Dynamic Changes in Circulating Tumor Fraction as a Predictor of Real-World Clinical Outcomes in Solid Tumor Malignancy Patients Treated with Immunotherapy.

INTRODUCTION: A dynamic molecular biomarker that can identify early efficacy of immune checkpoint inhibitor (ICI) therapy remains an unmet clinical need. Here we evaluate if a novel circulating tumor DNA (ctDNA) assay, xM, used for treatment response monitoring (TRM), that quantifies changes in ctDNA tumor fraction (TF), can predict outcome benefits in patients treated with ICI alone or in combination with chemotherapy in a real-world (RW) cohort. METHODS: This retrospective study consisted of patients with advanced cancer from the Tempus de-identified clinical genomic database who received longitudinal liquid-based next-generation sequencing. Eligible patients had a blood sample ≤ 40 days prior to the start of ICI initiation and an on-treatment blood sample 15-180 days post ICI initiation. TF was calculated via an ensemble algorithm that utilizes TF estimates derived from variants and copy number information. Patients with molecular response (MR) were defined as patients with a ≥ 50% decrease in TF between tests. In the subset of patients with rw-imaging data between 2 and 18 weeks of ICI initiation, the predictive value of MR in addition to rw-imaging was compared to a model of rw-imaging alone. RESULTS: The evaluable cohort (N = 86) was composed of 14 solid cancer types. Patients received either ICI monotherapy (38.4%, N = 33) or ICI in combination with chemotherapy (61.6%, N = 53). Patients with MR had significantly longer rw-overall survival (rwOS) (hazard ratio (HR) 0.4, P = 0.004) and rw-progression free survival (rwPFS) (HR 0.4, P = 0.005) than patients with molecular non-response (nMR). Similar results were seen in the ICI monotherapy subcohort; HR 0.2, P = 0.02 for rwOS and HR 0.2, P = 0.01 for rwPFS. In the subset of patients with matched rw-imaging data (N = 51), a model incorporating both MR and rw-imaging was superior in predicting rwOS than rw-imaging alone (P = 0.02). CONCLUSIONS: xM used for TRM is a novel serial quantitative TF algorithm that can be used clinically to evaluate ICI therapy efficacy.

Longitudinal Shifts of Solid Tumor and Liquid Biopsy Sequencing Concordance in Metastatic Breast Cancer.

Tissue-based next-generation sequencing (NGS) in metastatic breast cancer (mBC) is limited by the inability to noninvasively track tumor evolution. Cell-free DNA (cfDNA) NGS has made sequential testing feasible; however, the relationship between cfDNA and tissue-based testing in mBC is not well understood. Here, we evaluate concordance between tissue and cfDNA NGS relative to cfDNA sampling frequency in a large, clinically annotated mBC data set. METHODS: Tempus LENS was used to analyze deidentified records of mBC cases with Tempus xT (tissue) and xF (cfDNA) sequencing results. Then, various metrics of concordance were assessed within overlapping probe regions of the tissue and cfDNA assays (104 genes), focusing on pathogenic variants. Variant allele frequencies of discordant and concordant pathogenic variants were also compared. Analyses were stratified by mBC subtype and time between tests. RESULTS: Records from 300 paired tissue and liquid biopsies were analyzed. Median time between tissue and blood collection was 78.5 days (standard deviation = 642.99). The median number of pathogenic variants/patient was one for cfDNA and two for tissue. Across the cohort, 77.8% of pathogenic tissue variants were found in cfDNA and 75.7% of pathogenic cfDNA variants were found in tissue when tests were ≤ 7 days apart, which decreased to 50.3% and 51.8%, respectively, for > 365 days. Furthermore, the median patient-level variant concordance was 67% when tests were ≤7 days apart and 30%-37% when > 30 days. The median variant allele frequencies of discordant variants were generally lower than those of concordant variants within the same time frame. CONCLUSION: We observed high concordances between tissue and cfDNA results that generally decreased with longer durations between tests. Thus, cfDNA NGS reliably measures tissue genomics and is likely beneficial for longitudinal monitoring of molecular changes in mBC.

Prototheca wickerhamii breast implant infection after reconstructive surgery: a new level of complexity.

