Gunther Bastert

Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients

Previous studies with animal tumors showed that bone marrow (BM) is a privileged site where potentially lethal tumor cells are controlled in a dormant state by the immune system. Here, we investigated BM of breast cancer patients with respect to tumor cell content, immune activation status and memory T‐cell content. BM‐derived cells from primary operated breast cancer patients (n = 90) were compared with those from healthy donors (n = 10) and also with cells from respective blood samples. Cytokeratin 19‐positive tumor cells were detected by nested polymerase chain reaction. Three‐color flow cytometry was used to identify numbers and activation state of T cells, natural killer (NK) cells, monocytes/macrophages and subsets by a panel of monoclonal antibodies (mAbs). The proportion of memory T cells among the CD4 and CD8 T cells was much higher in BM of cancer patients than in healthy donors (p < 0.001). The extent of memory T‐cell increase was related to the size of the primary tumor. Patient‐derived BM memory CD8 T cells could be shown to contain specific HLA‐A2/Her‐2/neu369–377 tetramer binding cells. Patients with disseminated tumor cells in their BM had more memory CD4 T cells and more CD56+ CD8+ cells than patients with tumor cell‐negative BM. Only some of the immunological changes seen in BM samples of cancer patients were also detectable in peripheral blood samples. Our hypothesis that BM is a special compartment for immunological memory and tumor dormancy is supported by the above findings. The overall results reveal that BM is a valuable additional compartment for immune diagnosis in pathological conditions and possibly for follow‐up treatment strategies.

Her-2/neu Gene Amplification and Protein Expression in Primary Male Breast Cancer

The role of HER-2/neu in male breast cancer is not well defined. The purpose of the current study was to measure the frequency of HER-2/neu expression in primary male breast cancer, to demonstrate HER-2/neu gene amplification in cases found to be positive for protein overexpression, and to correlate HER-2/neu positivity with clinicopathological variables. Formalin-fixed, paraffin-embedded archival material from 99 primary male breast carcinomas was evaluated by immunohistochemistry (IHC) using the HercepTest™ (DAKO Corp., Hamburg, Germany). Scoring was performed according to established guidelines. All cases demonstrating HER-2/neu staining by IHC (1+/2+ and 3+) were analyzed for HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) utilizing the PathVysion™ assay (Vysis Corp., Downers Grove, Illinois) to assess HER-2/neu amplification status. The immunohistochemical staining of the HER-2/neu protein revealed HER-2/neu positivity in 15/99 (15.1%) cases, eight tumors showed 2+ and 7 tumors 3+ staining. HER-2/neu gene amplification was observed in 11/99 cases (11,1%), and all of the 3+ and 4/8 from the 2+ cases were amplified. HER-2/neu gene amplification/protein overexpression did not correlate with tumor state, histological grade or estrogen/progesterone receptor status nor the axillary lymph node status. This is the first comprehensive study of HER-2/neu gene amplification by FISH analysis in primary male breast cancer. Compared to female primary breast cancer the percentage of HER-2/neu positivity in our study was lower. Our data provide first evidence for HER-2/neu gene amplification in male breast cancer. Further studies should be addressed on the potential application of the monoclonal rhuMAB HER-2/neu antibody for treatment of HER-2/neu positive male breast cancer.

Breast cancer in young women (≤35 years): Genomic aberrations detected by comparative genomic hybridization

Sporadic breast cancer in young women is different from the one in older patients regarding pathological features and aggressiveness of the tumors, but the spectrum of genetic alterations are largely unknown. We used comparative genomic hybridization (CGH) to analyze DNA copy number changes in 88 tumor samples from women ≤35 years of age. Findings were compared to histopathological data including tumor type, grading, lymph nodes and metastasis. Genomic gains clustered to chromosome arms 1q (64.8%), 8q (61.4%), 17q (50.0%), 20q (33.0%), 3q (20.5%), 1p (17.0%), 5p (17.0%) and 15q (17%). Losses were commonly located on 8p (19.3 %), 11q (11.4%), 16q (11.4%), 17p (11.4%) and 18q (10.2%). A comparison with published CGH data from breast carcinomas of similar type and grade showed the following differences: (1) gains were much more frequent than losses, and (2) losses on 8p22–p23 were more prevalent in patients with positive lymph node metastasis (p = 0.02), and Grade III tumors were associated with gains on the long arm of chromosome 8 (p = 0.01). Therefore, alterations in these genomic regions may be responsible for the reduced survival of patients with early onset breast cancer.

