Treatment and outcome in metastatic lobular breast cancer in the prospective German research platform OPAL.

PURPOSE: Evidence about routine treatment and outcome of patients with invasive lobular cancer (ILC) is limited, especially regarding metastatic disease. Here we present prospective real-world data of patients with metastatic ILC (mILC) as compared to patients with metastatic invasive ductal cancer (mIDC) receiving systemic therapy in routine care in Germany. METHODS: Prospective data on patient and tumor characteristics, treatments, and outcomes of patients with mILC (n = 466) and mIDC (n = 2100), recruited between 2007 and 2021 into the Tumor Registry Breast Cancer/OPAL were analyzed. RESULTS: Compared to mIDCs, patients with mILC were older at start of first-line treatment (median 69 vs. 63 years) and had more often lower grade (G1/G2: 72.8% vs. 51.2%), hormone receptor (HR)-positive (83.7% vs. 73.2%) and less often HER2-positive (14.2% vs. 28.6%) tumors, which metastasized more frequently to the bone (19.7% vs. 14.5%) or peritoneum (9.9% vs. 2.0%), and less frequently to the lungs (0.9% vs. 4.0%). Median OS of patients with mILC (n = 209) and mIDC (n = 1158) was 30.2 months [95% confidence interval (CI) 25.3, 36.0] and 33.7 months [95% CI 30.3, 37.9], respectively. Multivariate survival analysis did not show a significant prognostic impact of the histological subtype [HR mILC vs. mIDC 1.18 (95% CI 0.97-1.42)]. CONCLUSION: Overall, our real-world data confirm clinicopathological differences between mILC and mIDC breast cancer patients. Despite patients with mILC presenting with some favorable prognostic factors, ILC histopathology was not associated with a better clinical outcome in multivariate analysis, suggesting the need for more tailored treatment strategies for patients with the lobular subtype.

Treatment of primary mediastinal large B cell lymphoma with an alternating chemotherapy regimen based on high-dose methotrexate.

Primary mediastinal large B cell lymphomas (MLCL) differ from other diffuse large cell lymphomas, leading to a description as a separate entity in the current World Health Organization classification. Dose intensification improves long-term results, but no standard therapy has been established so far. We investigated the use of a high-dose methotrexate-based alternating chemotherapy regimen (B-ALL protocol of the German ALL study group) followed by consolidative mediastinal radiotherapy first as a single-center trial, then later as a prospective multicenter trial in 44 patients with a median age of 33 years. Response rates exceeded 90% with an overall survival rate of 80% in the single-center group (8.6 years median follow-up) and 82% in the multicenter group (2.5 years follow-up).Short-term toxicity was manageable, but required hospitalization: the rates of grade 3 or 4 toxicity were 20% (for mucositis), 42% (for neutropenia), 29% (for thrombocytopenia), and 9% (for neutropenic fever). No relapse occurred more than 2 years after diagnosis and initiation of treatment, but unfortunately, no patient with overt progression or relapse within these 2 years could be salvaged. Future directions in the treatment of MLCL will not focus on further dose intensification, but rather on the incorporation of (radio)immunotherapy as a therapeutic tool and gene expression profiling as well as positron emission tomography-computed tomography as stratifying tools.

Reduced-toxicity conditioning with fludarabine and treosulfan prior to allogeneic stem cell transplantation in multiple myeloma.

In recent years, reduced-intensity conditioning (RIC) regimens before allogeneic stem cell transplantation (SCT) are increasingly used in patients not eligible for conventional conditioning. We did a retrospective, multicenter analysis to assess the feasibility of conditioning with fludarabine and treosulfan before allogeneic SCT in multiple myeloma patients. Thirty-four patients with a median age of 51.5 years were included in the analysis. All patients underwent myeloablation after conditioning followed by stable engraftment, and 29 of 31 evaluable patients (94%) showed early complete hematopoietic chimerism. Non-hematological toxicities were limited and encompassed mainly fever in neutropenia and infections. Grade II-IV acute and chronic graft-versus-host disease was observed in 33 and 39%, respectively. With a median follow-up of 708 days (range 60-1729 days), the median progression-free survival was 180 days. The treatment-related mortality was 10% on day 100 and 25% after 1 year. The median overall survival has not yet been reached. Our data indicate that conditioning with fludarabine and treosulfan before allogeneic SCT is feasible in intensively pretreated multiple myeloma patients and leads to stable engraftment and complete hematopoietic chimerism. Randomized trials are warranted to determine if this approach might be incorporated in an algorithm of multiple myeloma treatment.

