X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

FMR1 low sub-genotype does not rescue BRCA1/2-mutated human embryos and does not explain primary ovarian insufficiency among BRCA1/2-carriers.

STUDY QUESTION: Can we confirm in our population whether FMRI low sub-genotypes are associated with BRCA1/2 mutations, as recently proposed? SUMMARY ANSWER: Our results indicate that the distribution of the FMR1 sub-genotypes in female BRCA1/2-mutation carriers is significantly different from what has been reported previously and resembles that of the control population. FMRI low sub-genotypes are not associated with BRCA1/2 mutations and this association is also absent among male mutation carriers. WHAT IS KNOWN ALREADY: Recently, BRCA1 mutations were reported to be associated with primary ovarian insufficiency (POI) in female carriers. In animal models, BRCA2-deficiency also results in impaired oogenesis. A recent study has reported that the POI in BRCA1/2-mutation carriers is most likely due to low FMR1 sub-genotype (CGG n < 26) and the authors also suggested that low sub-genotypes of the FMR1 gene might be important to rescue the BRCA1/2 embryos, which would otherwise be embryonically-lethal. STUDY DESIGN, SIZE, DURATION: This retrospective study was performed in October and November of 2012, using genetic material of 464 patients who underwent genetic screening in our centre in the past. PARTICIPANTS/MATERIALS, SETTING, METHODS: We tested the FMR1 sub-genotypes in 60 female and 29 males with either BRCA1 or BRCA2 mutations and 375 controls by PCR amplification and size fragment analysis. MAIN RESULTS: We did not find any evidence for an association of FMR1 low sub-genotypes and BRCA1/2 mutations. LIMITATIONS, REASONS FOR CAUTION: This association study assumes that the female BRCA1/2 population tested has POI. WIDER IMPLICATIONS OF THE FINDINGS: Low FMR1 sub-genotypes are not responsible for the presumed rescue of embryos with BRCA1/2 mutations. Furthermore, the molecular mechanism of the POI in BRCA1/2-female carriers is not likely to be associated with low FMR1 sub-genotype. STUDY FUNDING/COMPETING INTEREST(S): The Department of Clinical Genetics of the Maastricht University Medical Centre supported the study. The authors do not have any competing interests to declare.

Somatic mosaicism in a mother of two children with Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder characterized by intellectual disability, unusual face and breathing abnormalities and can be caused by haploinsufficiency of TCF4. The majority of cases are sporadic. Somatic mosaicism was reported infrequently. We report on a proband with typical manifestations of PTHS and his younger brother with a less striking phenotype. In both, a heterozygous frameshift mutation (c.1901_1909delinsA, p.Ala634AspfsX67) was found in exon 19 of TCF4. The same mutation was found at low levels in DNA extracted from the mother's blood, urine and saliva. This report of familial recurrence with somatic mosaicism in a healthy mother has important consequences for genetic counseling. We suggest careful studies in parents of other patients with PTHS to determine the frequency of germline and somatic mosaicism for TCF4 mutations.

Ovarian cancer susceptibility alleles and risk of ovarian cancer in BRCA1 and BRCA2 mutation carriers.

Germline mutations in BRCA1 and BRCA2 are associated with increased risks of breast and ovarian cancer. A genome-wide association study (GWAS) identified six alleles associated with risk of ovarian cancer for women in the general population. We evaluated four of these loci as potential modifiers of ovarian cancer risk for BRCA1 and BRCA2 mutation carriers. Four single-nucleotide polymorphisms (SNPs), rs10088218 (at 8q24), rs2665390 (at 3q25), rs717852 (at 2q31), and rs9303542 (at 17q21), were genotyped in 12,599 BRCA1 and 7,132 BRCA2 carriers, including 2,678 ovarian cancer cases. Associations were evaluated within a retrospective cohort approach. All four loci were associated with ovarian cancer risk in BRCA2 carriers; rs10088218 per-allele hazard ratio (HR) = 0.81 (95% CI: 0.67-0.98) P-trend = 0.033, rs2665390 HR = 1.48 (95% CI: 1.21-1.83) P-trend = 1.8 × 10(-4), rs717852 HR = 1.25 (95% CI: 1.10-1.42) P-trend = 6.6 × 10(-4), rs9303542 HR = 1.16 (95% CI: 1.02-1.33) P-trend = 0.026. Two loci were associated with ovarian cancer risk in BRCA1 carriers; rs10088218 per-allele HR = 0.89 (95% CI: 0.81-0.99) P-trend = 0.029, rs2665390 HR = 1.25 (95% CI: 1.10-1.42) P-trend = 6.1 × 10(-4). The HR estimates for the remaining loci were consistent with odds ratio estimates for the general population. The identification of multiple loci modifying ovarian cancer risk may be useful for counseling women with BRCA1 and BRCA2 mutations regarding their risk of ovarian cancer.

