Correction to: Prognostic Assessment of Non-functioning Neuroendocrine Pancreatic Neoplasms as a Basis for Risk-Adapted Resection Strategies.

This article contains parts of the doctoral thesis of F. Meyer.

Prognostic Assessment of Non-functioning Neuroendocrine Pancreatic Neoplasms as a Basis for Risk-Adapted Resection Strategies.

BACKGROUND: In contrast to exocrine pancreatic carcinomas, prognosis and treatment of pancreatic neuroendocrine neoplasms (PNEN) are significantly different. The variable growth pattern and associated clinical situation of functioning and non-functioning PNEN demand an individualized surgical approach. However, due to the scarce evidence associated with the rare disease, guidelines lack detailed recommendations for indication and for the required extent of surgical resection. METHODS: In a retrospective single-center study from 1990 to 2018, 239 patients with PNEN were identified. Clinical data were collected in the MaDoc database of the University Medical Center Mainz. A total of 155 non-functional PNEN were selected for further analysis. RESULTS: According to the classification of NET by the WHO in 2017, 28.8% (n = 40) of the tumors were G1, 61.9% (n = 86) G2, and 9.4% (n = 13) G3. In 73 patients, hepatic metastases were present. Sixty patients had lymph node metastasis. An R0 resection was achieved in 98 cases, an R1 situation in 10 cases. Five times, a tumor debulking was carried out (R2) and 5 times the operation was aborted without any resection because of the advanced tumor stage. A relapse occurred in 29 patients. Different prognostic factors (grade, tumor size, age) were analyzed. Grade-dependent 10-year overall survival rates were 79.5% (grade 1) and 60.1% (grade 2), respectively. The survival rate of grade 3 patients was limited to 66.7% after 13 months. CONCLUSION: In our study, patients with non-functioning PNEN had a longer overall survival after successful R0 resection. The risk analysis confirmed a Ki-67 cutoff value of 5%, which divided a high- and low-risk group. Patients with a PNEC G3 (Ki-67 index > 50%) had a very poor prognosis.

[Autologous Bypass to Pedal Side Branches to Avoid a Major Amputation]. / Der autologe Bypass zu den pedalen Seitenästen zur Vermeidung der Majoramputation.

Background: In critical limb ischaemia (CLI), a pedal vein graft bypass offers good long-term results regarding function and limb salvage. However, some cases require bypasses to branches of pedal arteries based on angiographic findings. Methods: In a retrospective database we analysed all patients who received a vein graft bypass to branches of pedal arteries for treatment of critical limb ischaemia. Results: From January 1998 to June 2014 we performed bypasses to branches of pedal arteries in 72 patients (59 men and 13 women) out of a total of 534 patients who underwent pedal bypass surgery. The proximal bypass anastomosis was above the knee in 30 cases and below the knee in 42 patients. In 6 cases the bypass connection was made to the lateral tarsal artery, in 15 cases it was made to the lateral and in 24 cases to the medial plantar artery. In 27 patients a direct connection was made to the plantar bifurcation. All reconstructions were completely autologous. The limb salvage rate after 5 and 10 years was 82 %. Conclusion: A bypass to branches of pedal arteries is a procedure recommendable for limb salvage in cases of critical ischaemia where arteries with a larger diameter are no longer available.

[ASCO-update 2015 - highlights of the 51. meeting of the american society of clinical oncology/ASCO 2015]. / ASCO-Update 2015 - Neuigkeiten vom 51. Meeting der American Society of Clinical Oncology/ASCO 2015.

The field of gastrointestinal oncology is rapidly developing, on the one hand through the identification of novel molecular targets and therapeutic principles, on the other hand through the establishment and improvement of multidisciplinary treatment strategies. The following manuscript summarizes the most important trial results of the ASCO Meeting 2015 for gastrointestinal cancers. Besides trials on perioperative treatment of esophageal-, pancreatic- and colon cancer, we will present impressive data on new therapeutic strategies such as immunotherapy in gastric-, liver and microsatellite instable colorectal cancer. The trials will be put into context by the authors.

Surgical Treatment of NEN of Small Bowel: A Retrospective Analysis.