Allogeneic hematopoietic cell transplantation following conditioning with 90Y-ibritumomab-tiuxetan.

Radioimmunotherapy (RIT) of relapsed lymphoma is gaining increasing importance. Especially the commercially available anti-CD20 antibody 90Y-ibritumomab tiuxetan is currently under investigation in various trials including dose escalation and autologous hematopoietic progenitor cell support. It is not clear, however, whether the implementation of this radiolabeled antibody into another treatment option for relapsed or poor risk lymphoma patients-allogeneic hematopoietic cell transplantation-interferes with or delays successful engraftment. This study reports encouraging results with 2 relapsed lymphoma patients (1 transformed marginal zone lymphoma and 1 mantle cell lymphoma) who underwent allogeneic hematopoietic cell transplantation from HLA-matched donors. The conditioning regimen consisted of Rituximab 250 mg m(-2) on days -21 and -14, 0.4 mCi kg(-1) body weight 90Y-ibritumomab tiuxetan on day -14 and fludarabine (30 mg m(-2)) plus cyclophosphamide (500 mg m(-2)) on days -7 to -3. The data demonstrate that engraftment is fast and reliable with leukocytes >1 x 10(9) L(-1) on day 12 and platelets >50 x 10(9) L(-1) on day 10. Thus, the incorporation of radioimmunotherapy into allogeneic transplant protocols combines established modalities with proven anti-lymphoma activity and, hence, offers an attractive new therapeutic option for relapsed lymphoma patients.

Stem cell mobilization in multiple myeloma patients: do we need an age-adjusted regimen for the elderly?

The upper age limit for autologous progenitor cell transplantation in multiple myeloma patients is increasing continuously. We examined whether this shift in the age of pretreated myeloma patients requires modification of mobilization regimen. We compared retrospectively 21 consecutive progenitor cell mobilizations in 15 pts < 60 years (median age 56, range 37-59) with 33 consecutive mobilizations in 23 pts > 60 years (median age 65, range 60-73) of age. The number of CD34 positive circulating cells before scheduled leukapheresis was a mean of 67,935 cells/mL (SEM +/- 17,614) in the younger population and a mean of 19,069 (SEM +/- 5,396) for older pts (P = 0.0027). In patients >60 years, 13/33 mobilizations (including 2 patients with 2 failing attempts) were not successful (39%), compared to 6/21 mobilizations (29%, including 1 patient with 3 failing attempts) in the younger population. The increased number of progenitor cells in the grafts of younger patients led to a more rapid regeneration of leukocytes and platelets after stem cell infusion. Our data show that stem cell mobilization in older multiple myeloma patients is inferior compared to a younger patient population. There is a trend towards more leukapheresis until the target stem cell dose has been collected, and the decreased number of progenitor cells in the actual graft delays engraftment of leukocytes and platelets. The overall number of unsuccessful mobilization attempts, however, did not differ significantly between both age groups. A special "age-adjusted" increase in the dose of growth factors seems unjustified. Improvements in timing of leukapheresis, growth factor application, and mobilizing chemotherapy regimen as well as the use of alternative cytokines should be investigated for both age groups.

Culture of haematopoietic cells in a 3-D bioreactor made of Al2O3 or apatite foam.

Foams of Al2O3 and apatite ceramics with interconnecting pores were produced using a new technique. The surfaces of the ceramics served as substrates for the culture of human peripheral and bone marrow derived stem cells. Up to 27 days the cells were kept in culture where they proliferated and developed into different morphologies consistent with bone marrow cell lines.

Physical performance, depression, immune status and fatigue in patients with hematological malignancies after treatment.