Interferon-alpha as maintenance therapy in patients with multiple myeloma.

BACKGROUND: The effect of interferon-alpha 2b (IFN-alpha-2b) on progression-free and overall survival as well as quality of life (QoL) was studied in mainly elderly patients with multiple myeloma (MM), who reached a plateau phase after melphalan/prednisone induction. PATIENTS AND METHODS: In an open phase III trial, 262 patients, median age 69 years (range 34-91), received at least 10 monthly courses of melphalan/prednisone followed by response evaluation. Plateau phase was reached by 128 patients. Next, 90 patients were randomized between IFN-alpha-2b and no maintenance therapy. Reasons for non-randomization were: refusal (18), concomitant disease (nine), protocol violation (six), WHO performance status >2 (four) and allogeneic transplantation (one) RESULTS: At a median follow-up from diagnosis of 97 months (0-140) for those patients alive, IFN-alpha-2b therapy was associated with improved progression-free survival (median 13.5 versus 8.4 months from randomization), although this did not translate in a better overall survival (41 versus 38.4 months). One-third of patients discontinued IFN-alpha due to toxicity. No differences were observed between patient groups in QoL. CONCLUSIONS: IFN maintenance therapy in MM prolongs progression-free survival and, provided that the burden of toxicity is not too high, does not adversely affect QoL.

Rett syndrome in females with CTS hot spot deletions: a disorder profile.

From a series of 107 females with Rett syndrome (RTT), we describe the long-term history of ten females with a deletion in the C-terminus of the MECP2 gene. We observed that their disorder profile is clinically recognizable with time and different from other atypical and milder RTT phenotypes. In females with hot spot deletions in the C-terminus, dystonia is present from childhood and results in a serious spine deformation in spite of preventive measures. Their adaptive behavior is surprisingly better preserved and in contrast with the typical decline in motor functioning. The delineation of disorder profiles by long-term clinical observation can teach us about genotype/phenotype relationships and eventually about the effect of epigenetic phenomena on the final phenotype.

Alveolar soft-part sarcoma responding to interferon alpha-2b.

A 23-year-old woman with an alveolar soft-part sarcoma of her calf with pulmonary metastases unresponsive to chemotherapy is described. Interferon (IFN) alpha-2b induced an impressive tumour response still ongoing after IFN treatment had to be stopped because of a psychosis. An explanation of this effect is still speculative.

[A gastric signet ring cell carcinoma as the first expression of a breast carcinoma]. / Een zegelringcelcarcinoom van de maag als eerste uiting van een mammacarcinoom.

A 73-year-old woman presented with dull pain in the epigastric region, a rapid feeling of fullness upon eating and a weight loss of 10 kg in 6 months. Further examination showed linitis plastica due to a signet ring cell carcinoma in the stomach, multiple bone metastases, and an occult, small breast tumour. Immunohistochemical comparison of the tumours strongly suggested that all cases involved a metastasised breast carcinoma. At check-up after one year of tamoxifen treatment, the complaints had disappeared and the activity of the tumour marker had dropped. Gastric metastases from breast carcinoma are rare. Nevertheless, this possibility should be kept in mind in women presenting with malignancies of the stomach and mastopathy. Hormonal treatment and chemotherapy may result in reasonable palliation.

[Non-traumatic myositis ossificans circumscripta in the teres major muscle]. / Niet-traumatisch ontstane myositis ossificans circumscripta in de M. teres major.