Bisphosphonates and the prevention of metastasis

Bisphosphonates have been used successfully for many years in the treatment of hypercalcemia and to reduce skeletal‐related complications of metastases. In the first years of bisphosphonate use, the efficacy of these substances was thought to lie purely in the inhibition of osetoclasts. However, there is recent evidence to suggest that an antitumor effect also may play a role. As well as having an apoptotic and antiproliferative effect on osteoclasts, bisphosphonates may exert a similar influence on macrophages and tumor cells.

The effect of Leboyer delivery on blood viscosity and other hemorheologic parameters in term neonates

OBJECTIVEnThis study was done to compare postnatal alterations in blood viscosity, hematocrit value, plasma viscosity, red blood cell aggregation, and red blood cell deformability in term neonates undergoing both early umbilical cord clamping and delivery according to the Leboyer method.nnnSTUDY DESIGNnThe umbilical cords of 15 healthy, term infants were clamped within 10 seconds of birth (early cord clamping), and 15 infants delivered according to the Leboyer method were placed on the mothers abdomen, and the umbilical cords were clamped 3 minutes after birth. Hemorheologic parameters were studied in umbilical cord blood at 2 hours, 24 hours, and 5 days from the time of delivery.nnnRESULTSnThe residual fetal placental blood volume decreased from 45 +/- 8 ml/kg (x +/- SD) after early cord clamping to 25 +/- 5 ml/kg after delivery by the Leboyer method. After Leboyer-method delivery, the hematocrit value rose from 48% +/- 5% at birth to 58% +/- 6% 2 hours after delivery, 56% +/- 7% at 24 hours, and 54% +/- 8% after 5 days. Blood viscosity in the Leboyer-method group increased by 32% within the first 2 hours but did not change significantly during the following 5 days. Plasma viscosity, red blood cell aggregation, and red blood cell deformability were not affected by the mode of cord clamping.nnnCONCLUSIONSnDelivery by the Leboyer method leads to a significant increase in blood viscosity as a result of increasing hematocrit value, whereas other hemorheologic parameters are similar to those of infants with early cord clamping.

Efficient engraftment of human primary breast cancer transplants in nonconditioned NOD/Scid mice

We describe a new human tumor xenotransplant animal model that is highly efficient for engraftment, does not need host conditioning and is suitable for in vivo studies of human tumors. Pieces of 61 freshly operated primary breast tumors were implanted into 172 irradiated and 228 nonconditioned NOD/Scid mice. A high mortality was observed in irradiated but not in nonconditioned recipients. More than 90% of analyzed implanted breast cancer specimens engrafted in the NOD/Scid mice irrespective of pretreatment. The tumors were vascularized within 3 days of implantation and maintained original histomorphology as well as expression patterns of tumor markers (cytokeratin and MUC1) and cytokines (tumor necrosis factor alpha (TNF‐α), interleukin‐4 (IL‐4) and IL‐10) released by adjacent stromal cells. A majority of tumors grew slowly, locally infiltrating host tissue, whereas some grew aggressively, developing large, fatal tumor masses and metastases within regional lymph nodes. Tumor progression in mice correlated with stage, grade, proliferation index and hormone receptor status of primary tumors. The reproducible growth behavior and preservation of characteristic features suggest that this new xenotransplant model is relevant and can be recommended for testing new anticancer therapies.

Sensitive detection of micrometastases in bone marrow from patients with breast cancer using immunomagnetic isolation of tumor cells in combination with reverse transcriptase/polymerase chain reaction for cytokeratin-19.

Abstract We report a highly sensitive method to detect rare human breast cancer cells, which combines an immunomagnetic separation (IMS) using antibody BM2 against MUC-1 with cytokeratin-19 (CK19) and the reverse transcriptase/polymerase chain reaction (RT-PCR). The IMS-RT-PCR technique allows the detection of 1 tumor cell/107–108 mononuclear cells. This is at least ten times more sensitive than CK19 RT-PCR alone, or immunocytochemistry. All 117 peripheral blood and 8 bone marrow samples obtained from healthy donors as negative controls were positive for β2-microglobulin by RT-PCR but negative for CK19 by IMS-RT-PCR or RT-PCR alone. Out of 26 bone marrow samples from breast cancer patients, 18 had CK19 transcripts detectable by IMS-RT-PCR. In contrast, only 14 and 13 samples from the 26 were found to be positive by RT-PCR alone or by routine immunocytochemical staining. In conclusion, IMS-RT-PCR for CK19 is a highly sensitive and specific method for detecting very low numbers of micrometastatic breast cancer cells in bone marrow amidst an excess of non-malignant cells. For the early diagnosis of disseminating disease, this assay is more efficient than RT-PCR alone and routine immunocytochemistry.