BACKGROUND: Neuroendocrine Neoplasms of the small intestine have been noticed more frequently over the past 35 years. They constitute about 25% of all NENs and 29% of all tumors of the small intestine. Due to the predominantly indolent nature and overall good prognosis, the benefit of surgical treatment is still debated. METHODS: In a retrospective study, data of 83 surgically treated patients with neuroendocrine neoplasms of the small intestine, 48 males and 35 females with a median age of 62 years (range 25-86 years) were analyzed. Patient data were documented in the MaDoc database for neuroendocrine tumors of the University Medical Center of Mainz. IBM SPSS Statistics 20 was used for statistical analysis. Kaplan-Meier survival curves and Log-Rank tests, censoring patients at the time of last follow-up, were used to compare the overall survival depending on potential prognostic factors (stage, grade, surgical treatment). RESULTS: At the time of diagnoses, the most common clinical symptoms were abdominal pain (n = 31, 37.3%), bowel obstruction (n = 11, 13.3%), bowel perforation and peritonitis (n = 3, 3.6%), gastrointestinal bleeding (n = 9, 10.8%), weight loss (n = 11, 13.3%), and carcinoid syndrome (n = 27, 32.5%). 65 patients (78.3%) had lymph node metastasis and in 58 patients (69.9%) distant metastasis were present. Segmental bowel resection (44) was the most common surgical procedure, followed by right hemi-colectomy (32) and explorative laparotomy (7). In most patients (78.9%), lymphadenectomy (systematic/selective) was performed. The 5-year survival of patients who underwent a systematic or a selective lymphadenectomy differed significantly (82.2 vs. 40.0%). The overall 3-, 5-, and 10-year survival rates were 88.2, 80.3, and 71.0%, respectively. CONCLUSION: Mesenteric lymph node metastases are almost invariably present and have significant impact on patients' prognosis. Systematic lymphadenectomy prevents complications and improves the survival. Early surgical treatment should be the goal in order to prevent complications.

Prognostic factors in stage III-IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study.

BACKGROUND: The clinical course of advanced adrenocortical carcinoma (ACC) is heterogeneous. Our study aimed primarily to refine and make headway in the prognostic stratification of advanced ACC. PATIENTS AND METHODS: Patients with advanced ENSAT ACC (stage III or stage IV) at diagnosis registered between 2000 and 2009 in the ENSAT database were enrolled. The primary end point was overall survival (OS). Parameters of potential prognostic relevance were selected. Univariate and multivariate analyses were carried out: model 1 'before surgery'; model 2 'post-surgery'. RESULTS: Four hundred and forty-four patients with advanced ENSAT ACC (stage III: 210; stage IV: 234) were analyzed. After a median follow-up of 55.2 months, the median OS was 24 months. A modified ENSAT (mENSAT) classification was validated: stage III (invasion of surrounding tissues/organs or the vena renalis/cava) and stage IVa, IVb, IVc (2, 3 or >3 metastatic organs, including N, respectively). Two- or 5-year OS was 73%, 46%, 26% and 15% or 50%, 15%, 14% and 2% for stages III, IVa, IVb and IVc, respectively. In the multivariate analysis, mENSAT stages (stages IVa, IVb, or IVc, respectively) were significantly correlated with OS (P < 0.0001), as well as additional parameters: age ≥ 50 years (P < 0.0001), tumor- or hormone-related symptoms (P = 0.01 and 0.03, respectively) in model 1 but also the R status (P = 0.001) and Grade (Weiss >6 and/or Ki67 ≥ 20%, P = 0.06) in model 2. CONCLUSION: The mENSAT classification and GRAS parameters (Grade, R status, Age and Symptoms) were found to best stratify the prognosis of patients with advanced ACC.

[Neuroendocrine neoplasms of the breast]. / Neuroendokrine Neoplasien der Mamma.

