Impact of uPA/PAI-1 and disseminated cytokeratin-positive cells in breast cancer.

BACKGROUND: The protease uPA and its inhibitor PAI-1 play major roles in hemostasis and are also involved in cancer progression. This is mainly caused by their ability to degrade extracellular matrix-facilitating tumor cell migration. This study aimed to investigate the impact of uPA/PAI-1 and disseminated cytokeratin-positive cells (dCK+) on the outcome and the existence of synergistic effects. METHODS: We retrospectively analyzed a cohort of 480 breast cancer cases with known uPA/PAI-1 and dCK+ status. uPA/PAI-1 was tested on fresh tumor samples using a commercial ELISA test. Bone marrow aspirates were investigated immunocytochemically for CK18. RESULTS: DCK+ cells were identified in 23% of cases. uPA positivity was significantly associated with the occurrence of dCK+ cells (P = 0.028). uPA and PAI-1 were significantly associated with outcome in the subgroup of early-stage cases without chemotherapy. DCK+ cells alone were not prognostic. However, we found synergistic effects. In the subgroup of node-negative cases with and without chemotherapy, the prognostic impact of uPA and PAI-1 was enhanced in cases with additional dCK-positivity (triple +). In cases without chemotherapy, triple-positive status was independently prognostic (HR: 9.3 CI: 1.1-75) next to T stage. CONCLUSIONS: uPA and PAI-1 seem to influence the metastatic potential of dCK+ cells, which underlines its important role in tumor progression.

Correction: Patients with Acute Myeloid Leukemia Admitted to Intensive Care Units: Outcome Analysis and Risk Prediction.

[This corrects the article DOI: 10.1371/journal.pone.0160871.].

The hypomorphic TERT A1062T variant is associated with increased treatment-related toxicity in acute myeloid leukemia.

Hypomorphic germline variants in TERT, the gene encoding the reverse transcriptase component of the human telomerase complex, occur with a frequency of 3-5% in acute myeloid leukemia. We analyzed the clinical and prognostic impact of the most common TERT A1062T variant in younger patients with acute myeloid leukemia intensively treated within two prospective multicenter trials. Four hundred and twenty patients (age 17-60 years) were analyzed for the TERT A1062T variant by direct sequencing. Fifteen patients (3.6%) carried the TERT A1062T variant. Patients with the TERT A1062T variant had a trend towards less favorable and more intermediate 2/adverse karyotypes/genotypes according to the European Leukemia Net classification. In univariate and multivariate analysis, patients with the TERT A1062T variant had a significantly inferior overall survival compared to wild-type patients (6-year overall survival 20 vs. 41%, p = 0.005). Patients with the TERT A1062T variant showed a high rate of treatment-related mortality: 5/15 (33%) died during induction therapy or in complete remission as compared to 62/405 (15%) of the wild-type patients. In patients with the TERT variant, 14/15 (93%) suffered from non-hematological/non-infectious grade 3/4 adverse events (mostly hepatic and/or mucosal) as compared to 216/405 (53%) wild-type patients (p = 0.006). In multivariate analysis, the TERT A1062T variant was an independent risk factor predicting for adverse events during induction chemotherapy. In conclusion, the TERT A1062T variant is an independent negative prognostic factor in younger patients with acute myeloid leukemia and seems to predispose those patients to treatment-related toxicity.

Circulating cytokeratin-positive cells and tumor budding in colorectal cancer.

