Current treatment of mantle cell lymphoma: results of a national survey and consensus meeting.

In most patients, mantle cell lymphoma (MCL) shows an aggressive clinical course with a continuous relapse pattern and a median survival of only 3-5 years. In the current study generation of the European MCL Network, the addition of high-dose Ara-C to R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-like regimen followed by myeloablative consolidation achieved a significant improvement of progression-free survival in younger patients. In elderly patients, rituximab maintenance led to a marked prolongation of remission duration. Emerging strategies include mammalian target of rapamycin (mTOR) inhibitors, proteasome inhibitors, immune modulatory drugs, Bruton's tyrosine kinase inhibitors and others, all based on the dysregulated control of cell cycle machinery and impairment of several apoptotic pathways. Combination strategies are currently being investigated in numerous trials, but their introduction into clinical practice and current treatment algorithms remains a challenge. In the current survey, the application of the molecular targeted compounds were collected and evaluated by a representative national network of 14 haematological institutions. Optimised strategies are recommended for clinical routine. Future studies will apply individualised approaches according to the molecular risk profile of the patient.

Triple antifungal therapy for severe systemic candidiasis allowed performance of allogeneic stem cell transplantation.

Systemic candidiasis is a rare but life threatening complication in immunosuppressed patients undergoing allogeneic SCT. Combination of new antifungal agents may improve outcome in this patient population. Here, triple anti-mycotic therapy is described in an relapsed ALL patient in urgent need of allogeneic bone marrow transplantation. The patient with T-cell acute lymphoblastic leukemia of thymic differentiation achieved remission after treatment according to the German ALL protocol 07/03. Two months after the consolidation therapy relapse occurred requiring high dose chemotherapy with allogeneic stem cell transplantation. One day after start of the conditioning regimen the patient showed skin manifestations typical for septic mycosis and blood cultures became positive for Candida krusei while on fluconazole prophylaxis. Because of the limited sensibility of fluconazole resistant candida species to liposomal amphotericin B and the high mortality rate in patients with systemic candidiasis, voriconazole was added immediately to liposomal amphotericin B. Since fever did not resolve and the conditioning therapy for allogeneic transplantation was not yet completed caspofungin was added. Skin manifestation responded to this triple anti-mycotic combination and peripheral blood stem cells from an unrelated donor were transplanted. With the triple antifungal therapy the patient finally became afebrile, skin manifestations showed complete resolution and blood cultures became negative. Three months after the onset of systemic candidiasis the patient was fully active with no signs of fungal infection and in haematological and molecular remission. Mycotic sepsis at the start of myeloablative conditioning therapy in heavily pretreated acute leukemia patients is usually considered as not allowing successful allogeneic transplantation. Thus this case demonstrates, that allogeneic stem cell transplantation is feasible in patients presenting with systemic candidiasis if combined antifungal therapy with liposomal amphotericin B, caspofungin and voriconazole is given.

An acquired G-CSF receptor mutation results in increased proliferation of CMML cells from a patient with severe congenital neutropenia.

Severe congenital neutropenia (CN) is characterized by a maturation arrest of myelopoiesis at the promyelocyte stage. Treatment with pharmacological doses of recombinant human granulocyte colony-stimulating factor (rh-G-CSF) stimulates neutrophil production and decreases the risk of major infectious complications. However, approximately 15% of CN patients develop myeloid malignancies that have been associated with somatic mutations in the G-CSF receptor (G-CSFR) and RAS genes as well as with acquired monosomy 7. We report a CN patient with chronic myelomonocytic leukemia (CMML) who never received rh-G-CSF. Molecular analysis demonstrated a somatic G-CSFR mutation (C2390T), which led to expression of a truncated G-CSFR protein in the CMML. Normal G-CSFR expression was unexpectedly absent in primary and cultured CMML. In addition, CMML cells showed monosomy 7 and an oncogenic NRAS mutation. In vitro culture revealed a G-CSF-dependent proliferation of CMML cells, which subsequently differentiated along the monocytic/macrophage lineage. Our results provide direct evidence for the in vivo expression of a truncated G-CSFR in leukemic cells, which emerged in the absence of rh-G-CSF treatment and transduces proliferative signals.

Successful autologous stem cell transplantation in a severely immunocompromised patient with relapsed AIDS-related B-cell lymphoma.

There is now evidence that the tolerability and response to systemic chemotherapy in HIV-infected patients with AIDS-related lymphoma (ARL) is significantly improved by highly active antiretroviral therapy. Here we report an severely immunocompromised AIDS patient with recurrent ARL who was successfully treated with autologous stem cell transplantation (ASCT). We also review the current literature of ASCT in HIV-infected patients.

