The role of endometrial staining for CD138 as a marker of chronic endometritis in predicting live birth.

PURPOSE: Chronic endometritis (CE) is diagnosed via endometrial biopsy and staining for plasma cells. A threshold plasma cell count that identifies CE and predicts pregnancy outcomes has not been established, and the prevalence of plasma cells in the general infertile population is unknown. The purpose of this study was to determine the prevalence of plasma cells in the general infertile population and whether a threshold exists which predicts live birth. METHODS: Endometrial samples were obtained prospectively from 80 women undergoing IVF, embedded in paraffin, and stained for plasma cells using mouse mono-clonal antibody for CD138. Slides were reviewed at 20× magnification and 10 random images captured. Three reviewers graded each image for plasma cells. Participants underwent single, euploid, and frozen blastocyst transfer. RESULTS: Forty-nine percent of samples had ≥1 plasma cell across 10 HPFs, 11% had ≥5 cells across 10 HPFs, and 4% had ≥10 cells across 10 HPFs. There was no difference in prevalence between those who did and did not achieve live birth. Using thresholds of 1, 5, and 10 plasma cells per 10 HPFs, there were no differences in implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates between patients with and without CE. CONCLUSION: Endometrial plasma cells are present in half the general infertile population and do not predict implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates at low levels.

Scaling of nanofiltration membranes used for chromium(III) ions recovery from salt solutions.

The effect of membranes' structure on the efficiency of chromium(III) ions recovery from salt solution at low pH and the efficiency of chemical cleaning of these membranes were analyzed in this work. The nanofiltration membranes (DL and HL) used in this study were provided by GE Osmonics. The DL membrane had an irregular, dense support layer structure, while the HL membrane had a loose one. In the case of the DL membrane, it was found that, under tested solutions, the layer of mineral scale formed on the surface gradually decreases the membrane permeability coefficient. In the case of the DL membrane, the scaling was observed only on the surface. On the other hand, a small roughness (118Å) and low density charge (zeta potential at level -4) of the HL membrane causes an uneven growth in deposits and, consequently, irregular nature of the surface structure which hinders the removal of accumulated sediment from the tested membranes' surface. Additionally, the loose structure of the support layer of HL membrane contributes to its internal scaling. Consequently, the permanently loose structure of the HL membrane permeability coefficient was observed.

Long-term results of treatment of advanced dermatofibrosarcoma protuberans (DFSP) with imatinib mesylate - The impact of fibrosarcomatous transformation.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is rare, infiltrating dermal neoplasm, characterized by indolent growth and low probability of metastases. The first effective systemic therapy in DFSP introduced into clinical practice was imatinib, demonstrating high activity in advanced cases. The aim of the study was to perform an analysis of patients with advanced DFSP treated with imatinib, with or without surgery, in routine clinical practice with long-term follow-up. PATIENTS AND METHODS: We analyzed the data of 31 Caucasian patients (14 male, 17 female; median age 56 years) with locally advanced/initially inoperable and/or metastatic DFSP who started therapy with imatinib at initial dose 800 mg daily between 12/2004 and 07/2014. All diagnoses were confirmed cytogenetically for the presence of specific COL1A1-PDGFB fusion. Median follow-up time was 5.3 years. RESULTS: Metastases were present in 15 cases (8 - lungs, 5 - soft tissue, 2 - lymph nodes). Fibrosarcomatous transformation (FS-DFSP) was confirmed in 16 patients (52%). 5-year progression-free survival (PFS) rate was 58% (median 6.8 years), 5-year overall survival (OS) rate was 64% (median time for OS was not reached). The shorter PFS and OS correlated with FS-DFSP and presence of metastatic disease. 5-year PFS rate was 93% for classic DFSP and 33% for FS-DFSP. The best overall responses were: 21 partial responses (68%, including 8 FS-DFSP, but the responses were shorter than for classic DFSP), 6 stable disease (19%) and 4 progressive diseases (13%). Thirteen patients (47%) underwent resection of residual disease and nine of them remained free of disease, although imatinib was discontinued. Median survival after progression on imatinib was 19 months, and longer survival were observed only in cases were rescue surgery/radiotherapy was possible. CONCLUSIONS: Our results indicate the long-term activity of imatinib in therapy of inoperable and/or metastatic cases of DFSP, including FS-DFSP. Some DFSP patients initially evaluated as unresectable/metastatic or necessitating mutilating surgery turned resectable after imatinib therapy and this rational approach leading to complete remission maybe potentially curative.