AIM: To investigate whether circulating cytokeratin-positive (CK+) cells in the mesenteric blood of resected colorectal specimens are prognostic and correlate with tumor budding. METHODS: Fifty-six colorectal specimens were collected between 9/2007 and 7/2008. Blood from the mesenteric vein was drawn immediately after receiving the fresh and unfixed specimens in the pathology department. After separation of the mononuclear cells by Ficoll-Hypaque density-gradient centrifugation, cytological smears were immunocytochemically stained for CK18. Tumor budding was evaluated on slides stained for pan-cytokeratin. The identification of ≥ 30 buds/1.3 mm2 was defined as high grade budding. RESULTS: CK+ cells and clusters were identified in 29 (48%) and 14 (25%) of the samples, respectively. Two cells were identified in one of three non-malignant cases. Clusters were found exclusively in malignant cases. The occurrence of CK+ cells or clusters was not associated with any of the evaluated clinicopathological factors, including surgical technique and tumor budding. Moreover, the occurrence of CK+ cells or clusters had no influence on the cancer-specific survival [75 mo (CI: 61; 88) vs 83 mo (CI: 72; 95) and 80 mo (CI: 63; 98) vs 79 mo (CI: 69; 89), respectively]. CONCLUSION: CK+ cells and showed neither prognostic significance nor an association with tumor budding. It is very likely that CK18-staining is not specific enough to identify the relevant cells.

Patients with Acute Myeloid Leukemia Admitted to Intensive Care Units: Outcome Analysis and Risk Prediction.

BACKGROUND: This retrospective, multicenter study aimed to reveal risk predictors for mortality in the intensive care unit (ICU) as well as survival after ICU discharge in patients with acute myeloid leukemia (AML) requiring treatment in the ICU. METHODS AND RESULTS: Multivariate analysis of data for 187 adults with AML treated in the ICU in one institution revealed the following as independent prognostic factors for death in the ICU: arterial oxygen partial pressure below 72 mmHg, active AML and systemic inflammatory response syndrome upon ICU admission, and need for hemodialysis and mechanical ventilation in the ICU. Based on these variables, we developed an ICU mortality score and validated the score in an independent cohort of 264 patients treated in the ICU in three additional tertiary hospitals. Compared with the Simplified Acute Physiology Score (SAPS) II, the Logistic Organ Dysfunction (LOD) score, and the Sequential Organ Failure Assessment (SOFA) score, our score yielded a better prediction of ICU mortality in the receiver operator characteristics (ROC) analysis (AUC = 0.913 vs. AUC = 0.710 [SAPS II], AUC = 0.708 [LOD], and 0.770 [SOFA] in the training cohort; AUC = 0.841 for the developed score vs. AUC = 0.730 [SAPSII], AUC = 0.773 [LOD], and 0.783 [SOFA] in the validation cohort). Factors predicting decreased survival after ICU discharge were as follows: relapse or refractory disease, previous allogeneic stem cell transplantation, time between hospital admission and ICU admission, time spent in ICU, impaired diuresis, Glasgow Coma Scale <8 and hematocrit of ≥25% at ICU admission. Based on these factors, an ICU survival score was created and used for risk stratification into three risk groups. This stratification discriminated distinct survival rates after ICU discharge. CONCLUSIONS: Our data emphasize that although individual risks differ widely depending on the patient and disease status, a substantial portion of critically ill patients with AML benefit from intensive care.

Reliable single cell array CGH for clinical samples.

BACKGROUND: Disseminated cancer cells (DCCs) and circulating tumor cells (CTCs) are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA) technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.

Influence of molecular subgroups on outcome of acute myeloid leukemia with normal karyotype in 141 patients undergoing salvage allogeneic stem cell transplantation in primary induction failure or beyond first relapse.