We report the first published case of Prototheca wickerhamii breast implant infection. This occurred after mastectomy, chemotherapy, radiotherapy, breast reconstruction, implant revisions and breast seroma aspirations and was preceded by polymicrobial infection. Definitive treatment required implant removal and intravenous liposomal amphotericin B. The management of breast prosthesis infections is discussed.

Validation of a liquid biopsy assay with molecular and clinical profiling of circulating tumor DNA.

Liquid biopsy is a valuable precision oncology tool that is increasingly used as a non-invasive approach to identify biomarkers, detect resistance mutations, monitor disease burden, and identify early recurrence. The Tempus xF liquid biopsy assay is a 105-gene, hybrid-capture, next-generation sequencing (NGS) assay that detects single-nucleotide variants, insertions/deletions, copy number variants, and chromosomal rearrangements. Here, we present extensive validation studies of the xF assay using reference standards, cell lines, and patient samples that establish high sensitivity, specificity, and accuracy in variant detection. The Tempus xF assay is highly concordant with orthogonal methods, including ddPCR, tumor tissue-based NGS assays, and another commercial plasma-based NGS assay. Using matched samples, we developed a dynamic filtering method to account for germline mutations and clonal hematopoiesis, while significantly decreasing the number of false-positive variants reported. Additionally, we calculated accurate circulating tumor fraction estimates (ctFEs) using the Off-Target Tumor Estimation Routine (OTTER) algorithm for targeted-panel sequencing. In a cohort of 1,000 randomly selected cancer patients who underwent xF testing, we found that ctFEs correlated with disease burden and clinical outcomes. These results highlight the potential of serial testing to monitor treatment efficacy and disease course, providing strong support for incorporating liquid biopsy in the management of patients with advanced disease.

Case 291: Amyloid-associated Cystic Lung Disease and Coexistent Mucosa-associated Lymphoid Tissue Lymphoma.

History A 46-year-old woman with known mixed connective tissue disease with clinical features of scleroderma and polymyositis and who was not on specific medications was referred to our institution to assess for interstitial lung disease due to her predisposing condition. She was a nonsmoker, had no respiratory symptoms, and enjoyed good exercise tolerance. She did not have any cutaneous lesions or renal disease. There was no family history of pulmonary or systemic disease. Her routine blood test results revealed a white blood cell count of 4.6 × 109/L (normal range, [4.4-10.1] × 109/L), a hemoglobin level of 7.76 mmol/L (normal range, 7.26-9.18 mmol/L), a platelet count of 189 × 109/L (normal range, [170-380] × 109/L), a bilirubin level of 8 mmol/L (normal range, <19 mmol/L), and a creatinine level of 63 mmol/L (normal range, 45-82 mmol/L), all within normal limits. Lung function tests at presentation yielded normal results, with a diffusing capacity for carbon monoxide of 95% and a forced vital capacity of 2.29 (98% predicted value). However, this patient had an elevated serum globulin level of 47 g/L (normal range, 26-32 g/L) and an erythrocyte sedimentation rate of 36 mm/h (normal range, 0-20 mm/h), while C-reactive protein level was normal at less than 0.35 mg/dL. She was seropositive for antinuclear (titer >1/720), anti-Ro, anti-La, and anti-extractable nuclear antigen antibodies. Chest radiography and CT were performed at presentation and 14-year follow-up. PET/CT was performed at 7- and 13-year follow-up. Throughout this 14-year follow-up period, she remained completely free of respiratory symptoms and continued to go for a brisk walk every day. At 14-year follow-up, there was no substantial change in serum laboratory values, but a lung function test revealed her diffusing capacity for carbon monoxide had decreased to 52%, while her forced vital capacity remained good at 95%; these findings were suggestive of interval development of restrictive lung function.

Proof of concept: Screening for REM sleep behaviour disorder with a minimal set of sensors.

