Application of Controlled Shear Stresses on the Erythrocyte Membrane as a New Approach to Promote Molecule Encapsulation.

Human red blood cells (RBCs) have a remarkable capacity to undergo reversible membrane swelling. Resealed erythrocytes have been proposed as carriers and bioreactors to be used in the treatment of various diseases. This work is aimed at developing a setup allowing the encapsulation of test molecules into erythrocytes by inducing reversible pore formation on the RBC membrane through the application of controlled mechanical shear stresses. The designed setup consists of two reservoirs connected by a glass capillary. Each reservoir is connected to a compressor; during the tests, the reservoirs were in turn pressurized to promote erythrocyte flow through the capillary. The setup was filled with a suspension of erythrocytes, phosphate buffer, and FITC-dextran. Dextran was chosen as the diffusive molecule to check membrane pore dimensions. Samples of the suspension were withdrawn at scheduled times while the setup was operating. Flow cytometry and stereo-optical microscopy analyses were used to evaluate the erythrocyte dextran uptake. The setup was shown to be safe, well controlled, and adjustable. The outcomes of the experimental tests showed significant dextran uptake by RBCs up to 8%. Microscopy observations highlighted the formation of echinocytes in the analyzed samples. Erythrocytes from different donors showed different reactions to mechanical stresses. The experimental outcomes proved the possibility to encapsulate test molecules into erythrocytes by applying controlled mechanical shear stresses on the RBC membrane, encouraging further studies.

Biological quality control for extracorporeal photochemotherapy: Assessing mononuclear cell apoptosis levels in ECP bags of chronic GvHD patients.

Extracorporeal photochemotherapy (ECP) is a treatment approved by the FDA for cutaneous T-cell lymphoma, and it is currently used off-label for graft-versus-host disease (GvHD) and other conditions. In agreement with good practices for the therapeutic use of human cells, quality control has to be performed to validate the ECP procedure with the off-line technique. Since no gold-standard biological test is available, we assessed the apoptosis generated in the ECP bag using a flow cytometric analysis. Thirty-one ECP procedures performed on 13 patients with chronic GvHD were studied by monitoring the induction of mononuclear cell (MNC) apoptosis using annexin V/propidium iodide double staining; residual lymphocyte proliferation to standard mitogens was also measured in 17 of the procedures. The kinetics of apoptosis was analyzed at different times in MNCs untreated or treated with 8-methoxy-psoralen plus ultraviolet A; the variation (ΔAPOPTOSIS ) after 24 h revealed the efficacy of the treatment. In 88.6% of the 31 ECP procedures, ΔAPOPTOSIS was >15% (the "alerting" threshold for ΔAPOPTOSIS was set at 15% on the basis of our data); in the remainder (19.4%), the increment in apoptosis was lower. In four procedures, the proliferation assay was useful for assessing the effect of ECP on the apheretic bag. In conclusion, both flow cytometric assays enabled a biologically significant result to be obtained. In our opinion, the apoptosis test-being faster and easier than the proliferation test-could be a reliable way to validate ECP procedures.

PALB2 sequencing in Italian familial breast cancer cases reveals a high-risk mutation recurrent in the province of Bergamo.

PURPOSE: Monoallelic germ-line deleterious mutations of PALB2 (partner and localizer of BRCA2) are associated with breast cancer risk and have been found in several populations, with carrier frequencies of ~1-2%. Initially, these mutations were considered to have moderate penetrance, but accumulating evidence now indicates that they are associated with much higher risk. METHODS: In this study, we sequenced the PALB2 coding regions unlinked to BRCA (breast cancer) genes in 575 probands from Italian breast cancer families recruited in Milan. RESULTS: We found 12 carriers (2.1%) of deleterious mutations, and none of the mutations was found in 784 controls collected in Milan. One of these mutations, the c.1027C>T (p.Gln343X), was found to be recurrent in the province of Bergamo in northern Italy, being detected in 6/113 (5.3%) familial breast cancer cases and 2/477 (0.4%) controls recruited in this area (Fisher's exact test: P < 0.01). CONCLUSIONS: Our data provide confirmatory findings that, in the Italian population also, deleterious mutations of PALB2 are relatively frequent predisposing factors for breast cancer and may be associated with high risk of the disease.

Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study.

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.

The lipid moiety of haemozoin (Malaria Pigment) and P. falciparum parasitised red blood cells bind synthetic and native endothelin-1.

Endothelin1 (ET-1) is a 21-amino acid peptide produced by the vascular endothelium under hypoxia, that acts locally as regulator of vascular tone and inflammation. The role of ET-1 in Plasmodium falciparum malaria is unknown, although tissue hypoxia is frequent as a result of the cytoadherence of parasitized red blood cell (pRBC) to the microvasculature. Here, we show that both synthetic and endothelial-derived ET-1 are removed by parasitized RBC (D10 and W2 strains, chloroquine sensitive, and resistant, resp.) and native haemozoin (HZ, malaria pigment), but not by normal RBC, delipidized HZ, or synthetic beta-haematin (BH). The effect is dose dependent, selective for ET-1, but not for its precursor, big ET-1, and not due to the proteolysis of ET-1. The results indicate that ET-1 binds to the lipids moiety of HZ and membranes of infected RBCs. These findings may help understanding the consequences of parasite sequestration in severe malaria.

Management of poor peripheral blood stem cell mobilization: incidence, predictive factors, alternative strategies and outcome. A retrospective analysis on 2177 patients from three major Italian institutions.

CD34+ peripheral blood hematopoietic stem cells (HSC) are usually collected following mobilization therapy accomplished by using growth factors (GF) such as rHuG-CSF or rHuGM-CSF with or without chemotherapy. A target dose of yielded CD34+ is usually prescribed by the attending physician depending on different protocols, which may include single or double transplantation. HSC collection usually is performed when at least 20 CD34+ HSC/microL are detected by means of flow cytometry. A cumulative dose of at least 2 x 10(6)/Kg/bw CD34+ HSC has been considered as the threshold to allow a prompt and persistent hematopoietic recovery. Unfortunately, this goal is not achieved by the totality of patients undergoing mobilization regimen. In fact, 5-46% of patients who underwent mobilization therapy fail HSC collection due to very low peripheral blood HSC CD34+ count. Patients' characteristics, including age, sex, stage of the underlying disease (complete or partial remission), diagnosis, previously administered radio/chemotherapy regimens, time-lapse from last chemotherapy before mobilization and mobilization schedule (including dose of GF) were considered as possibly predictive of poor or failed mobilization. We performed a retrospective analysis in 2177 patients from three large Italian academic institutions to assess the incidence of poor mobilizers within our patients' series. Therefore, a patient who fails a first mobilization (and when an HLA-compatible related on unrelated donor is not available) could undergo a second attempt either with different mobilization schedule or by using different GF, such as stem cell factor, growth hormone (GH), or more recently newly introduced drugs such as AMD3100, alone or in combination with rHuG- or -rHuGM-CSF. Thus, we investigated the fate of those who failed a first mobilization and subsequently underwent a second attempt or alternative therapeutic approaches.

Peripheral blood stem cell collection in multiple myeloma: a retrospective analysis of 6 years leukapheresis activity in 109 patients treated at the Istituto Nazionale dei Tumori of Milan.

Double autologous stem cell transplantation is the standard treatment in newly diagnosed multiple myeloma (MM) patients younger than 65 years; therefore, optimization of leukapheresis is crucial. We performed a retrospective analysis of 297 leukaphereses comparing semiautomated (V4.7 in 20% of collections) versus automated (V6.0 in 80%) Caridian (COBE) Spectra versions and analyzing the influence of M-protein on the outcome. Both methods gave comparable collection efficiencies (CE%) (53.4% vs. 55.7% in V6.0 and V4.7, respectively) with similar leukapheresis time and processed volume. Harvest volume was higher in V4.7 (P < 0.0001) with similar contamination of red blood cells (RBCs) (P = 0.77) and platelets (P = 0.09) when compared with V6.0. In patients with higher peripheral white blood cells (WBCs), V6.0 with adjusted harvest volume (<700 mL), achieved similar CD34(+) CE% (P = 0.39) and better enrichment of nucleated cells (P < 0.0,002) but higher RBCs (P < 0.0,001) and platelets contamination (P = 0.001), when compared with a larger cycle volume in patients with lower WBCs. In hard to mobilize patients, CD34(+) CE% was significantly more efficient with V4.7 than V6.0 (P < 0.0,001). CD34(+) CE% was unaffected by serologic M-protein, but platelet CE% was higher in the absence of M-protein (P = 0.0,003), without any reduction in peripheral patients platelets. We, therefore, conclude that in the setting of MM patients with a high WBCs count and/or low percentage of peripheral CD34(+) cells, collections with V4.7 or adjusted cycle volume V6.0 gave comparable result in CD34(+) CE%. RBCs and platelets contamination is higher if low cycle volume is chosen. In hard to mobilize patients, V4.7 is advisable.

