Reconstructing tumor history in breast cancer: signatures of mutational processes and response to neoadjuvant chemotherapy⋆.

BACKGROUND: Different endogenous and exogenous mutational processes act over the evolutionary history of a malignant tumor, driven by abnormal DNA editing, mutagens or age-related DNA alterations, among others, to generate the specific mutational landscape of each individual tumor. The signatures of these mutational processes can be identified in large genomic datasets. We investigated the hypothesis that genomic patterns of mutational signatures are associated with the clinical behavior of breast cancer, in particular chemotherapy response and survival, with a particular focus on therapy-resistant disease. PATIENTS AND METHODS: Whole exome sequencing was carried out in 405 pretherapeutic samples from the prospective neoadjuvant multicenter GeparSepto study. We analyzed 11 mutational signatures including biological processes such as APOBEC-mutagenesis, homologous recombination deficiency (HRD), mismatch repair deficiency and also age-related or tobacco-induced alterations. RESULTS: Different subgroups of breast carcinomas were defined mainly by differences in HRD-related and APOBEC-related mutational signatures and significant differences between hormone-receptor (HR)-negative and HR-positive tumors as well as correlations with age, Ki-67 and immunological parameters were observed. We could identify mutational processes that were linked to increased pathological complete response rates to neoadjuvant chemotherapy with high significance. In univariate analyses for HR-positive tumors signatures, S3 (HRD, P < 0.001) and S13 (APOBEC, P = 0.001) as well as exonic mutation rate (P = 0.002) were significantly correlated with increased pathological complete response rates. The signatures S3 (HRD, P = 0.006) and S4 (tobacco, P = 0.011) were prognostic for reduced disease-free survival of patients with chemotherapy-resistant tumors. CONCLUSION: The results of this investigation suggest that the clinical behavior of a tumor, in particular, response to neoadjuvant chemotherapy and disease-free survival of therapy-resistant tumors, could be predicted by the composition of mutational signatures as an indicator of the individual genomic history of a tumor. After additional validations, mutational signatures might be used to identify tumors with an increased response rate to neoadjuvant chemotherapy and to define therapy-resistant subgroups for future therapeutic interventions.

Induction of cytokine production from human monocytes stimulated with alginate.

Alginates are polysaccharides with gel-forming properties composed of 1,4-linked beta-D-mannuronic acid (M), alpha-L-guluronic acid (G), and alternating (MG) blocks. Alginate can be used as a matrix for implanted cells in vivo. In this study, we have examined the ability of alginates and their components to stimulate human monocytes to produce tumor necrosis factor-alpha, interleukin-6, and interleukin-1. Alginates stimulated the monocytes to produce high levels of all three cytokines. Low G alginates were approximately 10 times more potent in inducing cytokine production compared with high G alginates. The M-blocks and the MG-blocks, but not the G-blocks, stimulated the cytokine production. The results demonstrate that the mannuronic acid residues are the active cytokine inducers in alginates.

Prolonged pancreas preservation using a simplified UW solution containing polyethylene glycol.

The University of Wisconsin solution is considered the most effective universal flush and cold storage solution to date, and is now being widely applied clinically in organ transplantation. The results of this study show that Cardiosol, a modified cardioplegic solution containing 5% polyethylene glycol (PEG20M, MW 17,000 daltons), is significantly superior to UW (P less than 0.001) in the flush perfusion and hypothermic storage of pancreases for more than 36 hr prior to transplantation into streptozotocin-induced diabetic rats. When the pancreases were stored in Cardiosol, the 1-week survival rate was 7 of 10 (70%) after 24 hr of preservation; 7 of 12 (58%) after 36 hr; and 3 of 10 (30%) after 48 hr. In contrast, when the pancreases were stored in UW solution, the 1-week survival rate was 8 of 12 (67%) after 24 hr of preservation; after 36 hr, no animal survived (0 of 14). Intravenous glucose tolerance test K-values (decline in glucose concentration, percentage per minute) were normal in both groups receiving 24-hr-preserved pancreases, ranging from 2.60 to 4.16. Of interest, the peak insulin response 1 min following intravenous glucose was significantly higher (P less than 0.01) in the Cardiosol-preserved organs (303 +/- 29.8 microU/ml) (+/- SEM) than in the glands preserved in UW solution (112 +/- 47.9 microU/ml). We conclude that Cardiosol allows prolonged whole organ pancreas preservation in the rat transplant model.