Effect of interaction between anionic surfactants and poly(piperazine-amide) nanofiltration membranes used for chromium(III) recovery from saline solution.

The effect of the anionic surfactant on the permeation properties of the nanofiltration (NF) membranes used for chromium(III) recovery from saline solution at low pH have been presented in this paper. The membrane surface layer performance periodically modified by sodium dodecyl sulphate (SDS) solution has been studied with measurements of zeta potential, atomic force microscopy (AFM) and permeability coefficient of tested membranes. It was found that the membrane surface layer modification by SDS caused a substantial reduction in the possibility of separation of loose NF membrane characterized by a high density of positively charged groups activating under the effect of the low pH of the saline solutions (HL membrane). On the other hand, in the case of dense NF membranes characterized by a strong negatively charged surface (DL membrane) constituting used the SDS improves the separation of chloride and chromium(III) ions. In this case, the surfactant solution also provides a high membrane permeability coefficient behavior over a long period of use. DL membrane modification by SDS allowed both to retain the stable membrane working for a long period and to limit the frequency of the chemical cleaning of this membrane.

A four-year pathology review of the near total face transplant.

In December of 2008, our institution performed a near total face transplant. The patient was monitored for signs of rejection assessed by paired skin and mucosa biopsies. The results of histological review of 120 biopsies collected during the first 4 years posttransplant are discussed. All biopsies were stained with hematoxylin and eosin, periodic acid-Schiff, immunohistochemical and TUNEL assays and graded using the Banff 2007 classification. Grade III rejection was diagnosed clinically at weeks 45 and 66, posttransplant; week 45 was determined as folliculitis while the erythema episode at week 66 confirmed an acute rejection (AR) that required hospitalization. The mucosa frequently showed interface inflammation without clinical signs of rejection and was not present in skin biopsies. In all, 34 of the 45 mucosal biopsies (75%) showed these interface changes. Clinical symptoms concurred with skin pathology in two grade III rejections. The mucosa showed histologic signs of rejection more frequently, which may indicate: increased mucosal sensitivity to rejection, a different type or subtype of AR that is specific to the mucosa, or a nonspecific process such as a drug effect. With more data and world experience, the diagnosis of face transplant rejection will be better defined and the Banff classification enhanced.

Association of HLA haplotypes with paroxysmal nocturnal hemoglobinuria.

The pathologies of paroxysmal nocturnal hemoglobinuria (PNH) are primarily caused by somatic mutation in the PIG-A gene in hematopoietic stem cells resulting in glycosyl phosphatidylinositol deficiency and accumulation of phosphatidylinositol (PI) in plasma membranes. The mechanism of pathologic clone domination over normal hematopoietic clones in PNH patients is not yet understood. Forty-four PNH patients, including 9 with aplastic anemia traits (AA/PNH), 31 without full aplasia in bone marrow (de novo PNH, or dn/PNH), and 4 with unclassified PNH, and 200 ethnically matched controls were tested for the HLA A, B, C, DRB1, and DQB1 alleles and haplotype associations. The top block association analysis showed the primary association of PNH with 3 haplotype fragments: the class I fragment A*2501-Cw*1203-B*1801 (risk ratio [RR], 6.64; P=.00012), and 2 class II fragments: DRB1*1501-DQB1*0602 (RR, 7.09; P=.0000015) and DRB1*0401-DQB1*0301 (RR, 6.76, P=.0093). The stratified analysis revealed that the A*2501-Cw*1203-B*1801 haplotype associated preferentially with AA/PNH, and its component HLA molecule showed immunodominant antiapoptotic peptides derived from PI-activated phospholipase D; whereas the DRB1*1501-DQB1*0602 haplotype was associated strongly with dn/PNH and presented immunodominant class II-derived autopeptides. We concluded that certain HLA haplotypes were associated with PNH much more strongly than their allelic components. At least 3 HLA haplotype blocks (A*2501-Cw*1203-B*1801, DRB1*1501-DQB1*0602, and DRB1*0401-DQB1*0301) were primarily associated with PNH. Our results supported the hypothesis of the roles in AA/PNH of antiapoptotic and in dn/PNH of autoimmune mechanisms.