A 49-year-old male presented with a painful progressive swelling in his right axillar region, without further complaints, which had been present for 2 weeks. On radiological examination a peripheral circumferential zone of mineralisation was seen in the right teres major muscle. An incision biopsy specimen showed a lesion of fibroblastic tissue in which areas of osteoid and fragmented lamellar bone tissue, without signs of malignancy. The diagnosis was myositis ossificans circumscripta. This is a rare benign ossifying lesion in skeletal muscles, mostly caused by a trauma and with an average age of occurrence between 20 and 30 years old. It must be differentiated from extra-skeletal osteosarcoma. The pathogenesis is unknown. Because it is a benign and self-limiting disorder, surgical excision is only necessary in case of mechanical hindrance. The patient's swelling partially regressed and he had no further complaints.

D-TRP-6-LHRH (Triptorelin) is not effective in ovarian carcinoma: an EORTC Gynaecological Cancer Co-operative Group Study.

Between March and September 1988, 74 patients with progressive ovarian cancer after prior platinum-based therapy were treated with the luteinizing hormone-releasing hormone (LHRH) agonist Triptorelin (Decapeptyl degrees). Treatment consisted of i.m. injection of 3.75 mg of microencapsulated Triptorelin on days 1, 8 and 28 followed by 4-weekly injections until tumor progression. No objective responses were observed. Eleven out of 68 evaluable patients (16%) had stable disease. The median progression-free survival was 5 months in patients with disease stabilization and 2 months for all evaluable patients. The median survival for patients with disease stabilization was 17 months, whereas for all patients it was 4 months. The treatment was well tolerated; the only reported adverse events were incidental hot flushes. This study showed that the LHRH agonist Triptorelin has only modest efficacy in patients pretreated with platinum-containing chemotherapy.

Standard chemotherapy with or without high-dose chemotherapy for aggressive non-Hodgkin's lymphoma: randomized phase III EORTC study.

BACKGROUND: The long-term outcome for patients with aggressive non-Hodgkin's lymphoma (NHL) is poor. Consequently, the European Organization for Research and Treatment of Cancer Lymphoma Group designed a prospective randomized trial to investigate whether high-dose chemotherapy plus autologous bone marrow transplantation (ABMT) after standard combination chemotherapy improves long-term survival. METHODS: Patients aged 15-65 years with aggressive NHL received three cycles of CHVmP/BV polychemotherapy (i.e., a combination of cyclophosphamide, doxorubicin, teniposide, and prednisone, with bleomycin and vincristine added at mid-cycle). After these three cycles, patients with a complete or partial remission and at that time no lymphoma involvement in the bone marrow were randomly assigned to the ABMT arm (a further three cycles of CHVmP/BV followed by BEAC [i.e., a combination of carmustine, etoposide, cytarabine, and cyclophosphamide] chemotherapy and ABMT) or to the control arm (five more cycles of CHVmP/BV). All statistical tests are two-sided. RESULTS: From December 1990 through October 1998, 311 patients (median age = 44 years) were registered and received the first three cycles of CHVmP/BV, and 194 patients were randomly assigned to the treatment arms. Approximately 70% (140 patients) of these patients were of low or low-intermediate International Prognostic Index (IPI) risk. After a median follow-up of 53 months, an intention-to-treat analysis showed a time to disease progression and overall survival at 5 years of 61% (95% confidence interval [CI] = 51% to 72%) and 68% (95% CI = 57% to 79%), respectively, for the ABMT arm and 56% (95% CI = 45% to 67%) and 77% (95% CI = 67% to 86%), respectively, for the control arm. Differences between arms were not statistically significant. A subset analysis on IPI risk groups, although too small for reliable statistical analysis, yielded similar results. CONCLUSIONS: Standard combination therapies remain the best choice for most patients with aggressive NHL. We recommend that patients with IPI low or low-intermediate risk not be subjected to high-dose chemotherapy and ABMT as a first-line therapy.

Nadroparin versus dalteparin anticoagulation in high-volume, continuous venovenous hemofiltration: a double-blind, randomized, crossover study.