Genome-wide single nucleotide polymorphism analysis of lung cancer risk detects the KLF6 gene.

A genome-wide association analysis using the Affymetrix 100K SNP array was carried out in a case-control study of lung cancer. Allele frequencies were estimated initially in DNA pools. Significant differences in allele frequency detected in the SNP array analysis were first tested in the same DNA pools by pyrosequencing and then by individual genotyping. DNA pooling analysis identified rs10508266 SNP, located approximately 12.5kb from the 5'-end of the KLF6 gene, as a marker showing significant association with lung cancer risk. Since the SNP was in significant linkage disequilibrium with the KLF6 gene region, we analyzed an Italian population of 338 lung adenocarcinoma cases and 335 controls for the possible role of the reported functional rs3750861 SNP, located 15.6kb from the rs10508266 SNP. The rs3750861 affects expression of KLF6 splicing variants in prostate cancer and we found that its rare allele is associated with reduced lung cancer risk (odds ratio, 0.5; 95% CI, 0.3-0.8). A Norwegian replication series of 265 non small cell lung cancer cases, and 356 controls, however, did not confirm the association. In light of the reported functional involvement of the KLF6 gene in lung cancer and in other cancer types and to the functional nature of the rs3750861 SNP, our results suggest a potential involvement of KLF6 polymorphisms in lung cancer risk, although additional studies in large series are needed to confirm our findings and to elucidate the mechanism by which the KLF6 SNPs influence lung cancer risk.

A supportive care in cancer unit reduces costs and hospitalizations for transfusions in a comprehensive cancer center.

PURPOSE: Among patients with solid or hematologic malignancies undergoing oncologic therapies, blood product transfusions (BPT) are a relevant reason for planned/unplanned hospitalizations, as well as a possible cause of delay in administration of the oncologic therapies. Furthermore, they create additional costs for the healthcare system (HCS). The aim of this study was to compare the costs of performing BPT (erythrocytes and platelets) in medical units/wards to the costs derived from the administration of BPT in a dedicated outpatient supportive care in cancer unit (SCCU). METHODS: Costs were analyzed from June 3, 2009 (when the SCCU started), until December 2013. Four inpatient oncologic units (bone marrow transplantation, radiotherapy, medical oncology I and II) were compared to the SCCU. Data regarding the transfusions performed by the SCCU of the patients who were previously hospitalized for transfusions were extracted, checked, and analyzed through a cross-check on the tax codes. Therefore, patients were considered suitable for the analysis if they had received BPT in the SCCU after a previous hospitalization for transfusion in one of the 4 units/wards. The average daily cost deriving from blood product units and from the hospitalization in each ward (irrespective of pharmaceutical expenses) was compared with the average daily cost deriving from blood product units and from the management of patients in the SCCU. RESULTS: We analyzed 227 patients (112 female) with a mean age of 60 years (range 20-90) with hematologic malignancies in 79% of cases. The number of transfusions performed by the SCCU has grown constantly and consistently over the years, reaching 1,402 transfusions in 2013, thus exceeding the other considered units. The total savings for the HCS was €282.204.71, €151.182.85 in 2013 only. We saved €124.319,26 for each patient transfused at the SCCU. CONCLUSIONS: A dedicated outpatient SCCU, aimed at monitoring and treating cancer therapy-related toxicities and comorbidities and in which it is also possible to perform BPT promptly and effectively, reduces the number of hospitalizations and provides an economical benefit for HCS.