Based on molecular aberrations, in particular the NPM1 mutation (NPM1(mut)) and the FLT3 internal tandem duplication (Flt3-ITD), prognostic subgroups have been defined among patients with acute myeloid leukemia with normal karyotype. Whereas these subgroups are known to play an important role in outcome in first complete remission, and also in the indication for allogeneic stem cell transplantation, data are limited on their role after transplantation in advanced disease. To evaluate the role of molecular subgroups of acute myeloid leukemia with normal karyotype after allogeneic stem cell transplantation beyond first complete remission, we analyzed the data from 141 consecutive adults (median age: 51.0 years, range 18.4-69.3 years) who had received an allogeneic transplant either in primary induction failure or beyond first complete remission. A sequential regimen of cytoreductive chemotherapy (fludarabine, high-dose AraC, amsacrine) followed by reduced intensity conditioning (FLAMSA-RIC), was uniformly used for conditioning. After a median follow up of three years, overall survival from transplantation was 64 ± 4%, 53 ± 4% and 44 ± 5% at one, two and four years, respectively. Forty patients transplanted in primary induction failure achieved an encouraging 2-year survival of 69%. Among 101 patients transplanted beyond first complete remission, 2-year survival was 81% among patients with the NPM1(mut)/FLT3(wt) genotype in contrast to 43% in other genotypes. Higher numbers of transfused CD34(+) cells (hazard ratio 2.155, 95% confidence interval 0.263-0.964, P=0.039) and favorable genotype (hazard ratio 0.142, 95% confidence interval: 0.19-0.898, P=0.048) were associated with superior overall survival in multivariate analysis. In conclusion, patients with acute myeloid leukemia with normal karyotype can frequently be rescued after primary induction failure by allogeneic transplantation following FLAMSA-RIC. The prognostic role of NPM1(mut)/FLT3-ITD based subgroups was carried through after allogeneic stem cell transplantation beyond first complete remission.

Testing G-CSF responsiveness predicts the individual susceptibility to infection and consecutive treatment in recipients of high-dose chemotherapy.

The individual risk of infection and requirements for medical treatment after high-dose chemotherapy have been unpredictable. In this prospective, multicenter, open-label study we investigated the potential of granulocyte colony-stimulating factor (G-CSF) responsiveness as a predictor. A total of 168 patients with multiple myeloma or lymphoma received a single dose of subcutaneous G-CSF (lenograstim, 263 µg) after high-dose chemotherapy. Highly variable leukocyte peaks were measured and grouped as low (quartile 1; leukocytes 100-10 100/µL), medium (quartile 2; leukocytes > 10 100-18 300/µL), and high (quartiles 3/4; leukocytes > 18 300-44 800/µL). G-CSF responsiveness (low vs medium vs high) was inversely correlated with febrile neutropenia (77% vs 60% vs 48%; P = .0037); the rate of infection, including fever of unknown origin (91% vs 67% vs 54%; P < .0001); days with intravenous antibiotics (9 vs 6 vs 5; P < .0001); and antifungal therapy (P = .042). In multivariate analysis, G-CSF responsiveness remained the only factor significantly associated with infection (P = .016). In addition, G-CSF responsiveness was inversely correlated with grade 3/4 oral mucositis (67% vs 33% vs 23%; P < .0001). G-CSF responsiveness appears as a signature of the myeloid marrow reserve predicting defense against neutropenic infection after intensive chemotherapy. This study is registered at http://www.clinicaltrials.gov as NCT01085058.

Systematic comparison of sporadic and syndromic pancreatic islet cell tumors.

Pancreatic islet cell tumors (ICTs) occur as sporadic neoplasias or as a manifestation of multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau disease (VHL). Molecular classification of ICTs is mandatory for timely diagnosis and surveillance. Systematic comparison of VHL-ICTs and sporadic ICTs has been lacking. Our registry-based approaches used the German NET-Registry with 259 patients with neuroendocrine tumors (NETs), who were primarily diagnosed with NETs, and the German VHL-Registry with 485 molecular genetically confirmed patients who had undergone magnetic resonance imaging or computed tomography of the abdomen. All patients provided blood DNA for testing of the MEN1 and VHL genes for intragenic mutations and large deletions. In the NET-Registry, 9/101 patients (8.9%) with ICTs had germline mutations, 8 in MEN1 and 1 in VHL. In the VHL-Registry, prevalence of NETs was 52/487 (10.6%), and all were ICTs. Interestingly, of those with VHL p.R167W, 47% developed ICTs, compared to 2% of those with p.Y98H. In total, there were 92 truly sporadic, i.e. mutation-negative ICT patients. Comparing these with the 53 VHL-ICT patients, the statistically significant differences were predominance of female gender (P=0.01), multifocal ICTs (P=0.0029), and lower malignancy rate (P<0.001) in VHL-ICTs compared to sporadic cases. VHL was prevalent in <0.5% of NETs, while NETs occur in ∼10% of VHL, virtually exclusively as ICTs, which are rarely the first presentation. Patients with NETs should not be subjected to genetic testing of the VHL gene, unless they have multifocal ICTs, other VHL-associated tumors, and/or a family history for VHL.