OBJECTIVE: Rapid-Eye-Movement (REM) sleep behaviour disorder (RBD) is an early predictor of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. This study investigated the use of a minimal set of sensors to achieve effective screening for RBD in the population, integrating automated sleep staging (three state) followed by RBD detection without the need for cumbersome electroencephalogram (EEG) sensors. METHODS: Polysomnography signals from 50 participants with RBD and 50 age-matched healthy controls were used to evaluate this study. Three stage sleep classification was achieved using a random forest classifier and features derived from a combination of cost-effective and easy to use sensors, namely electrocardiogram (ECG), electrooculogram (EOG), and electromyogram (EMG) channels. Subsequently, RBD detection was achieved using established and new metrics derived from ECG and EMG channels. RESULTS: The EOG and EMG combination provided the optimal minimalist fully-automated performance, achieving 0.57 ± 0.19 kappa (3 stage) for sleep staging and an RBD detection accuracy of 0.90 ± 0.11, (sensitivity and specificity of 0.88 ± 0.13 and 0.92 ± 0.098, respectively). A single ECG sensor achieved three state sleep staging with 0.28 ± 0.06 kappa and RBD detection accuracy of 0.62 ± 0.10. CONCLUSIONS: This study demonstrates the feasibility of using signals from a single EOG and EMG sensor to detect RBD using fully-automated techniques. SIGNIFICANCE: This study proposes a cost-effective, practical, and simple RBD identification support tool using only two sensors (EMG and EOG); ideal for screening purposes.

Discrimination of pulmonary ground-glass opacity changes in COVID-19 and non-COVID-19 patients using CT radiomics analysis.

PURPOSE: The coronavirus disease 2019 (COVID-19) has evolved into a worldwide pandemic. CT although sensitive in detecting changes suffers from poor specificity in discrimination from other causes of ground glass opacities (GGOs). We aimed to develop and validate a CT-based radiomics model to differentiate COVID-19 from other causes of pulmonary GGOs. METHODS: We retrospectively included COVID-19 patients between 24/01/2020 and 31/03/2020 as case group and patients with pulmonary GGOs between 04/02/2012 and 31/03/2020 as a control group. Radiomics features were extracted from contoured GGOs by PyRadiomics. The least absolute shrinkage and selection operator method was used to establish the radiomics model. We assessed the performance using the area under the curve of the receiver operating characteristic curve (AUC). RESULTS: A total of 301 patients (age mean ±â€¯SD: 64 ±â€¯15 years; male: 52.8 %) from three hospitals were enrolled, including 33 COVID-19 patients in the case group and 268 patients with malignancies or pneumonia in the control group. Thirteen radiomics features out of 474 were selected to build the model. This model achieved an AUC of 0.905, accuracy of 89.5 %, sensitivity of 83.3 %, specificity of 90.0 % in the testing set. CONCLUSION: We developed a noninvasive radiomics model based on CT imaging for the diagnosis of COVID-19 based on GGO lesions, which could be a promising supplementary tool for improving specificity for COVID-19 in a population confounded by ground glass opacity changes from other etiologies.

A Case of Multiple Sclerosis-Like Relapsing Remitting Encephalomyelitis Following Allogeneic Hematopoietic Stem Cell Transplantation and a Review of the Published Literature.

Complications involving the central nervous system (CNS) occur in 9-14% of patients following allogeneic hematopoietic stem cell transplantation (HSCT), including stroke-like episodes, demyelination, encephalitis, and nonspecific neurological symptoms. Here we report a case of multiple sclerosis (MS) like relapsing remitting encephalomyelitis following allogeneic HSCT, which did not respond to disease modifying therapies (DMTs) and "domino" autologous HSCT. A 53-year-old male was treated with allogeneic HSCT for lymphoid blast transformation of chronic myeloid leukemia. Ten months later he presented with confusion, slurred speech, left sided facial weakness and ataxia. A magnetic resonance imaging brain scan showed multiple enhancing tumefactive lesions. Neuromyelitis optica (NMO) and myelin oligodendrocyte glycoprotein (MOG) antibodies were negative. After extensive investigations for infections, autoimmune disorders and recurrence of malignancy, he underwent brain biopsy, which showed a macrophage rich lesion with severe myelin loss but axonal preservation indicating a demyelinating pathology. Although his symptoms improved with corticosteroids, he relapsed five months later. In the absence of any systemic features suggesting graft versus host disease (GvHD), his presentation was thought to be compatible with MS. The illness followed an aggressive course that did not respond to glatiramer acetate and natalizumab. He was therefore treated with "domino" autologous HSCT, which also failed to induce long-term remission. Despite further treatment with ocrelizumab, he died of progressive disease. An autopsy limited to the examination of brain revealed multifocal destructive leukoencephalopathy with severe myelin and axonal loss. Immunohistochemistry showed macrophage located in the perivascular area, with no T or B lymphocytes. The appearance was unusual and not typical for chronic MS plaques. Reported cases of CNS demyelination following allogeneic HSCT are very limited in the literature, especially in relation to histopathological examination. Although the clinical disease course of our patient following allogeneic HSCT resembled an "MS-like" relapsing remitting encephalomyelitis, the autopsy examination did not show any evidence of active inflammation. The impact of DMTs and HSCT on the histological appearance of "MS-like" CNS pathologies is unknown. Therefore, reporting this and similar cases will improve our awareness and understanding of underlying disease mechanisms.