BACKGROUND: Fatigue is a frequent and severe problem after treatment of patients with hematological malignancies. This symptom has been associated with anemia, reduced physical performance, mood, endocrine disorders and impaired nutritional status. Recently, it has been suggested that fatigue can be related to a persistent activation of the immune system with increased production of proinflammatory cytokines. However, there is no conclusive evidence regarding the role of the immune system in the origin of fatigue in cancer patients. PATIENTS AND METHODS: We evaluated the correlation of fatigue with thyroid function, markers of immune activity [interleukin (IL)-1alpha, IL-1 soluble receptor, IL-6, C-reactive protein and neopterin], liver and kidney function, mood and physical ability in 71 patients with hematological malignancies. All patients had been free of relapse and not received treatment (chemotherapy, radiotherapy or immune modulators) for at least 3 months. RESULTS: Fatigue was related to depression (r=0.84; P<0.0001) and reduced performance status (r=-0.61; P<0.0001). However, there was no correlation between fatigue and thyroid, liver and kidney function, anemia, albumin concentration or markers of immune activity (all r-values <0.20; P>0.05). CONCLUSIONS: We conclude that fatigue in relapse-free patients with hematological malignancies is associated with depressive mood and reduced physical performance, but not with impairment of thyroid function, anemia or persistent activation of the immune system.

Efficacy and tolerability of alemtuzumab (CAMPATH-1H) in the salvage treatment of B-cell chronic lymphocytic leukemia--change of regimen needed?

We report on the response rate and tolerability of Alemtuzumab (Campath-1H) in a series of heavily pretreated patients with B-CLL with a special focus on treatment-related problems. All patients tested positive for CD52 on B-lymphocytes before entering the trial. Thirteen patients with B-chronic lymphocytic leukemia (B-CLL), 1 prolymphocytic leukemia (PLL), 1 mantle cell lymphoma (MCL) and 1 leukemic immunocytoma (IC) transformed into a high-grade NHL were included. Median age was 62 years (range 40-73), and pretreatment consisted of median 3 prior regimens (range 1-11). All patients received 3, 10 and 30 mg of Campath-1H on sequential days, and then were subsequently scheduled for 30 mg 3 times weekly. Nine out of 16 patients responded. One patient attained complete remission (CR), 8 patients achieved partial remission (PR), while 4 patients had stable disease (SD). Three patients had progressive disease (PD). Beginning with initiation of treatment recurrent profound leukopenia became evident in 13 out of 16 patients leading to treatment discontinuation. Severe nonhematological toxicity (WHO grade IV bronchospasm) occurred in the first patient of this series, who initially had no concomitant steroids. Therefore, we developed a steroid co-medication regimen for the first 4 Campath-1H applications with quick tapering thereafter. Following this regimen, no infusion associated side effects WHO grade > II were observed. Infectious complications leading to treatment discontinuation consisted of pulmonary aspergillosis in one and bacterial pneumonia in another case. One patient with refractory B-CLL and Pneumocystis carinii pneumonia plus CMV reactivation died. In summary, Campath-1H appears to be effective against leukemic low-grade B-NHL, also in advanced stage. In our series, application 3 times weekly was not possible due to hematotoxicity. We recommend, therefore, flexible time intervals depending on the leukocyte counts. Whether a cumulative dosage according to 3 x 30 mg Campath-1H for 12 weeks is needed still remains to be clarified.

Successful treatment of extracranially metastasized pineal gland germinoma with high-dose methotrexate.

Germinoma of the pineal gland is a rare disease usually confined to the brain which responds well to radiotherapy. Spinal seeding occurs in approximately 4% of cases and distant metastases are extremely rare. We report on a 27-year-old female with an intracranially metastasized pineal gland germinoma, meningeal carcinomatosis and distant bone metastases. Treatment was initiated with intrathecal methotrexate (MTX) and continued with high-dose intravenous MTX. The therapy was very well tolerated apart from reversible hepatic toxicity requiring a dose reduction. The patient was in complete remission after three courses followed by two consolidation cycles; the patient has now been in continuous complete remission for more than 22 months. This is the first report to show that MTX is a potent drug in treating pineal gland germinoma. Long-term side effects of radiotherapy such as reduced mental function or hypopituitarism can probably be avoided. Single-agent high-dose MTX may provide high efficacy with limited adverse effects, especially at a more advanced tumor stage with spinal seeding and extracranial disease.

Flow cytometric CD34+ determination in stem cell transplantation: before or after cryopreservation of grafts?