Prognostic impact of IDH2 mutations in cytogenetically normal acute myeloid leukemia.

Mutations in the nicotinamide adenine dinucleotide phosphate(+)-dependent isocitrate dehydrogenase gene 2 (IDH2) have recently been found in patients with acute myeloid leukemia (AML) as well as in patients with leukemic transformation of myeloproliferative neoplasms. We analyzed 272 adult patients with cytogenetically normal AML (CN-AML) for the presence of IDH2 mutations in codons R140 and R172. IDH2 mutations of amino acid 140 or 172 could be identified in 12.1% of CN-AML patients, with the majority of mutations (90%) occurring at position R140. The incidence of IDH2 mutations in AML patients with aberrant karyotypes (n = 130) was significantly lower (3.8%, P = .006). IDH2 mutations were mutually exclusive with mutations in IDH1. IDH2 mutation status alone or in combination with IDH1 mutations had no impact on response to therapy, overall survival, and relapse-free survival in patients with CN-AML. In conclusion, IDH2 mutations are frequently found in CN-AML, but in our analysis these mutations did not influence treatment outcome. This study was registered at www.clinicaltrials.gov as #NCT00209833.

Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid leukemia: SNP rs11554137 is an adverse prognostic factor.

PURPOSE: We assessed the prognostic impact of IDH1 R132 mutations and a known single nucleotide polymorphism (SNP) located in the same exon of the IDH1 gene in patients with cytogenetically normal acute myeloid leukemia (CN-AML) in the context of other prognostic markers. PATIENTS AND METHODS: IDH1 exon four was directly sequenced in 275 CN-AML patients from two subsequent AML multicenter treatment trials and 120 healthy volunteers. Moreover, mutations in NPM1, FLT3, CEBPA, and WT1 were analyzed, and mRNA expression of IDH1 was quantified. RESULTS: IDH1 R132 mutations were found in 10.9% of CN-AML patients. IDH1 SNP rs11554137 was found in 12% of CN-AML patients and 11.7% of healthy volunteers. IDH1 R132 mutations had no impact on prognosis. In contrast, IDH1 SNP rs11554137 was an adverse prognostic factor for overall survival in univariate and multivariate analysis. Other significant factors were age, NPM1/FLT3 mutational status, WT1 SNP rs16754, and platelet count. The impact of IDH1 SNP rs11554137 was most pronounced in the NPM1/FLT3 high-risk patients (either NPM1 wild-type or FLT3-internal tandem duplication positive). Patients with IDH1 SNP rs11554137 had a higher expression of IDH1 mRNA than patients with two wild-type alleles. CONCLUSION: IDH1 SNP rs11554137 but not IDH1 R132 mutations are associated with an inferior outcome in CN-AML.

Allogeneic hematopoietic stem cell transplantation (allo SCT) for chronic myeloid leukemia in the imatinib era: evaluation of its impact within a subgroup of the randomized German CML Study IV.

The role of allogeneic stem cell transplantation in chronic myeloid leukemia is being reevaluated. Whereas drug treatment has been shown to be superior in first-line treatment, data on allogeneic hematopoietic stem cell transplantation (allo SCT) as second-line therapy after imatinib failure are scarce. Using an interim safety analysis of the randomized German CML Study IV designed to optimize imatinib therapy by combination, dose escalation, and transplantation, we here report on 84 patients who underwent consecutive transplantation according to predefined criteria (low European Group for Blood and Marrow Transplantation [EBMT] score, imatinib failure, and advanced disease). Three-year survival after transplantation of 56 patients in chronic phase was 91% (median follow-up: 30 months). Transplantation-related mortality was 8%. In a matched pair comparison of patients who received a transplant and those who did not, survival was not different. Three-year survival after transplantation of 28 patients in advanced phase was 59%. Eighty-eight percent of patients who received a transplant achieved complete molecular remissions. We conclude that allo SCT could become the preferred second-line option after imatinib failure for suitable patients with a donor. The study is registered at the National Institutes of Health, http://clinicaltrials.gov: NCT00055874.