Association of the HLA-G gene polymorphism with multiple sclerosis in a Polish population.

Summary In this study, three polymorphic sites in the HLA-G gene: -725C>G>T, -716T>G and 14bp(indel) were genotyped. Significant differences were found between patients and controls in the alleles and genotypes for -725C>G>T and in three-point haplotypes. We observed also a significant difference in the age of disease onset between patients positive and negative for 14bp(ins). The results suggest that single nucleotide polymorphisms in the promoter of the HLA-G gene (mainly -725C>G>T), and 14bp(indel), or some genetic marker in tight linkage disequilibrium with them are associated with multiple sclerosis.

Hematopoietic stem cell engraftment and seeding permits multi-lymphoid chimerism in vascularized bone marrow transplants.

Vascularized bone marrow transplantation (VBMT) across a MHC barrier under a 7-day alphabeta-TCR mAb and CsA protocol facilitated multiple hematolymphoid chimerism via trafficking of the immature (CD90) bone marrow cells (BMC) between donor and recipient compartments. Early engraftment of donor BMC [BN(RT1(n))] into the recipient BM compartment [LEW(RT1(l))] was achieved at 1 week posttransplant and this was associated with active hematopoiesis within allografted bone and correlated with high chimerism in the hematolymphoid organs. Two-way trafficking between donor and recipient BM compartments was confirmed by the presence of recipient MHC class I cells (RT1(l)) within the allografted bone up to 3 weeks posttransplant. At 10 weeks posttransplant, decline of BMC viability in allografted bone corresponded with bone fibrosis and lack of hematopoiesis. In contrast, active hematopoiesis was present in the recipient bone as evidenced by the presence of donor-specific immature (CD90/RT1(n)) cells, which correlated with chimerism maintenance. Clonogenic activity of donor-origin cells (RT1(n)) engrafted into the host BM compartment was confirmed by colony-forming units (CFU) assay. These results confirm that hematolymphoid chimerism is developed early post-VBMT by T-cell lineage and despite allografted bone fibrosis chimerism maintenance is supported by B-cell linage and active hematopoiesis of donor-origin cells in the host BM compartment.

Donor-origin cell engraftment after intraosseous or intravenous bone marrow transplantation in a rat model.

We compared the effects of intraosseous BMT with those of standard i.v. BMT on the efficacy on donor-cell engraftment into the BM and lymphoid organs across an MHC barrier in rats. Twenty-four intraosseous and 24 i.v. BMTs were performed from 48 ACI (RT1(a)) donors to 48 Lewis (RT1(l)) recipients. Each transplant group received either intraosseous or i.v. BMT. Groups I and II served as controls without immunosuppression (n=16); groups III and IV received cyclosporine monotherapy (n=16); and V and VI received alphabeta-TCR monoclonal antibody and cyclosporine A (alphabeta-TCR/CsA) for 7 days (n=16). In each group, four rats received 35 x 10(6) transplanted bone marrow cells (BMCs) and four received 70 x 10(6) cells. All animals survived without GVHD. Mean (+/-s.d.) donor-cell engraftment into BM of recipients after intraosseous BMT was 7.9% (+/-1.3%) in recipients receiving alphabeta-TCR-CsA and 70 x 10(6) BMCs, and 4.2% (+/-1.4%) in recipients after i.v. transplantation. The seeding efficacy of donor cells into lymphoid tissue was greater after intraosseous BMT and alphabeta-TCR-CsA than after standard i.v. transplantation. In our model, intraosseous BMT facilitated donor-cell engraftment under short-term immunodepletive alphabeta-TCR/CsA protocol, which resulted in a temporary state of immune unresponsiveness.