Preclinical studies with Fc(gamma)R bispecific antibodies and granulocyte colony-stimulating factor-primed neutrophils as effector cells against HER-2/neu overexpressing breast cancer.

Immunotherapies directed to the proto-oncogene product HER-2/neu, which is overexpressed on a subset of breast and other carcinomas, currently receive considerable attention. We have investigated cell-mediated effector mechanisms of HER-2/neu antibodies against breast cancer cell lines. Compared to unfractionated control blood, whole blood from patients during granulocyte colony-stimulating factor (G-CSF) treatment exhibits significantly enhanced lysis (P < 0.001) of SK-BR-3 cells in the presence of HER-2/neu antibody 520C9. The extent of tumor cell killing correlated positively (r = 0.74) to polymorphonuclear neutrophil (PMN) blood counts. Fractionation of whole blood into plasma, mononuclear cells, and PMNs showed major killing capacity to reside in the granulocyte fraction. PMNs were efficiently cytolytic with a panel of HER-2/neu antibodies and against various breast cancer cell lines. Experiments with blocking antibodies to Fc(gamma)R documented Fc(gamma)RII (CD32) as the major trigger molecule for monoclonal antibody 502C9-mediated cytotoxicity. Killing via 520C9 was significantly influenced by an allotypic polymorphism of Fc(gamma)RIIa, the CD32 molecule expressed on PMNs. In reverse antibody-dependent cell-mediated cytotoxicity experiments with a panel of HER-2/neu-directed bispecific antibodies, Fc(gamma)RIII (CD16) proved to be an efficient trigger molecule in blood from healthy volunteers. During G-CSF treatment, however, Fc(gamma)RI (CD64)-expressed on monocytes and G-CSF primed, but not on healthy donor PMNs-became the predominant cytotoxic trigger molecule. Thus, G-CSF application increased effector cell numbers for HER-2/neu-directed immunotherapy, and G-CSF primed PMNs proved particularly effective with a [HER-2/neu x Fc(gamma)RI] bispecific antibody. These findings support clinical trials with HER-2/neu-directed antibodies in combination with G-CSF in breast cancer patients overexpressing HER-2/neu.

Rapid isolation of chromosomal breakpoints from patients with t(4;11) acute lymphoblastic leukemia: implications for basic and clinical research.

Chromosomal translocations t(4;11)(q21;q23) are associated with a group of acute lymphoblastic leukemias with very poor prognosis. From the complete sequences of the breakpoint cluster regions of the human MLL and AF-4 translocation partner genes, a novel set of 66 oligonucleotides that facilitates the rapid identification of translocation breakpoints by PCR analysis of genomic DNA was designed. For each breakpoint, a pair of optimally snited primers can be assigned, which improves the monitoring of the disease during treatment. Comparison of the breakpoints with the corresponding parental sequences also contributes to our better understanding of the illegitimate recombination events leading to these translocations.

Evaluating antibodies for their capacity to induce cell-mediated lysis of malignant B cells.

Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells, mononuclear cells and polymorphonuclear (PMN) cells from healthy donors were compared with Fc gammaRI (CD64)-expressing PMN cells from patients receiving granulocyte colony-stimulating factor (G-CSF) treatment. Of the 29 IgG workshop antibodies binding most strongly to the tested malignant human B-cell lines, only 3 consistently induced target cell lysis. These three antibodies were determined to be HLA DR reactive. Experiments with a panel of HLA class II antibodies showed the involvement of individual Fc gamma receptors on effector cells to be strongly dependent on the antibody isotype. We then compared killing mediated by chimeric IgG1 antibodies with that from Fc gammaRI-directed bispecific antibodies, targeting classical HLA class II, or the Lym-1 and Lym-2 antigens. The latter two are variant forms of HLA class II, which are highly expressed on the surface of malignant B cells but which are found only at low levels in normal cells. With blood from G-CSF-treated donors, bispecific antibodies showed enhanced killing compared to their chimeric IgG1 derivatives, because they were more effective in recruiting Fc gammaRI-expressing PMN cells. G-CSF- and Fc gammaRI-directed bispecific antibodies to HLA class II, therefore, seem to be an attractive combination for lymphoma therapy.

Azacitidine in combination with intensive induction chemotherapy in older patients with acute myeloid leukemia: The AML-AZA trial of the Study Alliance Leukemia.