Neuroendocrine neoplasms (NEN) of the breast are specific tumor entities. According to the literature up to 5% of breast neoplasms are malignant epithelial neoplasms of the breast. They are defined by a neuroendocrine (NE) architecture and cytology combined with an expression of the neuroendocrine vesicle markers chromogranin A and/or synaptophysin. The diagnosis is supplemented by the receptor status and the proliferative activity. According to the World Health Organization (WHO) classification of 2012 the following groups of NEN are distinguished: (1) invasive breast carcinoma with NE differentiation, (2) well-differentiated neuroendocrine tumor (NET) and (3) poorly differentiated small cell carcinoma (NEC). This review article focuses on (1) the definition and basic principles of diagnostics, (2) the history, nomenclature and WHO classification from 2003 and 2012, (3) the frequency of breast NEN, (4) the hereditary background and functional activity, (5) the expression of receptors and (6) the possible clinical implications. In addition, the first results of a retrospective single center study (n = 465 patients with breast cancer over a time period of 4 years) on the frequency of NEN of the breast at the Breast Center of the University Hospital Düsseldorf are presented. In this study a frequency of 4.5% of NEN was found based on a diagnostic cut-off of > 50% Chromogranin A and/or synaptophysin positive tumor cells.

Surgical therapy of neuroendocrine neoplasm with hepatic metastasis: patient selection and prognosis.

BACKGROUND: Patients with neuroendocrine neoplasms (NEN) develop hepatic metastases in 50-95 %. The aims of this study were to evaluate the outcome/prognosis of patients following hepatic surgery and to identify predictive factors for the selection of patient that benefit from hepatic tumor resection. PATIENTS AND METHODS: In a retrospective single-center study (1990 to 2014), 204 patients with hepatic metastasis of NEN were included. Ninety-four were subjected to various forms of liver resection. According to the overall survival, the influence of several prognostic factors like the Ki-67 index, stage of disease, and resection status was evaluated. RESULTS: The primary tumor was located in the small intestine (n = 73), pancreas (n = 58), colon (n = 26), esophagus or stomach (n = 9) and in 38 patients the primary site was unknown. The Ki-67 index was associated with significant different overall survival. Patients with an R0 resection (n = 38) of their hepatic metastasis had a very good 10-year survival of 90.4 %. Patients in whom an R1 (n = 23) or R2 (n = 33) resection of their hepatic metastasis could be achieved had a 10-year survival of 53.4 and 51.4 %, respectively. The majority of the patients (53.9 %) could not be resected and had a poor 10-year survival rate of 19.4 %. Partial or complete control of endocrine-related symptoms was achieved in all patients with functioning tumors following surgery. The overall 5- and 10-year survival rates were 77.9 and 65.2 %, respectively. CONCLUSION: Surgical resection of hepatic NEN metastases can reduce symptoms and improve the survival in selected patients with a Ki-67 index less than 20 %. The expected outcome has to be compared to the outcome of alternative treatment strategies. An R0 situation should be the aim of hepatic surgery, but also patients with R1 or R2 resection show a good survival benefit.

[Neuroendocrine neoplasms of the distal jejunum and ileum]. / Neuroendokrine Neoplasien des distalen Jejunums und Ileums.

Neuroendocrine neoplasms (NEN) of the distal jejunum and ileum derive from serotonin-producing enterochromaffin (EC) cells. Due to their low proliferation rate and their infiltrative growth, they are often discovered at an advanced disease stage when metastasis has already occurred. The biology of these tumours is different from other NEN of the digestive tract. In order to standardise and improve diagnosis and therapy, the guidelines for the diagnosis and clinical management of jejuno-ileal NEN as well as for the management of patients with liver and other distant metastases from NEN were revised by the European Neuroendocrine Tumour Society (ENETS) in 2012. This review focuses on aspects relevant for surgical pathology.

Resection strategies for neuroendocrine pancreatic neoplasms.

INTRODUCTION: Due to their rarity and lack of prospective trials, the optimal treatment of pancreatic neuroendocrine neoplasms (PNENs) is still debated. Recommendations gathered by retrospective analyses of patient data should be based on the new classification of neuroendocrine neoplasms. METHODS: In a retrospective single-center study (1990 to 2012), 127 patients with PNENs were included. Tumor stage and type of resections were analyzed to evaluate successful treatment strategies. RESULTS: Seventy-nine patients (62 %) were diagnosed with stage I or II, 48 patients (38 %) with stage III or IV disease; 49.6 % of all PNENs were nonfunctional. Surgical interventions consisted of 50 enucleations, 27 distal resections, and 2 partial duodenopancreatectomies in patients with stage I or II disease. Twenty-eight patients with stage III or IV disease received a distal resection and in 13 patients, a partial duodenopancreatectomy was carried out. Exploration with debulking was performed in seven patients in stages III and IV. Stage-dependent 10-year survival rates were 93.7 (stages I and II, n = 79) and 56.0 % (stages III and IV, n = 48). CONCLUSIONS: PNENs have a good prognosis if they are well-differentiated and resected completely. Organ-preserving resection does not impair the prognosis in selected cases with stage I or II. In case of hepatic metastasis and advanced tumor stage, surgical reduction can reduce symptoms and improve the survival.