A new method of bone marrow transplantation leads to extension of skin allograft survival.

Tolerance induction through allogeneic bone marrow transplantation is an alternative method to chronic immunosuppression in maintaining long-term allograft survival. In this article, we introduce a new method of bone marrow allotransplantation, which preserves its natural microenvironment and does not require marrow processing or recipient conditioning. A total of 43 skin graft transplantations were performed in nine experimental groups between isogeneic [Lewis to Lewis (LEW, RT1(1))] and allogeneic [Lewis x Brown Norway (LBN --> F1, RT1(1+n)) to Lewis] rats under 35-day protocol of alphabeta T-cell receptor (TCR) monoclonal antibody (mAb) and cyclosporine (CsA) protocol. Monotherapies combined with "crude" bone marrow transplantation resulted in extended survival up to 21 days under CsA and up to 10 days under alphabeta-TCR mAb protocol. The use of combined protocol of alphabeta-TCRmAb/CsA with crude bone marrow transplantation resulted in the extension of skin allograft survival up to 65 days (P < .05). This new simple method of "crude" bone marrow allotransplantation without recipient conditioning is a promising, minimally invasive technique with a potential for direct clinical application.

Different routes of donor-derived hematopoietic stem cell transplantation for donor-specific chimerism induction across MHC barrier.

Transplantation of donor-derived stem cells can improve organ allograft survival in animal models. This study was designed to investigate the effect of different routes of bone marrow cell (BMC) transplantation on donor-specific tolerance induction across MHC barrier under short-term CsA monotherapy and alphabetaTCR/CsA treatment protocols. Forty-eight BMC transplantations were performed between BN(RT1(n)) donors and LEW(RT1(1)) recipients. Intraosseous and intravenous BMC transplantation was studied in six groups of eight animals each receiving 35 x 10(6) (n = 4) and 70 x 10(6) (n = 4) bone marrow cells. Groups I and II (controls) received BMC transplantation but no treatment, groups III and IV CsA monotherapy, and groups V, VI alphabetaTCR/CsA protocol for 7 days. Flow cytometry monitored immunodepletion and donor-specific chimerism for MHC class I RT1(n)/CD4, RT1(n)/CD8 and RT1(n)/CD45RA antigens. All animals survived without graft-versus-host disease. At day 63 under CsA monotherapy a low level of chimerism for RT1(n)/CD4 was induced after intraosseous (1.9%) and intravenous (0.8%) transplantation of (70 x 10(6)) BMC. Under alphabetaTCR/CsA protocol chimerism for RT1(n)/CD4 revealed 6.5% and 0.9% in intraosseous and intravenous (70 x 10(6)) BMC transplantation, respectively. The total number of chimerism in intraosseous and intravenous (70 x 10(6)) BMC transplantation groups was 9.9% and 3.4%, respectively. Following intraosseous BMC transplantation under alphabetaTCR/CsA protocol chimerism was 50% higher in a group receiving 70 x 10(6) (9.9%) vs 35 x 10(6) (4.9%) BMC. Intraosseous transplantation of donor BMC under alphabetaTCR/CsA protocol was 75% more efficient in induction of donor-specific chimerism compared to intravenous transplantation.

Facial tissue allograft transplantation.