OBJECTIVES: To compare filter survival times during high-volume, continuous venovenous hemofiltration in patients with normal coagulation variables, using anti-factor Xa bioequivalent doses of nadroparin and dalteparin. To evaluate which other factors influence filter survival time. DESIGN: Randomized, prospective, double-blind, crossover study. SETTING: An 18-bed intensive care unit in a 530-bed teaching hospital. PATIENTS: Thirty-two critically ill patients with renal failure, treated with high-volume, continuous venovenous hemofiltration. INTERVENTIONS: High-volume, postdilutional continuous venovenous hemofiltration, with a standard blood flow rate of 200 mL/min and an ultrafiltrate volume of 100 L in 24 hrs, was performed with a highly permeable, large-surface cellulose triacetate membrane. Anticoagulation with anti-Xa bioequivalent doses of nadroparin and dalteparin was administered in the extracorporeal line before the filter. Blood was sampled for determination of coagulation variables before hemofiltration, 0.5, 2, 4, 6, and 12 hrs after starting the treatment, and at the end of the hemofiltration run. MEASUREMENTS AND MAIN RESULTS: Anti-Xa peak activity, time of anti-Xa peak activity, area under the curve for 0-3 hrs and filter survival time were not significantly different using nadroparin or dalteparin. When analyzing the patients according to the length of filter survival time, no relationship among anti-Xa peak activity, area under the curve for 0-3 hrs, and filter survival time was found. However, there was a strong trend toward a negative correlation between baseline platelet count and filter survival time (r2 = .11; p = .07). Mean blood urea nitrogen decreased from 81.0+/-31.9 to 41.1+/-21.2 mg/dL (p<.01) and mean creatinine decreased from 3.4+/-1.8 to 1.9+/-1.2 mg/dL (p<.01). There were no clinically important bleeding complications. CONCLUSIONS: Nadroparin and dalteparin are bioequivalent with respect to their anti-Xa activities. Using either drug, we did not find a difference in filter survival time during high-volume, continuous venovenous hemofiltration. No relationship between anti-Xa activity and filter survival time could be found. However, there is a strong trend toward a negative correlation between baseline platelet count and filter survival time. This suggests that during high-volume, continuous venovenous hemofiltration, patients with a higher baseline platelet count might need a different anticoagulation regimen to obtain longer filter survival times.

Urokinase-type-plasminogen-activator (uPA) production by human breast (myo) fibroblasts in vitro: influence of transforming growth factor-beta(1) (TGF beta(1)) compared with factor(s) released by human epithelial-carcinoma cells.

The urokinase-type plasminogen activator (uPA) may be considered as a key enzyme in the processes of cancer cell invasion and metastasis. Evidence has been presented that, in breast stroma, uPA is expressed predominantly by myofibroblasts located at the invasive areas of the tumor. To examine whether transforming growth factor type-1 (TGF beta(1)) produced by breast-carcinoma cells is a candidate responsible for the induction of uPA-producing myofibroblasts, we studied in vitro the capacity of normal and tumor-derived human breast fibroblasts to express uPA and the myofibroblast marker alpha-smooth-muscle actin in response to TGF beta(1). Next, we compared these influences with those elicited by factor(s) released by epithelial-cancer cells. In all 8 fibroblast strains tested, TGF beta(1) induced a similar concentration-dependent increase in the fraction of alpha-smooth-muscle-actin-positive fibroblasts. While uPA expression was decreased by TGF beta(1) in most of the fibroblast strains, 2 strains were relatively insensitive to TGF beta(1) in this respect. Although factors present in media conditioned by non-uPA-producing epithelial-tumor cells could trigger fibroblasts to become potent producers of uPA, the TGF beta(1) content of the conditioned media were linked to the differential effects of externally added TGF beta(1) with respect to uPA expression. The data demonstrate that, although fibroblasts may utilize TGF beta(1) secreted by tumor cells to differentiate into myofibroblasts, tumor cells secrete factor(s) other than TGF beta(1) ultimately responsible for the generation of powerful uPA-producing fibroblasts.

Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant.