Functional FGFR4 Gly388Arg polymorphism predicts prognosis in lung adenocarcinoma patients.

PURPOSE: Fibroblast growth factor receptor 4 (FGFR4) is a member of a family of transmembrane receptors with ligand-induced tyrosine kinase activity. The Gly388Arg polymorphism in the FGFR4 gene was reported to modulate cancer cell migration in vitro and to be associated with breast, colon, and prostate cancer prognostic parameters. The purpose of this study was to investigate the involvement of the FGFR4 polymorphism in lung tumorigenesis. PATIENTS AND METHODS: A case-control study was performed including 274 patients with histologically confirmed lung adenocarcinoma and 401 healthy control subjects from general population. mRNA expression analysis was carried out in healthy lung of cancer patients. RESULTS: Patients with the Arg/Arg or Gly/Arg genotype compared to those with a Gly/Gly genotype had an earlier age at cancer onset (median age, 60.2 v 63.4 years), higher proportion of poor clinical stage disease (hazard ratio [HR], 2.3; 95% CI, 1.4 to 3.9; P = .002), of nodal involvement (HR, 1.9; 95% CI, 1.1 to 3.2; P = .027), or of short-term survivors (HR, 1.6; 95% CI, 1.1 to 2.3; P = .008). In healthy lungs, FGFR4 did not show allele-specific expression and mRNA levels were not associated with genotype. CONCLUSION: This study suggests that FGFR4 Gly388Arg polymorphism may predict prognosis in lung adenocarcinoma.

Supratentorial primitive neuroectodermal tumors (S-PNET) in children: A prospective experience with adjuvant intensive chemotherapy and hyperfractionated accelerated radiotherapy.

PURPOSE: Supratentorial primitive neuroectodermal tumors (S-PNET) are rare and have a grim prognosis, frequently taking an aggressive course with local relapse and metastatic spread. We report the results of a mono-institutional therapeutic trial. METHODS AND MATERIALS: We enrolled 15 consecutive patients to preradiation chemotherapy (CT) consisting of high-dose methotrexate, high-dose etoposide, high-dose cyclophosphamide, and high-dose carboplatin, craniospinal irradiation (CSI) with hyperfractionated accelerated radiotherapy (HART) plus focal boost, maintenance with vincristine/lomustine or consolidation with high-dose thiotepa followed by autologous stem-cell rescue. RESULTS: Median age was 9 years; 7 were male, 8 female. Site of disease was pineal in 3, elsewhere in 12. Six patients were had no evidence of disease after surgery (NED). Of those with evidence of disease after surgery (ED), 2 had central nervous system spread. Of the 9 ED patients, 2 had complete response (CR) and 2 partial response (PR) after CT, 4 stable disease, and 1 progressive disease. Of the 7 ED patients before radiotherapy, 1 had CR, 4 PR, and 2 minor response, thus obtaining a 44% CR + PR after CT and 71% after HART. Because of rapid progression in 2 of the first 5 patients, high-dose thiotepa was systematically adopted after HART in the subsequent 10 patients. Six of 15 patients relapsed (4 locally, 1 locally with dissemination, 1 with dissemination) a mean of 6 months after starting CT, 2 developed second tumors; 5 of 6 relapsers died at a median of 13 months. Three-year progression-free survival, event-free survival, and overall survival were 54%, 34%, and 61%, respectively. CONCLUSION: Hyperfractionated accelerated RT was the main tool in obtaining responses in S-PNET; introducing the myeloablative phase improved the prognosis (3/10 vs. 3/5 relapses), though the outcome remained unsatisfactory despite the adoption of this intensive treatment.

Lack of terminally differentiated tumor-specific CD8+ T cells at tumor site in spite of antitumor immunity to self-antigens in human metastatic melanoma.