DNA methylation changes are a constant feature of acute myeloid leukemia. Hypomethylating drugs such as azacitidine are active in acute myeloid leukemia (AML) as monotherapy. Azacitidine monotherapy is not curative. The AML-AZA trial tested the hypothesis that DNA methyltransferase inhibitors such as azacitidine can improve chemotherapy outcome in AML. This randomized, controlled trial compared the efficacy of azacitidine applied before each cycle of intensive chemotherapy with chemotherapy alone in older patients with untreated AML. Event-free survival (EFS) was the primary end point. In total, 214 patients with a median age of 70 years were randomized to azacitidine/chemotherapy (arm-A) or chemotherapy (arm-B). More arm-A patients (39/105; 37%) than arm-B (25/109; 23%) showed adverse cytogenetics (P=0.057). Adverse events were more frequent in arm-A (15.44) versus 13.52 in arm-B, (P=0.26), but early death rates did not differ significantly (30-day mortality: 6% versus 5%, P=0.76). Median EFS was 6 months in both arms (P=0.96). Median overall survival was 15 months for patients in arm-A compared with 21 months in arm-B (P=0.35). Azacitidine added to standard chemotherapy increases toxicity in older patients with AML, but provides no additional benefit for unselected patients.

Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23).

We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;ll)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains five simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5'-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX differs significantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79-84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.

A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells.

Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.

A new fingerprint method for sequence analysis of chromosomal translocations at the genomic DNA level.

Chromosomal rearrangements constitute a significant feature of leukemogenesis and malignant transformation in general. Nucleotide patterns in the immediate vicinity of the break point may provide important information about the underlying causalities, eg illegitimate recombination events mediated by topoisomerase II, Alu repeats, or VDJ recombinase. In order to facilitate the determination of those DNA patterns, we developed a new fingerprint approach. In a first step, two DNA fragments were independently amplified by long distance PCR: the genomic region carrying the break point and the normal nonrearranged counterpart. Subsequently, both PCR products were digested with restriction enzymes, end-labelled with a fluorescent dye, and subjected to high resolution polyacrylamide gel electrophoresis. By comparing the restriction patterns of the rearranged and the nonrearranged PCR fragments, the break points could be easily localized within a size range coverable by a single sequencing reaction. Finally, the exact DNA sequence across the break point was directly determined. The 'fingerprint' technique is fast, reliable and enables the assay of multiple samples in parallel.

Molecular analysis of MLL-1/AF4 recombination in infant acute lymphoblastic leukemia.

We examined ten cases of acute lymphoblastic leukemia (ALL) in infants (less than 1 year of age) by RT-nested PCR for a MLL-1/AF4 rearrangement. Five patients revealed a positive result. The specific PCR product differed in size from approximately 380-670 bp indicating various splicing variants in the MLL-1/AF4 rearrangement. Three patients had a fusion between exon 6 of the MLL-1 gene and codon 362 of the known AF4 cDNA sequence. Moreover, in two patients more than one specific PCR product was detected, possibly due to alternative splicing. In the first case, sequencing of these products revealed a hybrid mRNA consisting of MLI-1 exon 7 or exon 8, respectively, fused to the AF4 gene at codon 348. In the second case with alternative splicing, again, exon 7 or 8 of the MLL-1 gene were fused to the AF4 gene as in case 1. The AF4 sequence involved in this patient, however, started at codon 362. The AF4 break was, therefore, identical to the three MLL-1/AF4 positive patients as described above. Moreover, we investigated all ten patients for the reciprocal mRNA transcript AF4/MLL-1 by a similar PCR approach. In none of these patients, including the five MLL-1/AF4 positive cases was a specific PCR product obtained. However, in the MV411 cell line bearing a t(4;11), which served as a positive control in our MLL-1/AF4-PCR assay, the reciprocal AF4/MLL-1 mRNA was detected. Our results indicate that a MLL-1/AF4 rearrangement occurs in about 50% of infants with ALL. In contrast, the reciprocal hybrid mRNA can only rarely be detected, if at all.

Rapid synthesis of hybrid RNA molecules associated with leukemia-specific chromosomal translocations.

