Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Daratumumab in multiple myeloma: experience of the multiple myeloma GIMEMA Lazio group.

Daratumumab (DARA) is a human IgG-K monoclonal antibody (MoAb) targeting CD38 that is approved alone or in combination with bortezomib and dexamethasone or lenalidomide and dexamethasone for relapsed or refractory MM (RRMM) in patients previously exposed or double refractory to proteasome inhibitors (PI) and immunomodulatory drugs (IMiDs). However, there are limited data on its clinical activity and tolerability in real-world patients. Therefore, in the present study, we aim to determine the efficacy and toxicity profile of daratumumab in a real-life setting. In this study, we report the experience of the multiple myeloma GIMEMA Lazio Group in 62 relapsed/refractory MM patients treated with daratumumab as monotherapy who had previously received at least two treatment lines including a PI and an IMiDs or had been double refractory. Patients received DARA 16 mg/kg intravenously weekly for 8 weeks, every 2 weeks for 16 weeks, and every 4 weeks until disease progression or unacceptable toxicity. The overall response rate to daratumumab was 46%. Median progression-free survival (PFS) and overall survival reached 2.7 and 22.4 months, respectively. DARA was generally well tolerated; however, 2 patients interrupted their therapy due to adverse events. Present real-life experience confirms that DARA monotherapy is an effective strategy for heavily pre-treated and refractory patients with multiple myeloma, with a favorable safety profile.

Management of meningeal solitary fibrous tumors/hemangiopericytoma; surgery alone or surgery plus postoperative radiotherapy?

INTRODUCTION: A meningeal solitary fibrous tumor (SFT), also called hemangiopericytoma, is a rare mesenchymal malignancy. Due to anatomic constrains, even after macroscopic complete surgery with curative intent, the local relapse risk is still relatively high, thus increasing the risk of dedifferentiation and metastatic spread. This study aims to better define the role of postoperative radiotherapy (RT) in meningeal SFTs. PATIENTS AND METHODS: A retrospective study was performed across seven sarcoma centers. Clinical information was retrieved from all adult patients with meningeal primary localized SFT treated between 1990 and 2018 with surgery alone (S) compared to those that also received postoperative RT (S + RT). Differences in treatment characteristics between subgroups were tested using independent samples t-test for continuous variables and chi-square tests for proportions. Local control (LC) and overall survival (OS) rates were calculated as time from start of treatment until progression or death from any cause. LC and OS in groups receiving S or S + RT were compared using Kaplan-Meier survival curves. RESULTS: Among a total of 48 patients, 7 (15%) underwent S and 41 (85%) underwent S + RT. Median FU was 65 months. LC was significantly associated with treatment. LC after S at 60 months was 60% versus 90% after S + RT (p = 0.052). Furthermore, R1 resection status was significantly associated with worse LC (HR 4.08, p = 0.038). OS was predominantly associated with the mitotic count (HR 3.10, p = 0.011). CONCLUSION: This retrospective study, investigating postoperative RT in primary localized meningeal SFT patients, suggests that combining RT to surgery in the management of this patient population may reduce the risk for local failures.

1,2,3-Triazole Bridge as Conformational Constrain in ß-Hairpin Peptides: Analysis of Hydrogen-Bonded Positions.

Conformational constrained ß-hairpin peptides are useful tool to modulate protein-protein interactions. A triazole bridge in hydrogen-bonded positions between two antiparallel strands induces a conformational stabilization of the ß-hairpin peptide. The entity of the stability of the ß-hairpin peptide depends on the length of the bridge.

Amplicon-based next-generation sequencing: an effective approach for the molecular diagnosis of epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of biopsy samples and transmission electron microscopy followed by single-candidate gene Sanger sequencing (SS), which are labour-intensive and expensive clinical pathways. OBJECTIVES: According to the recently published recommendations for the diagnosis and treatment of EB, the assessment of the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. We aimed to develop a customized, cost-effective amplicon panel for the complete and accurate sequencing of all the pathogenic genes already identified in EB, and to minimize the processing time required for the execution of the test and to refine the analysis pipeline to achieve cost-effective results from the perspective of a routine laboratory set-up. Next-generation sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the processing time and costs of EB diagnostics. MATERIALS AND METHODS: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on an Ion Torrent Personal Genome Machine platform. The panel was performed on 10 patients with known genetic diagnoses and was then employed in eight family trios with unknown molecular footprints. RESULTS: The panel was successful in finding the causative mutations in all 10 patients with known mutations, fully confirming the SS data and providing proof of concept of the sensitivity, specificity and accuracy of this procedure. In addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including ones that the SS missed or de novo mutations. CONCLUSIONS: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a cost- and time-effective 72-h procedure.