A hemifacial allograft transplant model was used to investigate the rationale for development of functional tolerance across an MHC barrier. Thirty hemiface transplantations were performed in five groups of six Lewis (RT1(1)) rat recipients each. Isografts were performed in group 1. Transplants were obtained from semiallogenic LBN(RT1(1+n)) in group 2 and from fully allogenic ACI(RT1(a)) in group 3 donors, which served as allograft rejection controls. Group 4 grafts using LBN donors and group 5 using ACI donors in addition received CsA monotherapy (16 mg/kg/d for 1 week) and maintained at 2 mg/kg/d. Signs of graft rejection were sought daily. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 8 days posttransplant. Eighty-three percent of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days, respectively. Flow cytometry at day 63 in LBN recipients showed the presence of donor-specific chimerism for MHC class I RT1(n) antigens, namely 3.39% CD4/RT1(n); 1.01% CD8/RT1(n) T-lymphocytes; and 3.54% CD45RA/RT1(n) B-lymphocytes. In ACI recipients the chimerism test revealed 10.55% CD4/RT1(a) and 4.59% of CD8/RT1(a) T-lymphocytes. MLR assay at day 160 posttransplant revealed suppressed responses against LBN donor antigens in group 4, but moderate reactivity to ACI donor antigens in group 5. Functional tolerance toward hemifacial allograft transplants induced across MHC barrier using a CsA monotherapy protocol was associated with the presence of donor-specific chimerism in T- and B-cell subpopulations.

B-cell lymphoproliferative syndrome and peripheral blood CD20+ cells expansion after hematopoietic stem cell transplantation: association with fludarabine and anti-thymocyte globulin containing conditioning regimen.

Fifty-eight patients who received hematopoietic stem cell transplants during a 3-year period in our unit were followed for the symptoms of posttransplant B-cell lymphoproliferative syndrome (B-cell PTLD). Three cases showed lymph node enlargement; in 14, there was an excess of B cells in the blood. Histochemical staining of lymph nodes revealed CD20+ cell expansion in two cases, and in one, CD38+ and CD138+ cells. Kappa and lambda staining revealed poly- or oligoclonal expansion, which was characterized by the presence of Ki67+ cells in 10% to 50% of cells. In 14 cases, an excess of CD20+ cells were observed in blood. Clinical analysis revealed that patients with B-cell expansion in blood and/or in lymph nodes frequently showed fever and that some subjects displayed arthralgia, hemolytic anemia, and hepatitis. LMP-1-positive cells were observed in lymph nodes as well as EBV copies, whereas only a proportion of patients with the excessive CD20+ cells in blood were EBV positive. Notably, lymph node enlargement and CD20+ blood excess occurred significantly more frequently among patients receiving a Fludarabine (Flu) and anti-thymocyte globulin (ATG) conditioning regimen than those whose treatment lacked Flu independent of whether they received ATG (0.80 vs 0.44; P =.036).

The role of multidrug resistance protein 1 (MRP1) in transport of fluorescent anions across the human erythrocyte membrane.

We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2',7'-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL). We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane. Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds. Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity. Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters. BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport. Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye. No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.

[Evaluation of the efficacy of cyclosporine A treatment in idiopathic nephrotic syndrome in children]. / Ocena skutecznosci cyklosporyny A w idiopatycznym zespole nerczycowym u dzieci.

This study reports the results of cyclosporine A (CsA) treatment in 106 children with idiopathic nephrotic syndrome. All of them had normal renal function. In 66 CsA was added to prednisone because of primary steroid-resistancy. Remission was obtained in 31 (47%) and improvement in 23 (35%). In 12 patients (18%) CsA was ineffective. Best results were obtained in youngest children (< 2 years of age) and those with minimal change disease (78% of remission). Renal function remained normal in all children in remission, while in 30% of those with improvement GFR decreased. In all children with persistent nephrotic syndrome chronic or end stage renal failure developed. 40 children were treated with CsA because of long-lasting steroid-dependency, the lack of effect of alkylating agents and steroid toxicity. In 16 of them prednisone was discontinued and in the rest given in very low doses. The steroid-toxicity effects disappeared or became less prominent in all of them. Appearance of albuminuria with decrease of the CsA dose and/or relapses of nephrotic syndrome during or after discontinuation of CsA treatment were quite frequent. For this reason the treatment course was prolonged, in some patients up to 55 months. This may result in chronic CsA nephrotoxicity. We performed second renal biopsy during remission in 28 children. The morphological signs of nephrotoxicity (grade I according to Habib and Niaudet) were considered in 5 (18%) of them.

Apoptosis of keratinocytes is associated with infiltration of CD8+ lymphocytes and accumulation of Ki67 antigen.