A number of gene arrangements have been described as characteristic abnormalities associated with different types of leukemia, and this list is still growing. In view of the biological, clinical and prognostic relevance of the pathological fusion products, techniques permitting their detection are of paramount importance in the clinical setting. In some instances, permanent leukemic cell lines carrying the abnormality of interest are available for the establishment and standardization of molecular assays. For a number of newly discovered gene rearrangements, however, this may not be the case. It is therefore of great interest for clinical laboratories to have alternative technical possibilities for the set-up of standardized molecular tests. This problem provided the stimulus to design a simple and rapid method for in vitro generation of chimeric RNA molecules corresponding to pathological fusion transcripts typical for chromosomal translocations in leukemias. Two separate fragments are generated in a four-primer multiplex PCR. Due to a PCR-generated overlap, a chimeric fragment can be synthesized in a second round of PCR. This PCR product is then purified with the help of magnetic beads. Due to the SP6 promotor sequence incorporated during the second round of PCR, transcription into RNA is easily facilitated while the template DNA is still bound to the solid phase. Following this strategy we were able to synthesize the fusion transcripts m-BCR/ABL, CBF beta/MYH11, and MLL/AFp1 which are the molecular equivalents of t(9;22)(q34,q11), inv16(p13;q22) and t(1;11)(p32;q23), respectively. The chimeric RNA will be useful as a control template in diagnostic RT-PCR strategies. It can also be further processed in translation systems leading to the corresponding chimeric oncoprotein. This approach can be easily used to create any hybrid RNA of interest.

Detection of four different 11q23 chromosomal abnormalities by multiplex-PCR and fluorescence-based automatic DNA-fragment analysis.

A nested polymerase chain reaction (PCR) protocol was developed for rapid detection of four different 11q23 abnormalities by a single PCR assay. During each of the two PCR rounds a sense primer located within exon 5 of the MLL gene at 11q23 was combined with four different antisense primers, each located within possible translocation partner genes at chromosomes 4, 6, 9, and 19, respectively. Except for the MLL primer all primers used during the second round of nested-PCR carried a characteristic fluorescence label at their 5'-end. Agarose gel analysis of the PCR products was sufficient to discriminate between the absence of any of the four MLL rearrangements and the presence of at least one of them. Discrimination of the four different MLL translocation partner genes was not possible by agarose gel analysis due to a molecular heterogeneity of the 11q23 breakpoints resulting in PCR products of variable size. For this reason, automatic fluorescence-based DNA-fragment analysis was used to exactly define the MLL translocation partner genes if a positive result had been obtained by agarose gel analysis. In patients with leukemia, this assay may enable a fast and highly sensitive detection of different 11q23 abnormalities, which usually correlate with poor clinical prognosis.

Incidence and clinical outcome of children with BCR/ABL-positive acute lymphoblastic leukemia (ALL). A prospective RT-PCR study based on 673 patients enrolled in the German pediatric multicenter therapy trials ALL-BFM-90 and CoALL-05-92.

A variety of oncogenes are activated by specific chromosomal translocations, which are associated with distinct subtypes of leukemia. The identification of these rearrangements provides critical diagnostic and prognostic information, which may contribute to the selection of specific anti-leukemic therapy. The translocation t(9;22), the equivalent of the BCR/ABL rearrangement, is associated with a poor prognosis. We therefore used RT-PCR to detect this molecular event in a prospective study including 890 children. 673 of them suffered from acute lymphoblastic leukemia (ALL) at primary diagnosis and a transcription of the chimeric gene was detected in 21 of 648 with a successful analysis (3.2%). All children were treated by one of the two German multicenter childhood ALL therapy studies ALL-BFM-90 or COALL-05-92, respectively. Comparison of clinical features between BCR/ABL-positive and -negative children showed no significant differences regarding WBC, percentage of blasts, splenomegaly, hepatomegaly and age. Immunophenotypic studies at diagnosis in 21 BCR/ABL-positive children identified common ALL in 16 patients (76.2%), pre-B-ALL in four (19.0%), and an early T-lineage ALL in one (4.8%). Coexpression of myeloid antigens (CD13 and/or CD33) was observed in six of 16 common ALL patients as well as in the one child with early T-lineage ALL phenotype. The type of breakpoint (m-BCR/ABL: n = 14; M-BCR/ABL: n = 7) showed no correlation with clinical parameters. A comparison of cytogenetic and molecular data was performed in 16 positive patients and was concordant in all of them. We analyzed the response to the prednisone pretreatment and found a higher incidence of poor responders among the BCR/ABL-positive children. Regarding the event-free survival (EFS) of BCR/ABL-positive (0.53) and -negative patients (0.79) after a follow-up of 2 years, significant differences (P < 0.05) between both groups could be demonstrated.

Leptin and neuropeptide Y gene expression in human placenta: ontogeny and evidence for similarities to hypothalamic regulation.