Oral flora of Python regius kept as pets.

UNLABELLED: This study was aimed at evaluating the oral bacterial flora of 60 Python regius kept as pets by culture and biochemical methods. All isolates were also submitted to antimicrobial susceptibility testing using the disc diffusion method. The oral cavity of snakes sampled harboured a wide range of Gram-negative bacteria mainly constituted by Pseudomonas spp., Morganella morganii, Acinetobacter calcoaceticus, Aeromonas hydrophila, but also by Salmonella spp. Staphylococcus spp. was the commonest Gram-positive isolates, and various anaerobic Clostridium species were also found. The most effective antimicrobial agents were enrofloxacin and ciprofloxacin, followed by doxycycline and gentamicin. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity of snakes sampled harboured a wide range of bacteria. Our results suggest that people who come in contact with snakes could be at risk of infection and should follow proper hygiene practices when handling these reptiles.

Alkaline phosphatase (alp) levels in multiple myeloma and solid cancers with bone lesions: Is there any difference?

INTRODUCTION: Bone involvement in Multiple Myeloma results from increased osteoclast formation and activity that occurs in proximity to myeloma cells. The role of Alkaline Phosphatse (ALP) in this process and the diagnostic significance of plasma levels in patients with MM are unclear. AIM: To compare plasma ALP levels in patients with MM and solid cancers and metastatic lesions to the bone. RESULTS: In this observational retrospective study we enrolled 901 patients were enrolled: 440 patients (49%) with Multiple Myeloma, 461 (51%) with solid cancers. All 901 patients had bone lesions. Among patients with Multiple Myeloma, ALP values were mainly in the range of normality than those observed in patients with solid cancers and bone lesions. This difference is independent of stage, number and type of bone lesions. CONCLUSION: This study suggests that plasma ALP has a different clinical significance in MM than in other neoplasms and could be used as a discriminating marker in presence of bone lesions. In particular, lower or normal values, should suggest further investigations such as urinary and serum electrophoresis, associated with bone marrow aspirate in case of the presence of a monoclonal component, in order to confirm or exclude a MM diagnosis.

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers.

Cytogenetic analyses were performed in two Curimatidae species (Steindachnerina insculpta and Cyphocharax modesta) from the Paranapanema and Tietê Rivers (São Paulo State, Brazil), showing a karyotype composed of 54 meta-submetacentric chromosomes in both species. Silver- and chromomycyn-staining and fluorescent in situ hybridization (FISH) using a 18S rDNA probe indicated that the nucleolar organizer regions (NORs) of both species are localized in the terminal region of the long arm of two metacentric chromosomes. Although a single NOR system was evidenced in both analyzed species, S. insculpta and C. modesta presented the nucleolar organizer regions in distinct chromosome pairs, indicating that these cistrons can be considered cytogenetic markers. Variation on the amount and distribution of the constitutive heterochromatin (C-bands) could also be detected between the two species - while S. insculpta presented few heterochromatic blocks, intensely stained C-bands were evidenced in C. modesta specially in the terminal region of the long arm of the NOR-bearing chromosomes. Although most Curimatidae species have been characterized by homogeneous karyotypes, isolated populations could be established under different environmental conditions leading to karyotype micro-structure variations specially related to the NORs localization and C-banding distribution. The obtained data were useful for the cytogenetic characterization and differentiation of S. insculpta and C. modesta and could be used in evolutionary inferences in the Curimatidae group.

A phase 2 study of three low-dose intensity subcutaneous bortezomib regimens in elderly frail patients with untreated multiple myeloma.