[Neuroendocrine neoplasms of the gastrointestinal tract. Classification, clinical presentation and diagnosis]. / Neuroendokrine Neoplasien des Gastrointestinaltrakts. Klassifikation, Klinik und Diagnose.

Neuroendocrine neoplasms of the digestive system represent a rare and heterogeneous group of malignancies with various clinical presentations and prognoses. The WHO classification for the year 2000 was updated in 2010 to take the histopathology and tumor biology of these tumors into account. Together with proliferation-based grading and the recently established staging system using the ENETS TNM classification, it forms the basis for the further diagnostic and therapeutic approach. Clinical presentation depends mainly on the primary site of the tumor and its functionality. Characteristic symptoms are seen only rarely, this being the reason these tumors are usually detected at an advanced stage. Approximately 30% of GEP-NEN are hormonally active and can cause a specific clinical syndrome. In addition to these specific hormones, chromogranin A is considered the most accurate general marker for the biochemical follow-up of these patients. In addition to commonly used radiological and endoscopic imaging modalities, somatostatin receptor-based functional imaging using either octreotide scintigraphy or novel PET-based techniques with specific isotopes such as Ga68-DOTA-octreotate play a crucial role in the detection of the primary tumor as well as in the evaluation of tumor extent and the selection of patients for receptor-based radionuclide therapy.

6-18F-fluoro-L-dihydroxyphenylalanine positron emission tomography is superior to 123I-metaiodobenzyl-guanidine scintigraphy in the detection of extraadrenal and hereditary pheochromocytomas and paragangliomas: correlation with vesicular monoamine transporter expression.

CONTEXT: Pheochromocytomas (PHEOs) and paragangliomas (PGLs) may be better detected by (18)F-fluorodihydroxyphenylalanine-positron emission tomography (FDOPA-PET) than (123)I-metaiodobenzyl-guanidine (123-I-MIBG) scintigraphy. OBJECTIVE: The objective of the study was to correlate functional imaging results with immunohistochemical, molecular-genetic, and biochemical findings. DESIGN AND SETTING: Thirty consecutive patients with suspected PHEO/PGL presenting at a tertiary referral centre were investigated in a prospective study. PATIENTS: Twenty-five patients had confirmed PHEO/PGL. Thirteen of 25 patients had a hereditary PHEO/PGL syndrome (two multiple endocrine neoplasia II, six succinate dehydrogenase complex, subunit D, two succinate dehydrogenase complex, subunit B, one von Hippel Lindau tumor suppressor protein, two Neurofibromatosis-1), and 12 of 25 were classified as sporadic. Five patients had hormonally inactive adrenal incidentalomas. MAIN OUTCOME MEASURES: In all patients computed tomography scan and/or magnetic resonance imaging as well as both 123-I-MIBG scintigraphy and FDOPA-PET were performed. Resected tumors were examined by immunohistochemistry for expression of the vesicular monoamine transporter (VMAT)-1 and -2 and other markers. RESULTS: A total of 64 lesions were found with both functional imaging modalities. FDOPA-PET detected 62 lesions, whereas only 34 lesions were detected by 123-I-MIBG scintigraphy. This resulted in an overall sensitivity and specificity for FDOPA-PET of 98 and 100% and for MIBG of 53 and 91%, respectively. Comparable sensitivities were found for adrenal and extraadrenal abdominal lesions (94 vs. 97%), whereas in thoracic/cervical lesions, the sensitivity for 123-I-MIBG scintigraphy (15%) was inferior to that of FDOPA-PET imaging (100%). Immunohistochemistry demonstrated a lack of VMAT-1 expression in all MIBG-negative tumors. Clinical predictors for MIBG negativity were a predominant norepinephrine/normetanephrine secretion, an age less than 45 yr, and a hereditary cause. CONCLUSION: FDOPA-PET is superior to 123-I-MIBG scintigraphy in patients with extraadrenal, predominantly noradrenaline-secreting, and hereditary types of PHEO/PGL. The lack of VMAT-1 expression predicts negativity for MIBG-scintigraphy.