Various attempts have been made to standardize and improve the reproducibility of flow cytometric determination of CD34+ hematopoietic progenitor cells. It is still not clear, however, whether the quantification of CD34+ cells in a stem cell graft should be done before or after cryopreservation. To address this issue, we investigated 78 unselected and 32 immunomagnetically selected autologous and allogeneic leukapheresis products (LA) before and after cryopreservation using pilot vials. Cell numbers were quantified within a Neubauer chamber, and CD34+ content was determined by flow cytometry; propidium iodide staining was used to exclude dead cells from analysis. Before freezing, the mean viable CD34 cell content in the unselected samples was 1.22% and increased after thawing to a mean of 2.16% of viable cells. Taking into account cell loss and cell death, the overall recovery of viable cells was 64.5%; all CD34+ cells could be recovered. Mean purity in the CD34-selected cell fraction was 85% (48-97) before and 91.3% (67-99) after thawing. The number of viable cells was 86.8% before and 86.1% after freezing with a 93.9% recovery of total cells. This leads to a mean 93.7% (SD +/- 23.1) recovery of viable cells and 100% (SD +/- 22.3) recovery of viable CD34+ cells. There was no significant difference in tolerance to freeze/thaw stress between cells from heavily pretreated autologous patients and healthy allogeneic donors. Our data show that freezing significantly increases the percentage of CD34(+) cells in unmanipulated LA, probably due to the death of granulocytes and mononuclear cells (MNCs). Nevertheless, the overall number of viable CD34+ cells in unselected as well as selected samples remains unchanged. Thus, CD34 data from different laboratories, for example, within multicenter trials, should be comparable independent of the different time points of acquisition.

MDR-1 expression and deletions of chromosomes 7 and 5(Q) separately indicate adverse prognosis in AML.

In order to assess any correlation between MDR-1 expression and chromosomal aberrations, and to define their impact on clinical outcome in newly diagnosed AML pts, we investigated bone marrow and peripheral blood samples of 49 consecutive pts admitted to our hospital. Monosomy 7, trisomy 8 and 5q- were evaluated by means of interphase fluorescence in situ hybridization (FISH). Monosomy 7 was present in 6 pts, trisomy 8 in 5 pts, and 5q- in 6 pts. More than one aberration was seen in 7 pts. Chromosomal aberrations were mostly found in older pts (12/14 >60 years; p=0.03) and in pts with CD34 positive leukemic blasts (13/14 coexpressed CD34, p=0.0004). In 25 pts also standard G-banding analysis was performed leading to concordant results regarding chromosomes 7, 8 and 5. Flow cytometry identifyed MDR-1 positivity (MDR+) in 16 pts. MDR-1 expression appeared to be a characteristic feature in CD34+ AML (12/16 were CD34+ and MDR+ pts; p=0.013). No correlation, however, was found between chromosomal aberrations and MDR-1 expression. Pts with aberrations of either chromosomes 7, 8 or 5 detected by FISH (FISH+) were predominantly resistant to induction therapy (6/8 pts, p=0.004). A lower rate of complete remission (CR) was also seen in pts with MDR-1 expression (p=0.006). MDR+/FISH+ pts (n=3) were all refractory to remission induction, while all MDR-/FISH- pts (n=19) achieved CR (p=0.0006). MDR-1+ as well as pts with aberrations of chromosomes 7, and 5(q) showed a significantly decreased probability of overall survival. In conclusion, MDR-1 expression as well as abnormalities of chromosomes 7, and 5(q) predict poor clinical outcome in AML. The identification of these prognostic factors provides useful information for risk adapted treatment strategies.

CD34 selected alloPBSCT and adoptive immunotherapy.

To circumvent aGVHD in the early phase after allogeneic stem cell transplantation but to provide GVL activity later on, we performed alloPBSCT with CD34+ selected grafts followed by delayed add-back of CD3+ T cells. Ten consecutive patients having an HLA-identical sibling donor were enrolled on to this trial. Four patients were in first CR of high-risk ALL, another four in first CR of AML, one was in second myeloid blast crisis of CML, and one was in PR of relapsed NHL. Conditioning consisted of 2 x 60 mg/kg CY plus 12 Gy TBI. G-CSF (Filgrastim) mobilized peripheral cells were CD34+ selected using the Isolex 300i system in nine patients and the CliniMacs system in one. Median CD34+ purity was 86%. A median of 2.8 x 10(6)/kg CD34+ cells were transplanted. The number of CD3+ cells in the allografts was 5.7 x 10(4)/kg (median) after Isolex 300i, and 0.2 x 10(4)/kg after CliniMacs. All patients received G-CSF (Filgrastim) and engrafted rapidly. Standard-dose CsA was administered, and until day +60 no aGVHD occurred. At that time point, seven patients received 2 x 10(6)/kg CD3+ cells while CsA had been tapered to 50% of the starting dose. One of these patients died after a second T cell boost given on day +90 without concomitant immunosuppression due to grade IV intestinal aGVHD. Three others developed cutaneous cGVHD. Taken together, T cell depletion by CD34+ selection does not impair rapid engraftment in the HLA-identical sibling donor setting. Using standard-dose CsA the risk for acute GVHD seems to be minimized. Add-back of 2 x 10(6)/kg CD3+ cells on day +60 with CsA protection is feasible. However, whether this is the optimal time point and number of T cells remain to be further elucidated.