This phase 2 trial evaluated three low-dose intensity subcutaneous bortezomib-based treatments in patients ⩾75 years with newly diagnosed multiple myeloma (MM). Patients received subcutaneous bortezomib plus oral prednisone (VP, N=51) or VP plus cyclophosphamide (VCP, N=51) or VP plus melphalan (VMP, N=50), followed by bortezomib maintenance, and half of the patients were frail. Response rate was 64% with VP, 67% with VCP and 86% with VMP, and very good partial response rate or better was 26%, 28.5% and 49%, respectively. Median progression-free survival was 14.0, 15.2 and 17.1 months, and 2-year OS was 60%, 70% and 76% in VP, VCP, VMP, respectively. At least one drug-related grade ⩾3 non-hematologic adverse event (AE) occurred in 22% of VP, 37% of VCP and 33% of VMP patients; the discontinuation rate for AEs was 12%, 14% and 20%, and the 6-month rate of toxicity-related deaths was 4%, 4% and 8%, respectively. The most common grade ⩾3 AEs included infections (8-20%), and constitutional (10-14%) and cardiovascular events (4-12%); peripheral neuropathy was limited (4-6%). Bortezomib maintenance was effective and feasible. VP, VCP and VMP regimens demonstrated no substantial difference. Yet, toxicity was higher with VMP, suggesting that a two-drug combination followed by maintenance should be preferred in frail patients.

Standard versus trans-epithelial collagen cross-linking in keratoconus patients suitable for standard collagen cross-linking.

PURPOSE: Evaluating the clinical results of trans-epithelial collagen cross-linking (CXL) and standard CXL in patients with progressive keratoconus. METHODS: This prospective study comprised 20 eyes of 20 patients with progressive keratoconus. Ten eyes were treated by standard CXL and ten by trans-epithelial cross-linking (TE-CXL, epithelium on) with 1 year of follow-up. All patients underwent complete ophthalmologic testing that included pre- and postoperative uncorrected visual acuity, corrected visual acuity, spherical error, spherical equivalent, corneal astigmatism, simulated maximum, minimum, and average keratometry, coma and spherical aberration, optical pachymetry, and endothelial cell density. Intra-and postoperative complications were recorded. The solution used for standard CXL comprised riboflavin 0.1% and dextran 20.0% (Ricrolin), while the solution for TE-CXL (Ricrolin, TE) comprised riboflavin 0.1%, dextran 15.0%, trometamol (Tris), and ethylenediaminetetraacetic acid. Ultraviolet-A treatment was performed with UV-X System at 3 mW/cm(2). RESULTS: In both the standard CXL group (ten patients, ten eyes; mean age, 30.4±7.3 years) and the TE-CXL group (ten patients, ten eyes; mean age, 28±3.8 years), uncorrected visual acuity and corrected visual acuity improved significantly after treatment. Furthermore, a significant improvement in topographic outcomes, spherical error, and spherical equivalent was observed in both groups at month 12 posttreatment. No significant variations were recorded in other parameters. No complications were noted. CONCLUSION: A 1-year follow-up showed stability of clinical and refractive outcomes after standard CXL and TE-CXL.

Long-term results of the GIMEMA VEL-03-096 trial in MM patients receiving VTD consolidation after ASCT: MRD kinetics' impact on survival.

Polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis is a useful prognostic tool in multiple myeloma (MM), although its long-term impact still needs to be addressed. This report presents the updated results of the GIMEMA-VEL-03-096 trial. Thirty-nine MM patients receiving bortezomib-thalidomide-dexamethasone after autologous transplantation were monitored for MRD by both nested and real-time quantitative-PCR until relapse. Our data confirm the strong impact of MRD on survival: overall survival was 72% at 8 years median follow-up for patients in major MRD response versus 48% for those experiencing MRD persistence (P=0.041). In addition, MRD kinetics resulted predictive for relapse: indeed median remission duration was not reached for patients in major MRD response, 38 months for those experiencing MRD reappearance and 9 months for patients with MRD persistence (P<0.001). Moreover: (1) 26 patients achieving major MRD response (67%) benefit of excellent disease control (median TNT: 42 months); (2) MRD reappearance heralds relapse, with a TNT comparable to that of MRD persistence (9 versus 10 months, P=0.706); (3) the median lag between MRD reappearance and need for salvage treatment is 9 months. These results suggest the usefulness of a long-term MRD monitoring in MM patients and the need for maintenance or pre-emptive treatments ensuring durable responses.

Costs of high-dose chemotherapy and peripheral blood progenitor cell autograft for breast cancer.