BACKGROUND: Microparticles from platelets and other cells have been extensively studied and characterized in vitro. Although the level of platelet-derived microparticles is elevated in a variety of diseases, including cardiac surgery, virtually nothing is known about their functions in vivo. The aim of the present study was to investigate the procoagulant properties of microparticles generated in vivo. METHODS AND RESULTS: In 6 patients at the end of cardiopulmonary bypass, 14.8 x 10(9)/L (median; range, 9.7 to 27.4 x 10(9)/L) platelet-derived microparticles were present in pericardial blood, whereas blood obtained from the systemic circulation contained 1.6 x 10(9)/L (median; range, 0.4 to 8.9 x 10(9)/L) of such microparticles, as determined by flow cytometry. Microparticles stained positively for phosphatidylserine as determined with labeled annexin V. In contrast to systemic blood, pericardial blood contained not only microparticles of platelet origin but also microparticles that originated from erythrocytes, monocytes, or granulocytes, and other hitherto unknown cellular sources. Plasma prepared from pericardial blood and to a lesser extent plasma from systemic blood obtained at the same time, stimulated formation of thrombin in vitro. This activity of pericardial plasma was lost after removal of its microparticles by high-speed centrifugation, whereas the corresponding microparticle pellet was strongly procoagulant. The generation of thrombin in vitro involved a tissue factor/factor VII-dependent and factor XII-independent pathway. CONCLUSIONS: This study is the first to demonstrate that microparticles generated in vivo can stimulate coagulation.

HIV-related thrombotic thrombocytopenic purpura: report of 2 cases and a review of the literature.

Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterised by the clinical pentad of microangiopathic haemolytic anaemia (MAHA), thrombocytopenia, renal failure, fluctuating neurologic signs, and fever. The aetiology of TTP is unknown, but associations with various underlying diseases, infections and drugs have been identified. One of these associations is with HIV infection. We describe the clinical picture, the laboratory results and the response to plasma therapy of two cases of HIV-associated TTP. In both patients, a longitudinal semiquantitative assessment of the numbers of schistocytes in blood was made, which correlated well with the more traditional parameters of disease activity. Since 1987, at least 49 patients with HIV-associated TTP have been reported. A case-analysis of the 38 patients who were described in sufficient detail and a review of the literature in the setting of HIV infection is presented. The most important conclusions from these combined data are: (1) TTP usually seems to occur in patients with a CD4+ lymphocyte count < 250 x 10(6).l(-1); (2) more than 50% of the patients present with TTP soon after or during an infectious or malignant disease; (3) plasma exchange is the therapy of choice, still resulting in mortality of 22%; (4) higher initial platelet count and creatinine level are correlated with an adverse outcome.

Etoposide in malignant pleural mesothelioma: two phase II trials of the EORTC Lung Cancer Cooperative Group.

Intravenous and oral etoposide (VP 16-213) were tested in two sequential phase II trials in chemotherapy-naive patients with malignant pleural mesothelioma. In the first trial, etoposide was given intravenously (i.v.) at a dose of 150 mg/m2 on days 1, 3 and 5 every 3 weeks. The second trial investigated a daily oral dose of 100 mg for 21 days followed by a 2-week treatment-free period, and then recycling. In both trials, the treatment was given until disease progression, intolerable toxicity or patient refusal. In the i.v. trial, 49 patients were included, 2 patients were ineligible. The oral trial recruited 45 patients, 4 patients were not eligible. In both trials, the main side-effects were moderate leucopenia, alopecia, nausea and vomiting. Two partial responses (4%) and three partial responses (7%) were reported in the i.v. and oral trials, respectively. The median survival was 29 weeks and 38 weeks in the i.v. and oral trials, respectively. In conclusion, further investigation of etoposide in malignant mesothelioma is not recommended.

Long-term treatment of transfusional iron overload with the oral iron chelator deferiprone (L1): a Dutch multicenter trial.