Rapid purification of islets using magnetic microspheres coated with anti-acinar cell monoclonal antibodies.

A simple, rapid method of islet purification is important in large-scale human islet isolation. We have previously identified monoclonal antibodies specific for acinar cells, but not islets, and described an immunologic method of purification by selective lysis of the acinar cells. An attractive alternative to lysis of the acinar cell is depletion by a magnetic immunomicrosphere technique. We report in this study a rapid, reproducible method of rat islet purification utilizing magnetic microspheres coated with acinar-cell-specific monoclonal antibodies. Pancreatic digestion with collagenase followed by depletion of acinar cells with the magnetic immunomicrospheres (MIMS) yields large numbers of intact islets. We compared the islets thus obtained with hand-picked (HP) islets (control) for yield, purity, in vitro insulin secretory capacities, and in vivo functional viability. The islet yield with the MIMS method (n = 35) was 72.7% that obtained with the HP method (n = 6) (378 +/- 8 vs. 519 +/- 31 islets per pancreas). The purity of the MIMS-isolated islets was 84 +/- 1.9%, ranging from 75-95%. Static glucose stimulation showed excellent function (2-3-fold increase of insulin release over basal levels) with no statistical difference in insulin secretion between MIMS and HP islets. Under microscopic examination, both groups revealed a well-preserved structure with healthy endocrine cells. When 1321 +/- 59 MIMS islets were transplanted into streptozotocin-induced diabetic rats (n = 10), normoglycemia (less than 200 mg/dl) was restored in all recipients following transplantation, and 100% of them remained normoglycemic on day 120 postgrafting. In summary, a rapid, consistent, and simple method of isolating viable, purified rat islets is described. The broad interspecies crossreactivity of the McAb suggests that this technique may be generally useful for islet purification in large mammalia, including man.

Successful reversal of spontaneous diabetes in dogs by intraperitoneal microencapsulated islets.

Long-term euglycemia by intraperitoneal transplantation of microencapsulated islets has not been described in the diabetic large animal model. In this study, we report the successful long-term reversal of diabetes by this method in spontaneous diabetic dogs. We have identified fundamental mechanism(s) associated with alginate-based microcapsule fibrosis, and have devised methods to ameliorate this problem. These include the use of purified alginate of low mannuronic acid content and cytokine suppression. Ten insulin-dependent, spontaneous diabetic dogs (insulin requirement 1-4 units/kg/day; absence of circulating C-peptide and diabetic K-values of 0.6 +/- 0.4) were entered into the study. Islets from mongrel donor pancreata were isolated and transplanted intraperitoneally either as free islet controls (n = 3) or as microencapsulated islet allografts (n = 7). In all seven encapsulated islet recipients, euglycemia was achieved within 24 hr (serum glucose failing from 304 +/- 117 to 116 +/- 72 mg/dl). IVGTT performed 14 days after islet transplant demonstrated normalization of K-values changing from a pretransplant level of 0.6 +/- 0.4 to 2.6 +/- 0.6. All animals receiving encapsulated islets remained euglycemic, free of the need for exogenous insulin, for a period of 63-172 days, with a median insulin-independence for 105 days. In contrast, recipients receiving free islets rejected their graft within seven days of implantation. In conclusion, this is the first report of long-term successful reversal of spontaneous diabetes in the large animal model by an intraperitoneal injection of encapsulated islets. The potential exists for this form of therapy to be explored in the treatment of type I diabetes in man.

Alginate polycation microcapsules. II. Some functional properties.

The main cause of alginate polycation capsule breakage under physiological conditions is probably the osmotic swelling of the alginate core owing to the Donnan equilibrium set up by the negative charges of the carboxyl groups not involved in cooperative binding of counterions in the junction zones of the network. In the present paper we show how capsules can be stabilized extensively by reducing their swelling capacity in various ways. Alginate polycation capsules with good chemical and mechanical stability have been made by controlling their swelling behaviour through selection of capsule material according to chemical structure and molecular weight, as well as by controlling the kinetics of the capsule formation. Stable capsules have been made either by increasing the strength of the polyanion-polycation membrane, or by keeping a low-swelling gel network in the core. The latter capsules are made from an alginate rich in guluronic acid both in the core and in an outer coating, and with anisotropic distribution of the polymer material in the core where the concentration at the surface is higher than that in the centre of the capsule. Some functional properties of these capsules, such as porosity, have also been studied.