The aim of the study was to analyze the real cost of single or tandem high-dose chemotherapy (HDC) and peripheral blood progenitor cell autologous transplant (PBPCT) in patients with breast cancer. We analyzed the costs of 40 PBPCT performed in 20 patients. Tandem transplant was planned for each patient. Resources used and direct costs were identified for each patient. The study was carried out using the hospital perspective and monetary values were reported in 1999 Euro. The mean cost of whole procedure for single transplant was 20,816.63 Euro, while the mean cost of tandem transplant was 38,770.83 Euro. The cost distribution in the two groups was similar: the most expensive phase of procedure was the supportive phase post transplant (about 60% of total cost), with the categories of cost most represented being professional fees (about 28%) and pharmacy (about 35%). Awaiting more convincing trials of the clinical advantage of HDC in breast cancer, our analytical evaluation of transplant costs for different therapeutic options, single or tandem, permits identification of the most expensive categories in order to intervene for cost savings.

Administration of low-dose interleukin-2 plus G-CSF/EPO early after autologous PBSC transplantation: effects on immune recovery and NK activity in a prospective study in women with breast and ovarian cancer.

This study evaluated the effects of low-dose IL-2 plus G-CSF/EPO on post-PBSC transplantation (PBSCT) immune-hematopoietic reconstitution and NK activity in patients with breast (BrCa) and ovarian cancer (OvCa). To this end, two consecutive series of patients were prospectively assigned to distinct post-PBSCT cytokine regimens (from day +1 to day +12) which consisted of G-CSF (5 microg/kg/day) plus EPO (150 IU/kg/every other day) in 17 patients (13 BrCa and 4 OvCa) or G-CSF/EPO plus IL-2 (2 x 10(5) IU/m(2)/day) in 15 patients (10 BrCa and 5 OvCa). Hematopoietic recovery and post-transplantation clinical courses were comparable in G-CSF/EPO- and in G-CSF/EPO plus IL-2-treated patients, without significant side-effects attributable to IL-2 administration. In the early and late post-transplant period a significantly higher PMN count was observed in G-CSF/EPO plus IL-2-treated patients (P = 0.034 and P = 0.040 on day +20 and +100, respectively). No significant differences were found between the two groups of patients in the kinetics of most lymphocyte subsets except naive CD45RA(+) T cells which had a delayed recovery in G-CSF/EPO plus IL-2 patients (P = 0.021 on day +100). No significant difference was observed between NK activity in the two different groups, albeit a significantly higher NK count was observed in G-CSF/EPO plus IL-2 series on day +20 (P = 0.020). These results demonstrate that low-dose IL-2 can be safely administered in combination with G-CSF/EPO early after PBSCT and that it exerts favorable effects on post-PBSCT myeloid reconstitution, but not on immune recovery.

Primary cutaneous follicle center cell lymphoma and limited stage follicular non-Hodgkin's lymphoma: a comparison of clinical and biological features.

Primary cutaneous follicular lymphoma (PCFL) and nodal follicular lymphoma (NFL) are different entities, which, nevertheless, exhibit common features. The aim of this study was to compare the clinico-biological characteristics and the outcome of patients with PCFL and with limited stage NFL. A group of 22 consecutive patients with PCFL presenting with single or multiple cutaneous lesions was compared to a group of 21 patients with limited stage NFL. The median age was 56 and 55 years, respectively. The histologic features were compared, as well as treatment modalities and response. Treatment of PCFL consisted of restricted field radiotherapy (RT), chemotherapy (CHT) and combined modalities (CM) in 12, 5 and 5 cases, respectively. Among the 21 patients with NFL, RT was employed in 13, CHT in 7 and the CM in one patient. The response to treatment was: 17 complete responses, CR (12 RT, 2 CHT, 3 CM), 3 partial responses, PR (1 CHT, 2 CM) and 2 non-responses, NR (2 CHT) in the PCFL group, while in the NFL group 18 patients attained CR (13 RT, 4 CHT, 1 CM) and 3 PR (3 CHT). The relapse rate was 22.7 and 28.5% in PCFL and NFL, respectively (median follow up of 48 and 50 months). The estimated 4 years event free survival (EFS) in the PCFL and NFL patients were 68 and 70%, respectively, (P = 0.83). The estimated 4 years overall survival (OS) in the PCFL and NFL groups were 90% and 100% respectively, (P = 0.254). Among the 22 patients with PCFL, there was a higher incidence of large cells than in NFL, although the differences were not significant (P = 0.08). In conclusion, despite histologic (higher proportion of large cells) and biologic differences, cutaneous and limited stage NFL show similar responses to treatment, with similar relapse rates, EFS and OS.