In 18 allogeneic blood/marrow transplanted (BMT) cases, skin biopsies were taken for acute GVHD diagnosis. In 14 cases clinical and histopathology data were consistent with acute GVHD with toxic lesions in four cases. All skin biopsies were stained for the presence of apoptotic cells (TUNEL technique). Nine patients' skin biopsies were positive for this stain. Apoptotic cells were seen in epidermal cells of the epidermis and in hair follicles. The presence of apoptotic cells was associated with (1) advanced stage (> or = II grade) acute GVHD (P = 0.015) and (2) heavy mononuclear cell infiltration (P = 0.015), predominantly of CD8+ cells (P = 0.025). Ki67 immunoreactivity of epidermal cells was different in the epidermis having and lacking apoptotic cells. Ki67 MoAb staining of epidermal cells revealed that nuclei were enlarged and stained stronger in biopsies with than biopsies without apoptotic cells (P = 0.008). Cyclin A positive cells were in a high proportion and cdc2-positive cells in a low proportion in patients' biopsies with acute GVHD and toxic lesions. In addition cyclin A accumulation was seen in keratinocytes in GVHD biopsies. Therefore, mitosis of skin cells is impaired, likely due to the genotoxic effect of conditioning, which renders the cells susceptible to acute GVHD-associated apoptotic damage.

[Granulomatous lung lesions after occupational exposure to glass fibers]. / Ziarniniakowe zapalenie pluc po ekspozycji zawodowej na wate szklana.

39 years old man with granulomatous lesions in both lungs caused by occupational contact with glass fibers was described. He has been working as an bricklayer-plasterer for 18 years and was in contact with lime, cement, plaster, asbestos, dust of coal and wood and with glass fibers. For the last two years before admission in 1993 he has had frequent bronchial infections. On admission he was in good general condition, his spirometric examination and blood gases were within normal limits. On chest x-ray disseminated lesions were found. Those lesions were of the round shapes on chest CT. Many sputum cultures for tubercle bacilli were negative. ANA and ANCA were not found in the serum. ACE was within normal limits. No precipitins to environmental antigens were found. Cancer metastases were suspected and lung biopsy during videothoracoscopy was done. Many foreign body type granulomas were found throughout the specimen. The character of the lesions was not typical for tuberculosis, sarcoidosis, extrinsic allergic alveolitis, silicosis or asbestosis. There are some reports concerning the possibility of development of such lesions after the exposition to glass fibers. We suspect that case is an example of such pathology. His occupational exposition was stopped in 1993 and he was observed without treatment. During the 5 years of observation (up till 1998) he was in good health with stable chest x-ray picture and results of respiratory system function.

IL-6 production in ovarian carcinoma is associated with histiotype and biological characteristics of the tumour and influences local immunity.

The presence of interleukin (IL)-6 in peritoneal carcinomatous fluid (PCF) and its effect on immune cells composition in PCF in patients with advanced ovarian carcinoma was studied. In 21 out of 30 ovarian carcinoma patients, PCF IL-6 levels were found to exceed those seen in PCFs of patients with gastrointestinal cancer. IL-6 activity was higher in serous/mucinous than in endometrioid and undifferentiated ovarian carcinoma PCF (P = 0.05). Ovarian carcinoma PCF IL-6 activities were correlated with serum C-reactive protein levels (r = 0.65, P = 0.0000, n = 25). Ovarian carcinoma PCF leucocyte profile differed from that in blood with respect to: (i) lower percentage of NK and CD8+ and (ii) higher percentage of B and CD45RO+, CD14+ and HLA-DR+ cells. The proportions of CD45RO+ in blood were correlated with IL-6 levels in PCF. Corresponding to PCF ovarian carcinoma tumours were stained for the presence of Ki-67 antigen and p53. The highest proportions of Ki-67+ cells and cells showing accumulation of p53 were seen in undifferentiated tumours. A low grade of p53 staining was seen in tumours associated with high IL-6 levels in PCF. It was evident that IL-6 production (i) depended on the histiotype of the tumour, (ii) influenced the local immune system in favour of accumulation of B, and T memory cells, and (iii) was higher in patients lacking p53 accumulation.