Activation of CTL-mediated antitumor immunity to self-epitopes expressed by neoplastic cells is thought to be prevented, at any stage of tumor progression, by tolerance mechanisms. In contrast, in 74 American Joint Committee on Cancer stages I-IV melanoma patients, we found that development of lymph node metastases is a key event triggering CD8(+) T-cell-mediated immunity to self-epitopes encoded by melanocyte differentiation antigens. This was shown by the increased peripheral precursor frequency to Melan-A/Mart-1, gp100, and tyrosinase epitopes in stage III and IV compared with stage I and II patients, and by accumulation of functional memory T cells directed to Melan-A/Mart-1(26-35) in tumor-invaded lymph nodes. However, in tumor-invaded lymph nodes of most patients, CD8(+) T cells directed to melanocyte differentiation antigens or to tumor-restricted antigens (MAGE-3 and NY-ESO-1 epitopes), showed a CCR7(+) CD45RA(+) CD27(+) CD28(+) perforin(-) "precursor" phenotype. Only in 7 of 23 cases antigen-specific CD8(+) T cells in invaded lymph nodes showed a predominant CCR7(-) CD45RA(-) CD27(+) CD28(-) perforin(+) "preterminally differentiated" phenotype. In the latter subset of patients, by immunohistochemistry in lymph node lesions, we found that CD8(+) T lymphocytes intermingling with the neoplastic tissue expressed a CCR7(-) CD45RO(+)/RA(-) phenotype, whereas CD4(+) lymphocytes did not infiltrate the tumor. Furthermore, perforin and granzyme B were expressed on a higher fraction of the CD8(+) cells surrounding the invading tumor compared with the lymphocytes infiltrating the neoplastic tissue. In addition, no evidence for tumor regression was found in such metastatic lesions, as documented by absence of neoplastic cell necrosis or apoptosis. These data indicate that neoplastic cells in the lymph nodes and/or increased tumor burden in metastatic disease activate CD8(+) T-cell-mediated antitumor immunity to self-epitopes. However, the paucity of terminally differentiated CD8(+) T cells at tumor site suggests that immunotherapy strategies may require not only the boosting of tumor immunity, but also effective means to promote CD8(+) T-cell differentiation in the neoplastic tissue.

Sequential chemotherapy, high-dose thiotepa, circulating progenitor cell rescue, and radiotherapy for childhood high-grade glioma.

Childhood malignant gliomas are rare, but their clinical behavior is almost as aggressive as in adults, with resistance to therapy, rapid progression, and not uncommonly, dissemination. Our study protocol incorporated sequential chemotherapy and high-dose thiotepa in the preradiant phase, followed by focal radiotherapy and maintenance with vincristine and lomustine for a total duration of one year. The induction treatment consisted of two courses of cisplatin (30 mg/m2) plus etoposide (150 mg/m2) x 3 days and of vincristine (1.4 mg/m2) plus cyclophosphamide (1.5 g/m2) plus high-dose methotrexate (8 g/m2), followed by high-dose thiotepa (300 mg/m2 x 3 doses), with harvesting of peripheral blood progenitor cells after the first cisplatin/etoposide course. From August 1996 to March 2003, 21 children, 14 females and 7 males, with a median age of 10 years were enrolled, 18 presenting with residual disease after surgery. Histologies were glioblastoma multiforme in 10, anaplastic astrocytoma in nine, and anaplastic oligodendroglioma in two; sites of origin were supratentorial areas in 17, spine in two, and posterior fossa in two. Of the 21 patients, 12 have died (10 after relapse, with a median time to progression for the whole series of 14 months; one with intratumoral bleeding at 40 months after diagnosis; and one affected by Turcot syndrome for duodenal cancer relapse). Four of 12 relapsed children had tumor dissemination. At a median follow-up of 57 months, overall survival and progression-free survival at four years were 43% and 46%, respectively. Sequential and high-dose chemotherapy can be afforded in front-line therapy of childhood malignant glioma without excessive morbidity and rather encouraging results.

FGFR4 Gly388Arg polymorphism and prognosis of breast and colorectal cancer.

A functional Gly388Arg variation in the FGFR4 gene has been reported to be associated with breast and colorectal cancer prognostic parameters. To further examine the functional role of this genetic polymorphism at the population level, we assessed the presence of the Arg388 allele in 142 breast carcinoma patients, 179 colorectal carcinoma patients and 220 general population controls with respect to an association with cancer prognosis and/or risk. No significant association with cancer risk, survival or any other prognostic parameters was observed in either breast or colorectal cancer. A pooled analysis of the present and published data on nodal status by FGFR4 genotypes revealed no association in either breast cancer [odds ratio (OR), 1.0; 95% confidence interval (CI), 0.7-1.4; 702 subjects] or colorectal cancer (OR, 1.4; 95% CI, 0.6-3.4; 260 cases). Thus, the FGFR4 polymorphism may not be relevant in predicting nodal involvement of breast cancer or colon cancer patients.