Efficacy and safety of simultaneous immunomagnetic CD34+ cell selection and breast cancer cell purging in peripheral blood progenitor cell samples used for hematopoietic rescue after high-dose therapy.

We have established a new simultaneous positive/negative selection procedure using the Baxter Isolex 300i system. We tested its tumor cell (TC) purging efficacy by tumor contamination tests ex vivo and its safety in a group of 17 breast cancer (BC) patients by measuring hematopoietic recovery after high-dose (HD) therapy and autologous stem cell rescue with the selected cells. Tumor contamination tests resulted in a TC depletion of 4.1-6.0 log steps. The CD34+ cell yield in this experimental setting was 38.9-91.5%, and the CD34+ cell purity was 86.0-96.0%. In a group of 17 BC patients (5 high-risk adjuvant, > or = 10 lymph nodes positive, and 12 metastatic), we processed leukapheresis products (LPs) by simultaneous positive/negative selection. In these clinical samples, the mean CD34+ cell yield was 56.2% (range, 14.0-80.1%), and the CD34+ cell purity was 94.5% (range, 69.0-99.8%). Additionally, we screened samples of the patients' LPs before and after the purging procedure for contaminating TC by immunocytochemistry. In 15 of 17 tested cases, TCs were detectable prior to the purging procedure. After the procedure, we could not detect residual TCs in 16 of 17 cases. In one case, we found a highly reduced number of TCs. Furthermore, we evaluated the times for hematopoietic reconstitution in a group of five BC patients in the high-risk adjuvant situation who underwent HD chemotherapy and hematopoietic rescue with positive/negative selected stem cells and compared it with our own data from 10 BC patients who, after identical HD therapy, received only positively selected CD34+ cells and 14 patients who, after identical HD therapy, received autografts purged by incubation with toxic ether lipids (ET-18-OCH3). In all groups, a leukocyte count of >2000 cells/microl was reached at day +10. A platelet count of > 50,000 cells/microl was reached at day +12 in the ET-18-OCH3 group and at day +14 in the other two groups. Furthermore, 12 patients with metastatic disease rescued with positive/negative selected stem cells after HD therapy also showed fast and comparable hematopoietic recovery. The new simultaneous immunomagnetic positive/negative selection using a closed system is effective and safe. Processing LPs leads to a similar CD34+ cell yield, a higher TC depletion compared to standard CD34+ cell selection, and no delay in hematopoietic recovery.

Culturing human umbilical cord blood: a comparison of mononuclear vs CD34+ selected cells.

We compared UCB mononuclear cells (MNC) with CD34+ selected cells in a serum-free static culture system. Cell number proliferation of MNCs was inferior to CD34+ selected cells. MNCs, however, showed a substantial increase from 0.94% CD34+ cells on day 0 to 5.8% on day 7, whereas in the CD34+ selected samples the CD34+ cell content declined continously from 62.2% on day 0 to 27.7% on day 7. The number of CFU-GM increased during culture of both cell fractions. Here, only the MNCs showed a substantial increase in clonogenicity on day 7 and day 14 to 11.1- and 4.1-fold input, respectively. This expansion of the CD34+ progenitor cell pool in the MNCs fraction was at least in part attributable to T cells, since the physical abrogation of T cells blocked this effect. Refeeding and reseeding of cells on day 7 had stimulating effects especially on the CD34+ cells, where cell number proliferation increased from 16.3-fold without to 58.1-fold on day 14. Also, we could find sporadic chromosomal aberrations in four of 100 metaphases examined after 7-20 days of ex vivo expansion. The significance of this observation needs to be clarified in a larger series.

[Clubbed fingers and arthralgia as a reversible paraneoplastic syndrome (Pierre-Marie-Bamberger syndrome) in non-small-cell bronchial carcinoma]. / Trommelschlegelfinger und Arthralgien als reversibles paraneoplastisches Syndrom (Pierre-Marie-Bamberger-Syndrom) beim nicht-kleinzelligen Bronchialkarzinom.