We performed an open, nonrandomized, multicenter phase-II trial to evaluate the efficacy and toxicity of 1 year of treatment with the oral iron chelator deferiprone in 38 mainly nonthalassemic patients with transfusional iron overload. Initial serum ferritin varied between 996 and 11.644 micrograms/l. Patients were treated with 3-6 g of deferiprone daily. Mean urinary iron excretion (UIE) in 36 evaluable patients was 21.0 mg/24 h and was significantly higher in the patients with thalassemia than in those with myelodysplasia. Negative iron balance was achieved in 20 patients (56%). The median duration of treatment was 10 months; due to side effects and other causes only 20 patients completed 1 year of treatment. Mean serum ferritin levels decreased from 3563 micrograms/l at the start of the trial to 2767 micrograms/l at 6 months (26 patients, p < 0.004) and to 2186 micrograms/l at 12 months (20 patients, p < 0.005). Serum ferritin levels normalized in two patients who were no longer transfusion dependent. Deferiprone was clearly not effective in three patients (two with myelofibrosis, one with myelodysplasia). One patient with myelodysplasia developed agranulocytosis after 12 months of treatment; this was rapidly reversible after stopping deferiprone. Three patients had a mild and transient decrease in white blood cell count. Other side effects leading to withdrawal from the trial consisted mainly of nausea (3 patients), arthralgia (2), and skin rash (1). No clinical signs of zinc deficiency were seen, although zinc excretion was increased in three patients. No changes were seen in liver enzymes, creatinine, antinuclear factor, T-cell subsets, cardiac function, visual acuity, and audiogram. Although our results confirm deferiprone as an effective iron chelator in patients with thalassemia and in some patients with other forms of iron overload, there is still some concern about the safety of this drug, which therefore, at this time, should be used exclusively in well-controlled clinical trials.

Salvage treatment for primary resistant acute myelogenous leukemia consisting of intermediate-dose cytosine arabinoside and interspaced continuous infusions of idarubicin: a phase-II study (no. 06901) of the EORTC Leukemia Cooperative Group.

Twenty-one patients with acute myeloid leukemia (AML) who failed to enter complete remission (CR) after first-line standard-dose remission-induction therapy with 7 days of cytarabine and 3 days of daunorubicin were treated with a salvage regimen containing intermediate-dose cytosine arabinoside (Ara-C) 2 x 500 mg/m2/day during 7 days in combination with continuous infusions of idarubicin 12 mg/m2/day on days 1, 3, and 5. Twenty patients were considered primary resistant, and one patient had a partial remission after two remission-induction courses. Overall, 11 patients (52%, 95% confidence interval: 30-74%) entered CR. Three patients died during hypoplasia and seven patients had resistant disease or a partial remission. The remission rate in this study compares favorably with the results obtained in similar patient categories. The toxicity of this salvage regimen was remarkably mild. No extramedullary toxicity was observed except for hepatic dysfunction in seven patients. The median duration of remission was 8.5 months, and ultimately, all complete remitters have relapsed except the patient who died from infectious complications after allogeneic bone marrow transplantation (BMT). This study shows that new intensive chemotherapy regimens may be effective after failure of primary treatment. Salvage regimens containing intermediate/high-dose Ara-C and/or alternative anthracyclines or anthracenes should be induced in the treatment of young patients with de novo AML.

Differential regulation of breast tumor cell proliferation by stromal fibroblasts of various breast tissue sources.

A stromal fibroblast-mediated paracrine regulation of epithelial tumor cell proliferation and differentiation plays an important role in the development and progression of breast tumors. We have studied the paracrine growth regulation of various phenotypically different breast cancer cell lines using conditioned serum-free media (C-SFM) from primary breast fibroblasts. Fibroblast cultures were established from malignant primary tumors and adjacent normal breast tissue, benign fibroadenomas, cosmetic reduction mammoplasties and breast skin tissues. All fibroblast-conditioned media were shown to stimulate the proliferation of breast cancer cell lines. However, the C-SFM-induced MCF-7 proliferative response was shown to be significantly higher than the proliferative response observed with any of the other cell lines tested. More importantly, the MCF-7 proliferative response obtained with malignant tumor tissue fibroblast C-SFM was shown to be significantly higher than the response to C-SFM from paired (and unpaired) normal adjacent breast tissue fibroblasts. The MCF-7 proliferative response to fibroblast C-SFM from normal tissue (adjacent to the tumor) was further shown to be comparable to the MCF-7 response using benign or reduction mammoplastic tissue fibroblast C-SFM. In addition, we show that IGFs are only partly responsible for the observed proliferative effect of the C-SFMs, while EGF, TGF alpha and basic-FGF are shown not to be involved. We conclude that stromal fibroblasts can differentially regulate breast cancer cell proliferation. Both the fibroblast's tissue source as well as the target tumor cell's phenotype will determine the extent of the proliferative response.