Boosting T cell-mediated immunity to tyrosinase by vaccinia virus-transduced, CD34(+)-derived dendritic cell vaccination: a phase I trial in metastatic melanoma.

PURPOSE: Six American Joint Committee on Cancer stage IV melanoma patients were enrolled into a Phase I study of vaccination with autologous CD34(+)-derived dendritic cells transduced with a modified vaccinia Ankara virus encoding human tyrosinase gene (MVA-hTyr). EXPERIMENTAL DESIGN: Patients received a first intravenous injection of 1 x 10(8) MVA-hTyr-transduced dendritic cells, followed by three s.c. injections at a 14-day interval. RESULTS: Treatment was well tolerated, except for low-grade fever (three of six patients), mild erythema at injection site (five of six), and vitiligo (two of six). A partial response, involving shrinkage of an s.c. nodule, later surgically removed, was observed in 1 patient, who then remained disease-free (>850 days). By human lymphocyte antigen tetramer analysis, significant and often long-lasting increases in frequency of T cells directed to tyrosinase(368-376) but not to gp100(209-217) were documented in periphery of 4 of 5 HLA-A*0201+ patients, a few days after vaccine administration. In addition, maturation phenotype of tyrosinase-specific T cell shifted toward the T effector memory/T terminally differentiate stages (CCR7(-)CD45RA(-/+)) in synchrony with the T-cell frequency peaks. By enzyme-linked immunospot in peripheral blood of five HLA-A*0201+ patients, we found that the vaccine could induce interferon gamma-releasing effector cells directed to HLA-A*0201/tyrosinase(368-376) and to vaccinia virus HLA-A*0201/H3L(184-192) epitopes. Moreover, an interferon gamma response after vaccination was elicited even against the HLA-DRB1-1501/tyrosinase(386-406) epitope in one out of two HLA-A* DRB1-01501+ patients. CONCLUSIONS: These results indicate that vaccination with MVA-hTyr-transduced dendritic cells is well tolerated, can possibly produce clinical responses, and activates tyrosinase- and vaccinia virus-specific T cells in vivo. These data suggest a broad utility of the MVA vector for targeting tumor-associated antigens to dendritic cells for tumor immunotherapy.

Germline mutations of the POU6F2 gene in Wilms tumors with loss of heterozygosity on chromosome 7p14.

Wilms tumor (WT) is a kidney malignancy of childhood characterized by highly heterogeneous genetic alterations. We previously reported the molecular and cytogenetic characterization of a WT (Case 30) carrying an interstitial deletion in chromosome 7p14 between markers D7S555 and D7S668. Loss of heterozygosity (LOH) analyses had revealed that this same region was lost in 8 out of 38 examined WTs, suggesting that the identified interval contains a putative tumor suppressor gene. To confirm this hypothesis, in this work, we analyzed an additional 35 WTs, four of which showed LOH in the region of interest. Furthermore, we were able to more accurately define the extension of the deletion in Case 30, mapping it within an interval not exceeding 390 kb, proximally to D7S555. To date, only a single expressed gene, POU6F2 (the POU domain, class 6, transcription factor 2; also known as RPF1), has been recognized in this interval. Sequencing of the gene in the 12 WTs showing LOH and in a corresponding numbers of WT cases without LOH, led to the identification of two germline nucleotide substitutions. The first occurred in the 5'-untranslated region, while the second caused an amino acid change in a glutamine repeat domain. These mutations, whose occurrence was not observed in more than 100 control subjects, were detected in two patients showing the loss of the constitutionally wild-type allele in tumor DNA. Together with the finding of the expression of the POU6F2 mouse homolog in both fetal and adult kidney, our observations suggest that the gene is a tumor suppressor and is involved in hereditary predisposition to WT.