Prodromal Parkinsonism and Neurodegenerative Risk Stratification in REM Sleep Behavior Disorder.

Objectives: Rapid eye movement (REM) sleep behavior disorder (RBD) is the most specific marker of prodromal alpha-synucleinopathies. We sought to delineate the baseline clinical characteristics of RBD and evaluate risk stratification models. Methods: Clinical assessments were performed in 171 RBD, 296 control, and 119 untreated Parkinson's (PD) participants. Putative risk measures were assessed as predictors of prodromal neurodegeneration, and Movement Disorders Society (MDS) criteria for prodromal PD were applied. Participants were screened for common leucine-rich repeat kinase 2 (LRRK2)/glucocerebrosidase gene (GBA) gene mutations. Results: Compared to controls, participants with RBD had higher rates of solvent exposure, head injury, smoking, obesity, and antidepressant use. GBA mutations were more common in RBD, but no LRRK2 mutations were found. RBD participants performed significantly worse than controls on Unified Parkinson's Disease Rating Scale (UPDRS)-III, timed "get-up-and-go", Flamingo test, Sniffin Sticks, and cognitive tests and had worse measures of constipation, quality of life (QOL), and orthostatic hypotension. For all these measures except UPDRS-III, RBD and PD participants were equally impaired. Depression, anxiety, and apathy were worse in RBD compared to PD participants. Stratification of people with RBD according to antidepressant use, obesity, and age altered the odds ratio (OR) of hyposmia compared to controls from 3.4 to 45.5. 74% (95% confidence interval [CI] 66%, 80%) of RBD participants met the MDS criteria for probable prodromal Parkinson's compared to 0.3% (95% CI 0.009%, 2%) of controls. Conclusions: RBD are impaired across a range of clinical measures consistent with prodromal PD and suggestive of a more severe nonmotor subtype. Clinical risk stratification has the potential to select higher risk patients for neuroprotective interventions.

Mitochondrial pathology in progressive cerebellar ataxia.

BACKGROUND: Mitochondrial disease can manifest as multi-organ disorder, often with neurological dysfunction. Cerebellar ataxia in isolation or in combination with other features can result from mitochondrial disease yet genetic testing using blood DNA is not sufficient to exclude this as a cause of ataxia. Muscle biopsy is a useful diagnostic tool for patients with ataxia suspected of mitochondrial disease. Our aim was to determine specific patient selection criteria for muscle biopsy to see how frequent mitochondrial mutations are responsible for progressive ataxia. We performed a two centre retrospective review of patients with unexplained progressive ataxia who underwent muscle biopsy for suspected mitochondrial disease between 2004 and 2014 (Sheffield and Newcastle Ataxia Centres). RESULTS: A total of 126 patients were identified; 26 assessed in Newcastle and 100 in Sheffield. Twenty-four patients had pure ataxia and 102 had ataxia with additional features. The total number of patients with histologically suspected and/or genetically confirmed mitochondrial disease was 29/126 (23 %). CONCLUSIONS: A large proportion of patients (23 %) with progressive ataxia who underwent muscle biopsy were found to have features of mitochondrial dysfunction, with molecular confirmation in some. Muscle biopsy is a helpful diagnostic tool for mitochondrial disease in patients with progressive ataxia.