Effects of Leboyer childbirth on left- and right systolic time intervals in healthy term neonates

The Leboyer birth method requires that the newborn infant is placed on the mothers abdomen and the cord is clamped when it stops pulsating. Since late cord-clamping may result in marked hypervolemia and polycythemia of the neonate, we studied right and left ventricular systolic time intervals by means of pulsed-Doppler echocardiography. Left and right ventricular preejection periods (LPEP, RPEP), right time peak velocity (RTPV), left and right ventricular ejection times (LVET, RVET), and ratio of RTPV/RVET(c) corrected for heart rate were studied in 15 fullterm neonates with early (< 10s) cord clamping and in 15 fullterm neonates delivered according to Leboyer (cord clamping after 3 min) on day 1 (2-4 h after birth) and day 5. After Leboyer birth hematocrit was significantly increased on day 1 (0.61 +/- 0.06 vs. 0.53 +/- 0.07) and on day 5 (0.57 +/- 0.02 vs. 0.50 +/- 0.07). Blood pressure was similar in both groups and increased by about 10% from day 1 to day 5. LVET and RVET were not affected by the mode of placental transfusion, thereby suggesting normal left and right ventricular function after Leboyer birth. The LPEP/LVET (0.36 +/- 0.09 vs. 0.30 +/- 0.08) and RPEP/RVET ratio (0.41 +/- 0.11 vs. 0.33 +/- 0.08) were significantly higher in the Leboyer group (p < 0.05) compared to the early cord clamped group suggesting higher systemic and pulmonary resistance. RPEP decreased significantly by 17% in the control group from day 1 to day 5 (p < 0.05), but did not change in the Leboyer group. In the Leboyer group RPEP/RVET ratio decreased significantly from day 1 to day 5, whereas the control values did not change during the first five days. RTPV:RVET(c) is inversely related to pulmonary artery pressure. A normal ratio is > 0.35, or greater. Mean ratio of RTPV : RVET(c) was significantly lower in the Leboyer group (0.31 +/- 0.08) on day 1 compared to the control group (0.41 +/- 0.09; p < 0.05), but did not differ on day 5. The results suggest that Leboyer delivery was associated with transiently increased pulmonary and systemic resistance, whereas right and left ventricular functions were not affected. This may be explained by increased blood viscosity due to increased hematocrit.

Analysis of sensitivity and specificity of cytokeratin 19 reverse transcriptase/polymerase chain reaction for detection of occult breast cancer in bone marrow and leukapheresis products

Purpose: The work aimed to evaluate the sensitivity and specificity of the cytokeratin (CK) 19 reverse transcriptase/polymerase chain reaction (RT-PCR) for the detection of occult breast cancer in bone marrow and leukapheresis products. Materials and methods: Peripheral blood and bone marrow samples, obtained from 96 and 8 healthy donors respectively, served as negative controls. A total of 115 bone marrow samples and 29 leukapheresis samples from routine patients with breast cancer were analysed by CK19 RT-PCR. The PCR results were compared with those from routine immunocytology for CK8, 18, 19. Results: The CK19 RT-PCR technique with primer pairs from Datta et al. (J Clin Oncol 12: 475–482, 1994), using an annealing temperature of 72°C, allowed the detection of one tumour cell in 107 mononuclear cells. None of the control samples (96 peripheral blood and 8 bone marrow) that were positive for β2-microglobulin by RT-PCR showed a signal for CK19. However, expression of CK19 mRNA was observed in 40.87% (70/115) of bone marrow and in 24.13% (7/29) of leukapheresis samples of patients with breast cancer. Standard immunocytology and PCR were combined for the detection of tumour cells. Five of the 65 bone marrow samples were found to be positive by CK19 RT-PCR, but were negative with the immunocytology method. Conclusion: RT-PCR using CK19-specific primers and optimal experimental conditions is a reliable and specific method for the detection of micrometastatic breast cancer cells.

Evaluating GA733-2 mRNA as a marker for the detection of micrometastatic breast cancer in peripheral blood and bone marrow

Abstract The GA733-2 gene encodes the epithelial glycoprotein 40, a homophilic cell-cell adhesion molecule, which is expressed on the surface of epithelial cells and associated with a variety of carcinomas, e.g. breast, colorectal and lung carcinomas. To test if it could serve as a tumor marker, we have analysed the expression of GA733-2 in bone marrow (BM) and peripheral blood (PB) from healthy donors, and of patients with haematological malignancies or breast cancer using reverse transcriptase-polymerase chain reaction (RT-PCR). The GA733-2 nested PCR was positive in 100% (8/8) of BM and 40% (16/40) of PB from healthy donors, in 100% (33/33) of BM from patients with breast cancer who had no evidence of distant metastases and also in BM and PB from patients with haematological malignancies. GA733-2 mRNA is not specific as a marker for the detection of breast cancer cells in BM and PB.