In vivo molecular imaging of somatostatin receptors in pancreatic islet cells and neuroendocrine tumors by miniaturized confocal laser-scanning fluorescence microscopy.

The aim of the study was to evaluate real time in vivo molecular imaging of somatostatin receptors (sstrs) using a handheld miniaturized confocal laser scan microscope (CLM) in conjunction with fluorescein-labeled octreotate (OcF) in healthy mice and murine models of neuroendocrine tumors. For CLM a small rigid probe (diameter 7 mm) with an integrated single line laser (488 nm) was used (optical slice thickness 7 mum; lateral resolution 0.7 mum). OcF was synthesized via Fmoc solid-phase peptide synthesis and purified by HPLC showing high-affinity binding to the sstr2 (IC(50) 6.2 nmol). For in vitro evaluation, rat and human pancreatic cancer cells were used and characterized with respect to its sstr subtype expression and functional properties. For in vivo confocal imaging, healthy mouse pancreatic islet and renal tubular cells as well as immunoincompetent nude mice harboring sstr-expressing tumors were evaluated. Incubation of sstr-positive cells with OcF showed a specific time- and dose-dependent staining of sstr-positive cells. CLM showed rapid internalization and homogenous cytoplasmatic distribution. After systemic application to mice (n = 8), specific time-dependent internalization and cytoplasmatic distribution into pancreatic islet cells and tubular cells of the renal cortex was recorded. After injection in tumor-harboring nude mice (n = 8), sstr-positive cells selectively displayed a cell surface and cytoplasmatic staining. CLM-targeted biopsies detected sstr-positive tumor cells with a sensitivity of 87.5% and a specificity of 100% as correlated with ex vivo immunohistochemistry. CLM with OcF permits real-time molecular, functional, and morphological imaging of sstr-expressing cell structures, allowing the specific visualization of pancreatic islet cells and neuroendocrine tumors in vivo.

In-vivo confocal real-time mini-microscopy in animal models of human inflammatory and neoplastic diseases.

BACKGROUND AND STUDY AIMS: Although various improvements in tissue imaging modalities have recently been achieved, in-vivo molecular and subsurface imaging in the field of gastroenterology remains a technical challenge. In this study we evaluated a newly developed, handheld, miniaturized confocal laser microscopy probe for real-time in-vivo molecular and subsurface imaging in rodent models of human disease. MATERIALS AND METHODS: The minimicroscope uses a 488-nm, single line laser for fluorophore excitation. The optical slice thickness is 7 microm, the lateral resolution 0.7 microm. The range of the z-axis is 0-250 microm below the tissue surface. Imaging was performed using different fluorescent staining protocols; 5-carboxyfluorescein-labeled octreotate was synthesized for targeted molecular imaging. RESULTS: Cellular and subcellular details of the gastrointestinal tract could be visualized in vivo at high resolution. Confocal real-time microscopy allowed in-vivo identification of tumor vessels and liver metastases, as well as diagnosis of focal hepatic inflammation, necrosis, and associated perfusion anomalies. Somatostatin-receptor targeting permitted in-vivo molecular staining of AR42-J-induced carcinoma and pancreatic islet cells. CONCLUSIONS: Confocal mini-microscopy allows rapid in-vivo molecular and subsurface imaging of normal and pathological tissue in the gastrointestinal tract at high resolution. Because this technology is applicable to humans, it might impact on future in-vivo microsocpic and molecular diagnosis of diseases such as cancer and inflammation.

The role of the insulin-like growth factor system in adrenocortical tumourigenesis.