Single nucleotide polymorphism in the mutational hotspot of WT1 predicts a favorable outcome in patients with cytogenetically normal acute myeloid leukemia.

PURPOSE: We assessed the prognostic impact of a known single nucleotide polymorphism (SNP) located in the mutational hotspot of WT1 in patients with cytogenetically normal acute myeloid leukemia (CN-AML) in the context of other prognostic markers. PATIENTS AND METHODS: WT1 exons 7 and 9 from 249 CN-AML patients from multicenter treatment trials AML-SHG Hannover 0199 (Clinical Trials Identifier NCT00209833) and 0295, and 50 healthy volunteers were analyzed by direct sequencing. NPM1, FLT3, CEBPA, and MLL were assessed for mutations and WT1 expression was quantified. RESULTS: The minor allele of SNP rs16754 (WT1(AG/GG)) was found in 25.7% of CN-AML patients' blasts and germline DNA and in 36% of healthy volunteers. Patient characteristics, frequencies of mutations, or WT1 expression levels were similarly distributed between patients homozygous for the major allele compared with patients heterozygous or homozygous for the minor allele. SNP rs16754 status was an independent predictor of relapse-free survival (RFS; hazard ratio [HR], 0.49; 95% CI, 0.3 to 0.81; P = .005) and overall survival (OS; HR, 0.44; 95% CI, 0.27 to 0.74; P = .002) in multivariate analysis. The favorable effect of SNP rs16754 was stronger in NPM1/FLT3-ITD (internal tandem duplication of the FLT3 gene) high-risk patients compared with NPM1/FLT3-ITD low-risk patients. Favorable prognosis could not be identified by any other known low-risk marker in half the patients with at least one minor allele (13% of all patients). No difference for complete remission rate, RFS, or OS was found between patients with or without acquired WT1 mutations. CONCLUSION: WT1 SNP rs16754 may be a novel independent favorable-risk marker in CN-AML patients that might improve risk and treatment stratification.

Perioperative activation of disseminated tumor cells in bone marrow of patients with prostate cancer.

PURPOSE: The outcome of prostate cancer is highly unpredictable. To assess the dynamics of systemic disease and to identify patients at high risk for early relapse we followed the fate of disseminated tumor cells in bone marrow for up to 10 years and genetically analyzed such cells isolated at various stages of disease. PATIENTS AND METHODS: Nine hundred bone marrow aspirates from 384 patients were stained using the monoclonal antibody A45-B/B3 directed against cytokeratins 8, 18, and 19. Log-rank statistics and Cox regression analysis were applied to determine the prognostic impact of positive cells detected before surgery (244 patients) and postoperatively (214 patients). Samples from primary tumors (n = 55) and single disseminated tumor cells (n = 100) were analyzed by comparative genomic hybridization. RESULTS: Detection of cytokeratin-positive cells before surgery was the strongest independent risk factor for metastasis within 48 months (P < .001; relative risk [RR], 5.5; 95% CI, 2.4 to 12.9). In contrast, cytokeratin-positive cells detected 6 months to 10 years after radical prostatectomy were consistently present in bone marrow with a prevalence of approximately 20% but had no influence on disease outcome. Characteristic genotypes of cytokeratin-positive cells were selected at manifestation of metastasis. CONCLUSION: Cytokeratin-positive cells in the bone marrow of prostate cancer patients are only prognostically relevant when detected before surgery. Because we could not identify significant genetic differences between pre- and postoperatively isolated tumor cells before manifestation of metastasis, we postulate the existence of perioperative stimuli that activate disseminated tumor cells. Patients with cytokeratin-positive cells in bone marrow before surgery may therefore benefit from adjuvant therapies.

Dose-escalated CHOP plus etoposide (MegaCHOEP) followed by repeated stem cell transplantation for primary treatment of aggressive high-risk non-Hodgkin lymphoma.