Alginate polycation microcapsules. I. Interaction between alginate and polycation.

The interactions between alginate and polycations have been studied by using different labelling techniques. Binding of poly-L-lysine (PLL) to alginate in the gel state is mainly governed by the amount of dissociable negative charges on the bead surface. PLL was found to bind more rapidly to gel beads made from alginate with a high content of mannuronic acid. The binding was enhanced by increasing the alginate concentration on the surface by making inhomogeneous beads. When the capsules were stored in the presence of cations with high affinity for alginate (Ca2+, Sr2+), PLL was washed off. Less PLL is bound to strontium alginate than to calcium alginate beads. Two mechanisms appear to be responsible for the binding of sodium alginate to alginate PLL capsules (coating): (i) an electrostatic interaction between the soluble coating material and excess positive charges on PLL on the surface; (ii) the formation of a calcium alginate gel on the surface owing to leaching of calcium ions from the core. The stability and efficiency of the coating as a function of molecular size and sequential structure of the coating polymer have also been investigated.

Vagal control of gastrin release in the dog: pathways for stimulation and inhibition.

The vagus has a dual effect on gastrin: it both stimulates and inhibits its release. To determine the gastric vagal pathways for these opposing effects, plasma gastrin and acid responses to meal (intragastric titration of 15% liver extract, pH 5.5) and to insulin (0.5 U regular insulin intravenously) were studied in seven dogs in three consecutive stages: a control stage, after antral vagotomy (AV), and after subsequent proximal gastric vagotomy (PGV). AV abolished the plasma gastrin response to insulin but had no effect on either basal or meal-stimulated gastrin release. Subsequent PGV caused significant elevation in basal plasma gastrin concentration, no further change in the gastrin response to insulin, but a significant increase in meal-stimulated gastrin release. AV decreased acid response to insulin nonsignificantly and had no effect on meal-stimulated acid secretion. Subsequent PGV reduced by 90% the acid response to insulin, had negligible effect on the gastric fistula acid response to meal, but increased Heidenhain pouch response sixfold. These studies show that vagal stimulation of gastrin release is mediated along direct antral vagal pathways, while vagal inhibition requires intact vagal fibers to the proximal stomach. The mechanism by which the fundic vagal pathways exert an inhibitory influence on the G cell in the antrum is yet to be elucidated.

Successful long-term exocrine and endocrine function of the autotransplanted pancreas in humans.

Segmental pancreatic autotransplantation has been performed to prevent the severe metabolic complications of total pancreatectomy. To date 15 segmental pancreatic autotransplants have been reported, 11 of which have been performed for relief of the abdominal pain of chronic pancreatitis. The major problem with segmental pancreatic graft relates to the handling of the pancreatic duct and its secretion. In all the reported cases, the autotransplanted duct was either ligated, stapled, or occluded with synthetic polymers. In this article we present a patient who has undergone a total pancreatectomy with segmental pancreatic autotransplantation and subsequent Roux-en-Y anastomosis to the transplanted duct. Physiologic studies indicate normal endocrine function 7 years following transplant. The patient is insulin-independent and tolerates a normal meal, requiring no oral pancreatic enzyme supplementation. To our knowledge this is the first long-term report of a patient with an autotransplanted pancreas who is presently both insulin sufficient and with intact exocrine function.

Immunocytochemical identification of monoclonal antibodies with binding activity to acinar cells but not islets.

The development of techniques for separating islets from acinar cells on a mass scale is a prerequisite to successful clinical attempts at islet transplantation. We have identified and partially characterized two blood group-reactive monoclonal antibodies (McAb), CAC1 and CAC2, with specific binding activity to acinar cells but not islets. These McAb are of the IgM subclass, and were found on immunocytochemical analysis to possess broad interspecies cross-reactivity, binding to antigens expressed in the acinar tissue of rats, dogs, and man. The antigens that these McAb recognize are glycolipid in nature and thus were not denatured by collagenase digestion of the pancreas. These properties, together with their ability to effect complement-mediated lysis, may make the McAb generally useful reagents in islet isolation and purification.