Autologous blood stem cell transplantation in malignant lymphomas: an Italian Cooperative Study.

Twenty-three patients with malignant lymphoma, (7 Hodgkin's, and 16 non-Hodgkin's) in different phases of disease were autografted in 4 Italian Haematology institutions using only chemotherapy-mobilized blood stem cells (BSC) collected by apheresis. Clinical and laboratory data were analysed centrally and showed mean collection yields of 8.1 x 10(8) kg mononuclear cells (MNC) (SE 0.5; range 2.6-13.8) and 24.1 x 10(4) kg CFU-GM (SE 7.4; range 1.4-162.9). The mean times required to attain 0.5 x 10(9)/l neutrophils and 50 x 10(9)/l platelets after marrow-ablative high-dose chemo+radiotherapy and BSC reinfusion were 14.9 days (SE 1.5; range 7-38) and 18.6 days (SE 2.6; range 6-49) respectively. The incidence of early deaths was < 5% and the requirement for support with blood product transfusion was moderate. The progression free survival (PFS) is > 50% at 3 years with a median follow-up of 17.3 months. Results were significantly better for patients autografted in remission. These results suggest that autologous blood stem cell transplantation (ABSCT) may be proposed for the primary treatment of poor prognosis malignant lymphomas. However, ABSCT needs to be compared with autologous bone marrow transplantation (ABMT) followed by infusion of growth factors to accelerate recovery.

Growth factor administration following autologous peripheral blood progenitor cell transplantation.

Hematopoietic growth factor (HGF) administration following autologous peripheral blood progenitor cell transplantation (APBPCT) is a current approach for shortening the duration of high-dose chemotherapy-induced transient peripheral pancytopenia. Several published clinical experiences and a retrospective study reported here show that recombinant human granulocyte colony stimulating factor (rhG-CSF) or recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) administration potentiates polymorphonuclear leukocyte (PMN) and white blood cell (WBC) recovery with some clinical benefits mainly related to the reduction of infectious complications during the shortened period of neutropenia. However, this therapeutic strategy does not produce any enhancement of platelet (PLT) recovery or potentiation of red cell production. Conversely, a recent phase I/II study carried out in our institution showed that the combined administration of rhG-CSF and recombinant human erythropoietin (rhEPO) is able to potentiate trilineage hematopoietic recovery with a reduction of PLT transfusions and to considerably simplify the clinical management of patients as compared to patients treated with APBPCT alone. The post-APBPCT administration of rhEPO with rhGM-CSF decreased the number of days with WBC < 1 x 10(9)/L but failed to produce any appreciable effect on PLT recovery. Both combined treatments significantly reduced the patients' hospital stay and allowed the abrogation of systemic antibiotic administration following APBPCT. A further group of patients were treated with the combined administration of rhEPO, rhG-CSF and rhGM-CSF; they did not show a faster hematopoietic recovery than rhG-CSF plus EPO treated patients and a consistent hyperthermia was observed in most patients as a prominent side effect. Future prospective randomized studies will clarify the efficacy of HGF administration following APBPCT. Moreover, further improvements in the hematopoietic support of transplanted patients may be obtained when stem cell factor, flt3/flk2 tyrosine kinase ligands or megakaryocyte growth and development factor will become clinically available.

Docetaxel and epirubicin plus G-CSF as mobilizing treatment to support high-dose chemotherapy in breast cancer.

