future instrument psychometric and clinimetric reviews. It a necessary first step in providing information to support decision making for clinicians and researchers selecting financial management skills instruments.

The NOTCH pathway in ß-cell growth and differentiation.

Beta-cell replacement represents the optimal therapy for type 1 diabetes. Efforts to manipulate ß-cell proliferation and differentiation could be advanced by a better understanding of the normal pathways regulating ß-cell development and renewal. NOTCH signaling is a highly conserved pathway which plays a central role in pancreas development. Cell-lineage tracing has revealed the reactivation of the NOTCH pathway in adult human ß cells cultured under conditions which induce cell proliferation and dedifferentiation. Inhibition of NOTCH signaling in dedifferentiated cells following ex vivo expansion has been shown to promote restoration of the ß-cell phenotype. This approach may increase the availability of functional ß cells for transplantation.

In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing.

OBJECTIVE: Expansion of insulin-producing beta-cells from adult human islets could alleviate donor shortage for cell-replacement therapy of diabetes. A major obstacle to development of effective expansion protocols is the rapid loss of beta-cell markers in the cultured cells. Here, we report a genetic cell-lineage tracing approach for following the fate of cultured beta-cells. RESEARCH DESIGN AND METHODS: Cells dissociated from isolated human islets were infected with two lentiviruses, one expressing Cre recombinase under control of the insulin promoter and the other, a reporter cassette with the structure cytomegalovirus promoter-loxP-DsRed2-loxP-eGFP. RESULTS: Beta-cells were efficiently and specifically labeled by the dual virus system. Label(+), insulin(-) cells derived from beta-cells were shown to proliferate for a maximum of 16 population doublings, with an approximate doubling time of 7 days. Isolated labeled cells could be expanded in the absence of other pancreas cell types if provided with medium conditioned by pancreatic non-beta-cells. Analysis of mouse islet cells by the same method revealed a much lower proliferation of labeled cells under similar culture conditions. CONCLUSIONS: Our findings provide direct evidence for survival and dedifferentiation of cultured adult human beta-cells and demonstrate that the dedifferentiated cells significantly proliferate in vitro. The findings confirm the difference between mouse and human beta-cell proliferation under our culture conditions. These findings demonstrate the feasibility of cell-specific labeling of cultured primary human cells using a genetic recombination approach that was previously restricted to transgenic animals.

HES-1 is involved in adaptation of adult human beta-cells to proliferation in vitro.

OBJECTIVE: In vitro expansion of beta-cells from adult human islets could solve the tissue shortage for cell replacement therapy of diabetes. Culture of human islet cells typically results in <16 cell doublings and loss of insulin expression. Using cell lineage tracing, we demonstrated that the expanded cell population included cells derived from beta-cells. Understanding the molecular mechanisms involved in beta-cell fate in vitro is crucial for optimizing expansion and redifferentiation of these cells. In the developing pancreas, important cell-fate decisions are regulated by NOTCH receptors, which signal through the hairy and enhancer of split (HES)-1 transcriptional regulator. Here, we investigated the role of the NOTCH signaling pathway in beta-cell dedifferentiation and proliferation in vitro. RESEARCH DESIGN AND METHODS: Isolated human islets were dissociated into single cells. beta-Cells were genetically labeled using a Cre-lox system delivered by lentiviruses. Cells were analyzed for changes in expression of components of the NOTCH pathway during the initial weeks in culture. HES-1 expression was inhibited by a small hairpin RNA (shRNA), and the effects on beta-cell phenotype were analyzed. RESULTS: Human beta-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and downregulation of the cell cycle inhibitor p57. Inhibition of HES-1 expression using shRNA resulted in significantly reduced beta-cell replication and dedifferentiation. CONCLUSIONS: These findings demonstrate that the NOTCH pathway is involved in determining beta-cell fate in vitro and suggest possible molecular targets for induction of beta-cell redifferentiation following in vitro expansion.

Expansion and redifferentiation of adult human pancreatic islet cells.

Beta-cell replacement represents the ultimate cure for type 1 diabetes, however it is limited by availability of organ donors. Adult human islets are difficult to propagate in culture, and efforts to expand them result in dedifferentiation. Here we describe conditions for expansion of adult human islet cells, as well as a way for their redifferentiation. Most cells in islets isolated from human pancreata were induced to replicate within the first week of culture in expansion medium. Cells were propagated for 16 population doublings, without a change in replication rate or noticeable cell mortality, representing an expansion of over 65,000-fold. Replication was accompanied by a decrease in expression of key beta-cell genes. Shift of the cells to differentiation medium containing betacellulin resulted in redifferentiation, as manifested by restoration of beta-cell gene expression and insulin content. These methods may allow transplantation of functional islet cells from single donors into multiple recipients.