Molecular mass distribution of sodium alginate by high-performance size-exclusion chromatography.

A sensitive high-performance size-exclusion chromatography (HPSEC) method with simple UV detection was developed for the molecular mass analysis of sodium alginate. It was used to evaluate alginates of varying molecular mass and the results were compared with the viscosity measurements. This HPSEC method was sensitive to serve as the stability indicating method for alginate after storage under different conditions. The information of relative molecular mass distribution of alginate was provided with reference to pullulan molecular mass standards. The comparison of the HPSEC chromatograms of alginate, pullulan and dextran revealed the effect of chemical composition of a polysaccharide and its effect on apparent molecular mass distribution.

Evidence of a noncholecystokinin stimulant of gallbladder contraction: comparison of fasting serum concentrations in healthy subjects and in patients with gallstones.

Some investigators have reported that patients with gallstones empty their gallbladders more rapidly than do healthy subjects. This may contribute to the formation of lithogenic bile. To date, cholecystokinin is considered the prime mediator of gallbladder contraction. Evidence exists that cholecystokinin may not be the major hormone accounting for gallbladder emptying. The purpose of this study was to demonstrate the existence of this noncholecystokinin substance in healthy persons and to compare its concentration with that in patients with cholesterol gallstones. Fasting serum levels from 15 healthy human subjects (8 women and 7 men, mean age 32 +/- 8 years) and 10 patients with cholesterol gallstones (5 women and 5 men, mean age 48 +/- 16 years) were studied. Using rabbit in vitro gallbladder bioassay and cholecystokinin-8 as standards, serum bioactivity was measured and expressed as cholecystokinin-8 equivalent bioactivity. The effectiveness of serum to contract the gallbladder was tested before and after removal of cholecystokinin from the serum. Cholecystokinin was removed from the serum samples by affinity chromatography with Sepharose 4B beads coated with cholecystokinin 5135 antibody. Gallbladder contractility from this treated serum thus reflects the action of a noncholecystokinin stimulant. The cholecystokinin-8 bioactivity equivalents in untreated samples from healthy subjects and from patients with gallstones were 2.9 +/- 0.3 and 7.6 +/- 0.7 ng/ml, respectively. The fact that bioactivity of serum persisted after removal of cholecystokinin in both groups of subjects provides evidence that a noncholecystokinin stimulant of gallbladder contraction exists. This substance is found in significantly higher concentrations in the fasting serum of patients with gallstones compared with healthy subjects. This finding may explain, at least in part, the increased gallbladder emptying rate in patients with gallstones and may account for the reduced bile salt pool size and, thus, formation of lithogenic bile.

Use of secretin in the roentgenologic and biochemical diagnosis of duodenal gastrinoma.

The use of secretin in the biochemical and roentgenologic diagnoses of a duodenal gastrinoma has been described. Preoperatively, the secretin test indicated that a gastrinoma and not a retained antrum was the cause of hypergastrinemia in a patient who had previously undergone Billroth II gastrectomy. Intravenous infusion of secretin during selective angiography resulted in greatly enhanced visualization of the tumor which allowed it to be localized to the duodenal stump. Several months postoperatively, the secretin test result had become negative, which presumably suggested that the tumor had been excised completely. Our experience has revealed that intravenous secretin might improve the diagnostic usefulness of selective angiography.

Early markers of pancreas transplant rejection.