The Role of Computed Tomography Scanning of the Thorax in the Initial Assessment of Gestational Trophoblastic Neoplasia.

OBJECTIVE: The aim of this study was to determine whether lesions found on computed tomography (CT) imaging of the thorax would affect FIGO (International Federation of Gynecology and Obstetrics) 2000 risk score and/or alter clinical management. METHODS: The Sheffield Trophoblastic Disease database was searched for all new patients registered for staging/scoring investigations between January 1, 2006, and June 30, 2010. The FIGO 2000 score was noted and then recalculated using information from CT scan reports. Where a change of risk score would have affected a patient's management, the case notes were further reviewed. RESULTS: 191 patients had undergone both modalities of imaging. Using standard FIGO scoring, 169 and 22 patients were noted to be at low and high risk, respectively. Using information from CT imaging, only a further 20 patients would have been reclassified as high risk. Fifteen of these "new" high-risk patients required salvage treatment with intravenous chemotherapy; all were cured. CONCLUSIONS: With no potential advantage in terms of patient outcome and significantly increased radiation dose, there is little justification for routine CT imaging of the thorax in the initial assessment of new patients with gestational trophoblastic neoplasia.

A genetic study of Wilson's disease in the United Kingdom.

Previous studies have failed to identify mutations in the Wilson's disease gene ATP7B in a significant number of clinically diagnosed cases. This has led to concerns about genetic heterogeneity for this condition but also suggested the presence of unusual mutational mechanisms. We now present our findings in 181 patients from the United Kingdom with clinically and biochemically confirmed Wilson's disease. A total of 116 different ATP7B mutations were detected, 32 of which are novel. The overall mutation detection frequency was 98%. The likelihood of mutations in genes other than ATP7B causing a Wilson's disease phenotype is therefore very low. We report the first cases with Wilson's disease due to segmental uniparental isodisomy as well as three patients with three ATP7B mutations and three families with Wilson's disease in two consecutive generations. We determined the genetic prevalence of Wilson's disease in the United Kingdom by sequencing the entire coding region and adjacent splice sites of ATP7B in 1000 control subjects. The frequency of all single nucleotide variants with in silico evidence of pathogenicity (Class 1 variant) was 0.056 or 0.040 if only those single nucleotide variants that had previously been reported as mutations in patients with Wilson's disease were included in the analysis (Class 2 variant). The frequency of heterozygote, putative or definite disease-associated ATP7B mutations was therefore considerably higher than the previously reported occurrence of 1:90 (or 0.011) for heterozygote ATP7B mutation carriers in the general population (P < 2.2 × 10(-16) for Class 1 variants or P < 5 × 10(-11) for Class 2 variants only). Subsequent exclusion of four Class 2 variants without additional in silico evidence of pathogenicity led to a further reduction of the mutation frequency to 0.024. Using this most conservative approach, the calculated frequency of individuals predicted to carry two mutant pathogenic ATP7B alleles is 1:7026 and thus still considerably higher than the typically reported prevalence of Wilson's disease of 1:30 000 (P = 0.00093). Our study provides strong evidence for monogenic inheritance of Wilson's disease. It also has major implications for ATP7B analysis in clinical practice, namely the need to consider unusual genetic mechanisms such as uniparental disomy or the possible presence of three ATP7B mutations. The marked discrepancy between the genetic prevalence and the number of clinically diagnosed cases of Wilson's disease may be due to both reduced penetrance of ATP7B mutations and failure to diagnose patients with this eminently treatable disorder.

Concurrent amyotrophic lateral sclerosis and cystic fibrosis supports common pathways of pathogenesis.

Abstract We present a case of concurrent cystic fibrosis (CF) and amyotrophic lateral sclerosis (ALS). To our knowledge this is the first reported coincidence of these two diseases. Although TDP-43 dysfunction has been linked to both pathologies, it does not appear to be pivotal in this individual who does not display TDP-43 mediated aberrant splicing of the CFTR gene or carry a mutation in the TARDBP gene. Alternative reasons for the coincidence are discussed including medication, infection, hypoxia and loss of function of the CFTR channel. Our findings await validation by others, but as the prognosis of CF improves then clinicians in both fields should be aware of the possibilities highlighted by this case.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

A rare complication of laparoscopic low anterior resection.