BACKGROUND: In order to combine an active regimen with a simultaneous efficient mobilization of peripheral blood precursor cells (PBPC), we explored the combination of Docetaxel 75 mg/m2 and Epirubicin 120 mg/m2 with G-CSF 5 mcg/Kg/day s.c. to mobilize PBPC in breast cancer patients to support high-dose chemotherapy (HDC). PATIENTS AND METHODS: Forty patients were enrolled: 27 high risk and 13 metastatic. The entire procedure, including chemotherapy and PBPC collection, was on an outpatient basis. RESULTS: The median day of starting apheresis was day +10 (range 10-12) and the average value of circulating CD34+ cells at peak was 175/microliter (range 33-403). The median yield of CD34+ cells per apheresis was 8.76 x 10(6)/Kg (range 1.83-27.87). None of the patients developed side effects which required hospitalization. All patients enrolled successively received HDC as consolidation treatment. High risk patients received one and metastatic patients two HDC with PBPC reinfusion. All patients obtained a complete engraftment. No significant differences between high-risk and metastatic patients were observed. CONCLUSIONS: Our study suggests that the combination of Docetaxel, Epirubicin, and G-CSF is feasible, safe and efficient outpatient mobilizing treatment for patients with breast cancer receiving HDC.

Therapy of 'high risk' myelodysplastic syndromes with an association of low-dose Ara-C, retinoic acid and 1,25-dihydroxyvitamin D3.

Forty-four patients with high risk primary myelodysplastic syndromes and an excess of marrow blasts were treated with a combination of low-dose Ara-C, retinoic acid and vitamin D3. Morphological subtypes were refractory anemia with excess of blasts (RAEB) in 16, RAEB in transformation (RAEB-T) in 20 and chronic myelomonocytic leukemia (CMML) in eight patients. The therapy was continued in responders until relapse or death. The results were compared to those of a matched control of 44 patients given a supportive therapy only. In the treated group the overall response rate was 50% (75% in RAEB, 50% in RAEB-T and 0% in CMML) and the survival was significantly better than in the control group (P < 0.025). Comparing separately each FAB subgroup gave statistical evidence that the treatment prolonged the survival in the RAEB-T subgroup only (P < 0.002). The median duration of response was 15 months and the survival in responders was statistically better than in non-responders (P < 0.0001). Myelosuppression has been the most important side effect, however, no death related to the treatment was observed. Our study suggests that patients with RAEB-T, who are not suitable candidates for aggressive chemotherapy, could benefit from our treatment schedule. The long duration of therapy seems to be of value for patients achieving a response in order to prolong the survival. The toxicity is acceptable and the therapy can be given on an outpatient basis.

Detection of breast cancer cells in the bone marrow or peripheral blood: methods and prognostic significance.

Tumor cells can reach every anatomic district, organ and tissue through the peripheral blood circulation. Tumor cell shedding is considered an early event in the multi-phase process of metastasis, and the possibility of detecting tumor cells in the bloodstream and/or bone marrow before clinical evidence of distant metastases needs to be explored. The use of new sophisticated diagnostic and investigative techniques has boosted the study of tumor cell contamination of bone marrow and peripheral blood. Molecular techniques, such as reverse-transcriptase polymerase chain reaction, may be useful tools to reach this target, but, today, immunocytochemistry is still considered the gold standard to assess new techniques to detect isolated tumor cells in hematopoietic tissue. Little is known about the biology of isolated tumor cells in the peripheral blood or bone marrow. Two crucial points need to be evaluated: the identification of specific markers of breast cancer cells with clonogenic potential and their biologic properties, and the prognostic impact of the detection of isolated tumor cells in the bone marrow or peripheral blood stem cell collections.

Progenitor cells purging: negative selection.

Disease relapse after autologous bone marrow transplantation (ABMT) may arise from residual tumor in the recipient and/or from cancer cells that are reinfused. The aim of purging by negative selection is to remove tumor cells from the marrow without adversely affecting the engraftment potential of the normal cell. We report the results of a study on fifty-six patients (pts) with non Hodgkin's lymphoma or acute leukemia submitted to ABMT after immunomagnetobead (IMB) purging (11 pts), Maphosphamide purging (31 pts) and no purging (14 pts). The IBM procedure involved one incubation of 3 monoclonal antibodies (CD10, CD19 and CD22) and two incubations with magnetic beads (Dynabeads M-450). The median recovery of mononuclear cells and CFU-GM was 40% and 45% after IMB purging and 84% and 5% after Maphosphamide purging respectively. The rate of leukocyte, neutrophils and platelets recovery following ABMT was similar in the three groups of pts, although platelet recovery was slow in patients received graft purged with Maphosphamide. Our study confirms the clinical feasibility of the IMB procedure, but only randomized studies will be able to definitely address the question of the clinical utility of purging.