BACKGROUND: The insulin-like growth factor (IGF) system plays a central role in the mechanism of transformation and tumourigenesis. Elevated levels of IGF-II and IGF-I have been found in adrenocortical carcinomas. MATERIAL AND METHODS: We examined binding characteristics and concentrations of both IGF-receptors in normal adult human adrenocortical glands, and compared them with the IGF-I receptor binding in adrenocortical rumours of various origins. The human IGF-I receptor was overexpressed in the mouse adrenocortical tumour cell line Y1, and growth studied in response to IGF stimulation. The influence of IGF-II on adrenal morphology and function was assessed in transgenic mice that postnatally overexpress IGF-II. RESULTS: While the abundance of the IGF-I receptor in adrenocortical hyperplasias and adenomas was similar to normal tissue, a strong overexpression of the intact IGF-I receptor was found in three out of four adrenocortical carcinomas. Y1 cells overexpressing the human IGF-I receptor respond to IGF-I with an increase in thymidine incorporation by 140%. Furthermore, the antiproliferative effect of ACTH is blunted. In transgenic mice postnatally overexpressing IGF-II, adrenal weight is increased, mainly due to a 50% increase in the number of zona fasciculata cells. Plasma corticosterone levels in these mice are twofold higher than in controls, in contrast to similar plasma ACTH levels, thus indicating a direct effect of IGF-II on adrenal cell hyperplasia and function. CONCLUSION: There is substantial evidence that the IGF-system is involved in adrenal growth and tumourigenesis. High local levels of IGF-II in combination with elevated IGF-I receptor concentrations would represent a significant growth advantage of the adrenocortical carcinoma cell and could contribute to a highly malignant phenotype. IGF-II overexpression alone seems not to be sufficient for malignant transformation.

Regulation of steroidogenesis by insulin-like growth factors (IGFs) in adult human adrenocortical cells: IGF-I and, more potently, IGF-II preferentially enhance androgen biosynthesis through interaction with the IGF-I receptor and IGF-binding proteins.

Although the effect of insulin-like growth factors (IGFs) in fetal adrenocortical cells has been investigated extensively, the role of the IGF system in the adult human adrenal gland remains unclear. In the present study we investigated the effect of recombinant human IGF-I and IGF-II on cortisol, dehydroepiandrosterone sulfate (DHEA-S) and cAMP synthesis in adult human adrenocortical cells in primary culture. Both IGFs stimulate basal as well as adrenocorticotropin (ACTH)-induced steroid secretion in a time- and dose-dependent fashion. While both IGFs (6.5 nM) induced only a moderate 2-fold increase in basal cortisol output after 48 h, the effect on basal DHEA-S secretion was significantly stronger, with a 2.7- and 3.7-fold stimulation by IGF-I and IGF-II respectively. Similarly, IGF-II enhanced ACTH-induced cortisol and DHEA-S secretion more potently than IGF-I. In dose-response experiments, the maximum stimulation of ACTH-induced DHEA-S secretion was induced by 1.6 nM IGF-I (2-fold increase) or IGF-II (2.9-fold increase), while the maximum response of cortisol secretion was elicited only at 13 nM IGF-I (2-fold increase) or IGF-II (2.5-fold increase). This resulted in a significant shift of the DHEA-S dose-response curves to the left, indicating a relative selective stimulation of androgen biosynthesis by physiologically low concentrations (0.4-3.2 nM) of IGF-II, and less potently by IGF-I. At all doses tested, the steroidogenic effect of IGF-II was significantly stronger than the effect of IGF-I. Although both IGF receptors are present in adult human adrenocortical cells, the steroidogenic effect of IGF-II is mediated through the IGF-I receptor, since [Arg54,55]IGF-II, which only binds to the IGF-I receptor, was equipotent with native IGF-II, whereas [Leu27]IGF-II, which preferentially binds to the type II IGF receptor, did not show any effect. In addition, [des1-3]IGF-I, which exhibits only minimal binding to IGFBPs, was significantly more potent than native IGF-I in stimulating adrenal steroid biosynthesis, and elicited almost the same maximum stimulatory effect as IGF-II and [des1-6]IGF-II. By Western ligand blotting of conditioned medium it was shown that adult human adrenocortical cells secrete various IGF-binding proteins (IGFBPs), which are induced differentially by treatment with ACTH. In conclusion, these results demonstrate that: (1)IGF-II stimulates basal as well as ACTH-induced DHEA-S and cortisol secretion from adult human adrenocortical cells more potently than IGF-I; (2) both IGFs predominantly stimulate androgen biosynthesis; (3) the steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor; (4) the different steroidogenic potency of IGF-I and IGF-II might be explained by interaction of these ligands with locally produced IGFBPs. These data indicate that the IGF system plays an important role in the regulation of the differentiated function of adult human adrenocortical cells.