We report a 70-year-old lady who suffered from carcinoma of rectum with the lowest border of the tumor at 5 cm from the anal verge. Preoperative staging did not show any distant metastasis. Laparoscopic low anterior resection with total mesorectal excision was performed. The rectum was transected with endoscopic stapler and a double stapling anastomosis with a circular stapler was attempted. The spear of the circular stapler pierced through the rectal stump and attempt to remove the spear by pulling the suture attached to the spear failed. A pair of strong laparoscopic forceps was used in an attempt to remove the spear. The spear broke during the attempt of removal. The stapler was withdrawn through the anus with a pair of laparoscopic forceps following it. A new circular stapler was used and the tip was tied to a suture and cotton tape. The pair of forceps, which was still at the anus, was used to pull the cotton tape and the suture through the original perforation at the rectal stump. The tip of the spear of the new stapler was guided through the original perforation of the rectal stump. Double stapling was then performed and the patient recovered uneventfully. This case showed that stapler complication in laparoscopic surgery could be salvaged without conversion.

Marijuana and chronic obstructive lung disease: a population-based study.

BACKGROUND: Our aim was to determine the combined and independent effects of tobacco and marijuana smoking on respiratory symptoms and chronic obstructive pulmonary disease (COPD) in the general population. METHOD: We surveyed a random sample of 878 people aged 40 years or older living in Vancouver, Canada, about their respiratory history and their history of tobacco and marijuana smoking. We performed spirometric testing before and after administration of 200 microg of salbutamol. We examined the association between tobacco and marijuana smoking and COPD. RESULTS: The prevalence of a history of smoking in this sample was 45.5% (95% confidence interval [CI] 42.2%-48.8%) for marijuana use and 53.1% (95% CI 49.8%-56.4%) for tobacco use. The prevalence of current smoking (in the past 12 months) was 14% for marijuana use and 14% for tobacco use. Compared with nonsmokers, participants who reported smoking only tobacco, but not those who reported smoking only marijuana, experienced more frequent respiratory symptoms (odds ratio [OR] 1.50, 95% CI 1.05-2.14) and were more likely to have COPD (OR 2.74, 95% CI 1.66-4.52). Concurrent use of marijuana and tobacco was associated with increased risk (adjusted for age, asthma and comorbidities) of respiratory symptoms (OR 2.39, 95% CI 1.58-3.62) and COPD (OR 2.90, 95% CI 1.53-5.51) if the lifetime dose of marijuana exceeded 50 marijuana cigarettes. The risks of respiratory symptoms and of COPD were related to a synergistic interaction between marijuana and tobacco. INTERPRETATION: Smoking both tobacco and marijuana synergistically increased the risk of respiratory symptoms and COPD. Smoking only marijuana was not associated with an increased risk of respiratory symptoms or COPD.

Polysaccharide peptide mediates apoptosis by up-regulating p21 gene and down-regulating cyclin D1 gene.

The use of herbal medicine is a common practice among Chinese women with breast cancer. Yunzhi (Voriolus versicolor), a substance that is regarded as a biological response modifier, is frequently used. The aim of the present study is to evaluate the anti-proliferative action of, Yunzhi, polysaccharide peptide (PSP), on breast cancer cells. Breast cancer cells (MDA-MB-231) were cultured with and without PSP for 7 days. Cell growth at 24, 72, 120 and 168 hours was measured by Cell Proliferation Reagent (WST-1). Cells treated with PSP were found to have a significant reduction in cell proliferation as compared to controls after 72 hours of incubation. This lasted for 168 hours. When the effect of PSP on apoptosis was studied by the TdT-mediated X-dUTP nick end-labeling (TUNEL) assay, we found that PSP had a significant effect upon apoptosis from 24 hours onward. Immunostaining showed that PSP increased p21 expression and decreased cyclin D1 expression. In conclusion, PSP is effective in inhibiting cell proliferation through apoptosis. The mechanism for the apoptosis may be through up-regulation of p21 and down-regulation of cyclin D1.