Feasibility, safety, and efficacy of a 4-course high-dose chemotherapy (HDT) protocol including autologous stem cell transplantation (SCT) after courses 2, 3, and 4 was investigated in 110 patients, aged 18 to 60 years, with primary diagnosis of aggressive NHL (aNHL), and lactic dehydrogenase (LDH) levels above normal. At dose level 1 (DL1), course 1 consisted of cyclophosphamide 1500 mg/m2, doxorubicin (Adriamycin) 70 mg/m2, vincristine 2 mg, etoposide 450 mg/m2, and prednisone 500 mg. With courses 2 and 3 cyclophosphamide and etoposide were escalated to 4500 mg/m2 and 600 mg/m2, respectively. With course 4 cyclophosphamide and etoposide were given at 6000 mg/m2 and 1000 mg/m2, respectively. At DL2 etoposide was further increased to 600, 960, 960, and 1480 mg/m2 with courses 1 to 4, respectively. Therapy as per protocol was completed by 81.8% of patients. Overall survival at 5 years was 67.2%, freedom from treatment failure (FFTF) was 62.1%, and treatment-related mortality was 4.5%. There was a trend to better FFTF at DL2 compared to DL1 (66.9% versus 54.2%). Repetitive HDT with escalated CHOP plus etoposide is feasible and effective treatment of patients with aNHL. DL2 of this therapy is being used in an ongoing phase 3 study.

A pooled analysis of bone marrow micrometastasis in breast cancer.

BACKGROUND: We assessed the prognostic significance of the presence of micrometastasis in the bone marrow at the time of diagnosis of breast cancer by means of a pooled analysis. METHODS: We combined individual patient data from nine studies involving 4703 patients with stage I, II, or III breast cancer. We evaluated patient outcomes over a 10-year follow-up period (median, 5.2 years), using a multivariable piecewise Cox regression model. RESULTS: Micrometastasis was detected in 30.6 percent of the patients. As compared with women without bone marrow micrometastasis, patients with bone marrow micrometastasis had larger tumors and tumors with a higher histologic grade and more often had lymph-node metastases and hormone receptor-negative tumors (P<0.001 for all variables). The presence of micrometastasis was a significant prognostic factor with respect to poor overall survival and breast-cancer-specific survival (univariate mortality ratios, 2.15 and 2.44, respectively; P<0.001 for both outcomes) and poor disease-free survival and distant-disease-free survival during the 10-year observation period (incidence-rate ratios, 2.13 and 2.33, respectively; P<0.001 for both outcomes). In the multivariable analysis, micrometastasis was an independent predictor of a poor outcome. In the univariate subgroup analysis, breast-cancer-specific survival among patients with micrometastasis was significantly shortened (P<0.001 for all comparisons) among those receiving adjuvant endocrine treatment (mortality ratio, 3.22) or cytotoxic therapy (mortality ratio, 2.32) and among patients who had tumors no larger than 2 cm in diameter without lymph-node metastasis and who did not receive systemic adjuvant therapy (mortality ratio, 3.65). CONCLUSIONS: The presence of micrometastasis in the bone marrow at the time of diagnosis of breast cancer is associated with a poor prognosis.

Evaluation of bone marrow in breast cancer patients: prediction of clinical outcome and response to therapy.

Blood-borne distant metastasis is the leading cause of cancer-related death in breast cancer. The onset of this fundamental process can now be assessed in cancer patients by using ultrasensitive immunocytochemical and molecular assays able to detect even single metastatic cells. To date, clinical studies with large study populations, validated immunoassays, and adequate follow-up time provide evidence for the independent prognostic value of the presence of disseminated tumor cells in the bone marrow. At present, bone marrow evaluation may therefore be considered a diagnostic tool for improved risk assessment with regard to distant relapse and death, especially among potentially curable breast cancer patients. Except for promising pilot studies, no data are available to support the immediate use of bone marrow evaluation for treatment monitoring in order to enable the prediction of response to adjuvant therapy.