To compare the predictive value of urinary amylase (UA), urinary insulin (UI), and urinary prostaglandin (PGE2), whole pancreas isografts or allografts from (ACI rat donors, RT1a) with bladder drainage of exocrine secretions were performed in Lewis rats (RT1(1)) with streptozotocin-induced diabetes. UA, UI, PGE2 and plasma glucose levels were measured daily. Euglycemia was restored on Postoperative Day 1 in all the recipients of isografts (N = 6) and was maintained for over a year. UI concentrations and PGE2 outputs were stable, with low levels ranging between 0.3 +/- 0.2 to 7 +/- 2 ng/ml and 56 +/- 15 to 164 +/- 48 ng/24 hr, respectively, while UA levels were significantly elevated compared to normal controls (> 1,000 U/ml vs 29 +/- 16 U/ml). In the allograft group (N = 12), rejection occurred on Days 7 through 9, with a mean graft survival time of 8.1 +/- 0.1 days. UA dropped from a post-transplant peak of 2,422 +/- 353 U/ml on Postoperative Day 4 to below 1,380 +/- 256 U/ml 3 days before rejection (Day -3). UI increased to 83 +/- 16 ng/ml (P < 0.05) on Day -6 and reached a post-transplant peak of 140 +/- 24 ng/ml on Day -5, while PGE2 output rose from a pretransplant level of 18 +/- 2 to 92 +/- 25 ng/24 hr on Postoperative Day 1, followed by a significant elevation on Day 4 (-4).(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose-insulin kinetics of the extravascular bioartificial pancreas. A study using microencapsulated rat islets.

The success of the extravascular bioartificial pancreas (BAP) is contingent on the rapid transfer of the glycemic signal across both an extravascular compartment and a semipermeable membrane and of insulin from the BAP to the recipient. To examine the possibility of microencapsulated islets such as the BAP to achieve satisfactory in vivo glucose-insulin kinetics, islets were isolated from Lewis rats, encapsulated in poly-L-lysine-alginate membranes, and isogenically transplanted into the peritoneal cavity of 14 streptozotocin induced diabetic rats. Fasting blood glucose (BG) was measured and intravenous glucose tolerance was tested at 8-10 weeks and compared with three control groups: 1) normal Lewis rats (n = 6); 2) untreated diabetic rats (n = 5); and 3) diabetic rats that received intraperitoneal implants of empty capsules (n = 4). Ten animals that received microencapsulated islets (5,271 +/- 431) promptly became normoglycemic, with a mean BG of 128 +/- 17 ng/dl 3 days after transplantation and maintained this level > 100 days. Intravenous glucose tolerance K-value for the group was 3.84 +/- 0.32 compared with 3.96 +/- 0.39 (p = 0.83) for the normal control group, and 0.60 +/- 0.12 (p < 0.01) and 0.40 +/- 0.15 (p < 0.01) for the diabetic control groups with and without empty capsules. The authors conclude from these results that, given sufficient beta-cell mass, a BAP without any vascular access can respond appropriately to an increase in blood glucose concentration, without overshoot hypoglycemia and within a lag lapse compatible with normal physiologic insulin delivery.(ABSTRACT TRUNCATED AT 250 WORDS)

Encapsulated islet cell therapy for the treatment of diabetes: intraperitoneal injection of islets.

Since conventional insulin therapy has failed to achieve tight glucose control, an alternative treatment is urgently needed to treat diabetes. The findings of the Diabetes Control and Complication Trial1 conclusively establish that improved glycemic control delays the onset and slows the progression of neuropathy, nephropathy, and retinopathy in insulin-dependent diabetic patients. The disease leads inexorably to one or more of the secondary complications, including renal failure, blindness, coronary and peripheral vascular occlusive disease. The challenge physicians face is to intervene before these secondary complications take their toll. Transplantation of encapsulated human islets to reverse diabetes by a minimally invasive or minor surgical procedure, without the risk of high-dose or life-long immunosuppression will be described here. In the near future, the shortage of donor human tissue will be overcome either by the use of encapsulated xenograft (porcine) islets or proliferated human islets. Thus, encapsulated islet therapy may become possible for the millions of diabetic patients who may benefit from islet cell transplantation.

Pancreas and islet-cell transplantation. Potential cure for diabetes.

In the past two decades, results of whole-organ pancreas transplantation have improved considerably. Patient survival rates of 95% to 100% and graft success rates of more than 70% at 1 year have been reported. After successful transplantation, exogenous insulin can be discontinued and the patient's quality of life improved. In the field of islet-cell transplantation, remarkable progress has been made in isolation techniques. However, the problem of rejection without serious sequelae remains. Immunoisolation of islet cells in microcapsules offers an exciting solution, but research is still in preliminary stages. Early results in large animal models are extremely encouraging.