HISTORY AND CLINICAL FINDINGS: During chemotherapy for a non-small-cell bronchial carcinoma with metastasis to the right femur (previously locally excised), a 34-year-old man suddenly developed severe, lasting joint pain in the ankle, knee, elbow and wrist without signs of increased warmth or swelling of these joints. At the time of diagnosis clubbed fingers had been noted. INVESTIGATIONS: Radiography of the hands showed bilateral periosteal hyperostoses. Computed tomography of the thorax revealed tumor progression. DIAGNOSIS, TREATMENT AND COURSE: The triad of clubbed fingers, periosteal hyperostoses and arthralgia/arthritis with the pulmonary findings established the diagnosis of hypertrophic pulmonary osteoarthritis (HPO; Marie-Bamberger syndrome). Initially there had merely been the signs of the much more frequent incomplete form with only the clubbed fingers, the complete form developing with progression of the disease during chemotherapy, joint pains dominating the symptoms. After right upper lobectomy (primary adenocarcinoma) the joint pains ceased and the finger clubbing regressed. CONCLUSIONS: Only 10% of non-small-cell bronchial carcinomas are associated with HPO. Conversely, such a tumor is found in 90% of HPO of recent onset and should therefore be sought of when searching for the primary tumor. The signs of HPO are reversible if the underlying disease is adequately treated.

Allogeneic blood stem cell transplantation in advanced hematologic cancers.

Allogeneic bone marrow transplantation for advanced hematologic cancer is associated with a high risk of early treatment-related morbidity and mortality. To determine the short-term benefits of allogeneic blood stem cell transplants when compared to bone marrow transplants, we reviewed outcomes of 74 adults with advanced hematologic cancer transplanted from HLA-matched related donors after conditioning with thiotepa, busulfan and cyclophosphamide. There were three cohorts: group 1 received bone marrow transplants with cyclosporine (CsA) and methotrexate (MTX) for GVHD prophylaxis; group 2 received bone marrow transplants with CsA and methylprednisolone (MP); and group 3 received blood stem cells with CsA and MP. All patients received filgrastim post-transplant. Median times (range) to neutrophils > or = 0.5 x 10(9)/l were 17 (8-30), 9 (8-16) and 10 (8-13) days post-transplant, and to platelets > or = 20 x 10(9)/l were 28 (14-100+), 19 (13-100+) and 14 (9-86) days post-transplant for groups 1, 2 and 3, respectively (P < 0.05 only for group 1 vs group 3 for both outcomes). Blood stem cell recipients had the least regimen-related toxicity, fewest early deaths and earliest discharge. There was no significant difference in acute GVHD between the three groups. One hundred and eighty-day survivals (95% CI) were 53% (35-72%), 32% (10-53%), and 68% (49-87%) for groups 1, 2 and 3, respectively (P < 0.05 only for group 2 vs group 3). For allogeneic transplantation, use of blood stem cell grafts has substantial advantages over marrow grafts.

Ex vivo expansion of human umbilical cord blood does not lead to co-expansion of contaminating maternal mononuclear cells.

One of the main limiting factors for increased use of human umbilical cord blood (UCB) in adult allogeneic transplantation is the small number of progenitor cells that can be collected and infused. Ex vivo expansion of UCB might help to overcome this limitation. Whether an expansion of UCB cells will also lead to co-expansion of contaminating maternal cells, and thus may alter graft characteristics and lead to an increased incidence of GVHD, has not been looked at so far. We initiated cultures with UCB mononuclear cells (MNC) in a standard medium containing stem cell factor (SCF), flt-3L, II-3, IL-6, EPO and G-CSF. To address the question of contaminating maternal cells we performed interphase FISH analysis of the X and Y chromosome simultaneously. Male (XY) cord blood samples were investigated for maternal (XX) cells at day 0 and at several time points during culture. We could not detect maternal cells in any of the nine samples studied when cultures were started at day 0. Culturing did not expand previously undetected maternal cells into a range that could be seen with FISH technology, as all samples remained negative for maternal cells throughout culture periods of 14 days. We then artificially contaminated male UCB with maternal mononuclear cells at concentrations of 5 and 15% at day 0. After 14 days, maternal MNC were still detectable, but the percentage was reduced to 1.7% and 6%, respectively. During culturing of CD34+-selected UCB the content of maternal cells also declined from a mean of 1.6% after contamination to 0.4% on day 7. Taken together we could show that maternal cells co-cultured with UCB do not co-expand and thus do not interfere with ex vivo expansion of UCB for adult allogeneic transplantation.

Comparative evaluation of integrin alpha- and beta-chain expression in colorectal carcinoma cell lines and in their tumours of origin.

The integrin family consists of broadly expressed cell surface adhesion receptors, each member of which is composed of a non-covalently linked alpha/beta heterodimer. Integrin receptors are involved in the interaction with matrix proteins and may contribute to invasion and metastasis of carcinomas. To examine the biological role integrins play in colorectal carcinoma we compared the expression of integrin alpha- and beta-subunits in situ and in vitro. Eight newly established cell lines derived from immunohistochemically characterized colorectal carcinomas together with two sublines obtained after nude mouse passage and the commonly used colon carcinoma lines HT-29, SW480, SW620, and COLO 205 were investigated by immunocytochemistry and flow cytometry. The carcinomas in situ expressed alpha 1-, alpha 2-, alpha 3-, alpha 6-, alpha v- and beta 1-subunits in variable amounts while being devoid of alpha 4, alpha 5, and beta 3. The individual integrin profile of the tumour in tissue was essentially maintained in vitro. However, a neo expression of the alpha 5 chain was found, together with an induction or increase in alpha 1, alpha 2, alpha 3, alpha v and beta 1 levels. No decrease in integrin subunit expression was observed. Standard-serum and serum-free medium revealed no striking differences in alpha- and beta-chain expression in the cell lines HT-29 and COLO 205. In serum-free medium, SW480 showed a slight increase of alpha 1 and alpha 5 and a decrease of alpha 3 and alpha v while SW620 expressed more alpha 1. We conclude that the great variability of adhesion receptor expression of the integrin family in colorectal carcinomas in situ is essentially maintained in vitro, although culture conditions which are only marginally influenced by serum factors unpredictably lead to some increase in expression or even induction of several integrin subunits.

Fibronectin and fibronectin-receptors of the integrin type in normal colon mucosa, adenoma and carcinoma.

Integrin alpha 3, alpha 4, alpha 5, and alpha v subunits heterodimerize with beta 1 and beta 3 chains to form functional fibronectin (FN) receptors. Two of them, alpha 4 beta 1 and alpha 5 beta 1, recognize FN as the only matrix molecule. Additional binding of laminin, collagen, and entactin is achieved by alpha 3 beta 1 while alpha v beta 1 and alpha v beta 3 also bind vitronectin. We investigated immunohistochemically the tissue distribution of FN and the expression of FN receptor alpha- and beta-chains in 20 normal colon tissues, 10 adenomas, 90 carcinomas, and 10 liver metastases derived thereof. In normal and adenomatous colon tissues FN was detected in association with reticular and fibrillar structures of the gut wall. The tumor stroma of carcinomas and liver metastases contained abnormally high amounts of FN which were detectable as chaotic deposits. Normal and adenomatous mucosa were alpha 3(+), alpha 4(-), alpha 5(-), alpha v(+/-), beta 1(+), beta 3(-) indicating that only promiscuous FN receptors were expressed. 21 of 90 carcinomas had a focal or complete loss of alpha 3 which was more often found in Dukes C/D tumors (p<0.005). The liver metastases more often had lower levels of alpha 3 expression compared to the primary tumor. The alpha v-expression was inconsistent and often weak. These losses of FN receptor constituents in carcinomas were not paralleled by any noticeable differences in stromal content or distribution pattern of FN, Enhanced expression/induction of FN receptors was found in only 2 of 90 carcinomas which focally neo-expressed the alpha 5-chain. This aberrant alpha 5 expression, however, was flow cytometrically found in 3/4 colon carcinoma cell lines which otherwise had receptor profiles corresponding to the in situ situation. FN-adherence of cell lines was variable. Anti-alpha 3 had no blocking effect on FN adherence of SW480, SW620, and COLO 205 but a minor effect on FN binding of HT-29. Anti-alpha 5 blocked FN adhesion whenever alpha 5 was expressed. Anti-av had no blocking effect while anti-beta 1 reduced FN adherence of all cell lines. The net biological effect(s) of aberrant expression of integrin type FN receptors in colon carcinomas is unpredictable as yet.