The objective of the present study was to examine the impact of preeclampsia on the relation of leptin and neuropeptide Y (NPY) gene expression in human placenta. A second goal was to monitor the change of leptin messenger RNA (mRNA) with increasing gestational age. Placental tissue was obtained from 17 premature deliveries, 18 term deliveries, and 10 mothers with preeclampsia. Gene expression of leptin, NPY, and two housekeeping genes (beta-actin and glyceraldehyde-3-phosphate dehydrogenase was quantified using real-time PCR. The leptin/beta-actin mRNA ratio was significantly higher in specimens of patients with preeclampsia than in those of gestational age-matched controls (0.63+/-0.23 vs. 0.09+/-0.04 relative U (RU); P = 0.03). NPY/beta-actin mRNA was significantly reduced in the preeclampsia group (0.003+/-0.001 vs. 0.026+/-0.008 RU in controls; P = 0.01). The NPY/leptin ratio was 0.11+/-0.09 for preeclamptic placenta samples and 1.7+/-0.6 RU for the controls (P = 0.02). The leptin/beta-actin ratio was significantly lower in placenta from premature deliveries than in term deliveries (0.02+/-0.004 vs. 0.12+/-0.05 RU; P = 0.01). Similar results were obtained for normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA. Our data suggest an increase of placental leptin production with gestational age. In patients with preeclampsia, elevated leptin expression goes along with suppressed NPY expression. This resembles hypothalamic regulation.

Flow cytometric discrimination between viable neutrophils, apoptotic neutrophils and eosinophils by double labelling of permeabilized blood granulocytes.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) is frequently used to detect apoptotic cells in tissues, cytospins and suspensions. Here we show that TUNEL staining of freshly isolated granulocytes results in non-specific positivity of a distinct population, which can be observed in the presence or absence of TdT. The morphological features of the false-positive cells examined in fluorescence microscopy suggest that the non-specifically stained cells are eosinophilic granulocytes. Granules of eosinophilic granulocytes were brightly stained by non-specific TUNEL reaction independent of TdT. This staining does not, therefore, indicate apoptosis and most likely reflects 'stickiness' of the permeabilized eosinophils. Immunofluorescence with phycoerythrin (PE)-labelled CD16 antibodies performed simultaneously with conventional TUNEL staining confirmed that the false-positive cells in TUNEL staining were CD16-negative eosinophils. In this report we describe a new procedure that allows: (i) the differentiation of neutrophilic and eosinophilic granulocytes in forward scatter versus log side scatter histograms after permeabilisation; (ii) the reliable discrimination between viable neutrophils, apoptotic neutrophils and eosinophilic granulocytes in cytofluorimetry.

Mechanisms of G-CSF- or GM-CSF-stimulated tumor cell killing by Fc receptor-directed bispecific antibodies.

Studies with gene-modified mice have recently reinforced the importance of Fc receptor-mediated effector mechanisms for the therapeutic efficacy of rituxan and herceptin - two clinically approved antibodies for the treatment of tumor patients. We investigated Fc receptor-dependent tumor cell killing by mononuclear and granulocytic effector cells - comparing human IgG1 antibodies against CD20 or HER-2/neu with their respective FcgammaRI (CD64)-, FcgammaRIII (CD16)-, or FcalphaRI (CD89)-directed bispecific derivatives. With blood from healthy donors as effector source, human IgG1 and FcgammaRIII (CD16)-directed bispecific antibodies proved most effective in recruiting mononuclear effector cells, whereas tumor cell killing by granulocytes was most potently triggered by FcalphaRI-directed bispecific constructs. Granulocyte-mediated tumor cell lysis was significantly enhanced when blood from G-CSF- or GM-CSF-treated patients was investigated. Interestingly, however, both myeloid growth factors improved effector cell recruitment by different mechanisms, which were furthermore dependent on the tumor target antigen, and on the selected cytotoxic Fc receptor.

Construction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR.

The exponential character of PCR amplification may compromise quantitative assays because it multiplies minor sample-to-sample variations. To overcome these problems, several authors have used recombinant standard DNA or RNA molecules to be spiked into the samples in a dilution series of known copy numbers before co-amplification by PCR. To obtain an equal efficacy of reverse transcription and PCR amplification, standard and template molecules should be highly homologous. However, the limited resolution of commonly used agarose gel electrophoresis requires rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR. Due to a much higher resolution, automatic post-PCR analyzing systems based on laser-induced fluorescence may help to overcome these difficulties. For using the capabilities of these systems in quantitative competitive RT-PCR, we developed a protocol to construct recombinant RNA standard molecules that only differ from the target sequence by a small deletion of 8 nucleotides. It is based on PCR-induced mutagenesis and solid-phase in vitro transcription. This protocol was applied to quantify multidrug resistance gene (MDRI) mRNA in malignant cells, but it can easily be adapted to any gene of interest.

Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation.

UNLABELLED: Conjugation chemistry and kit formulated binding of the NHS ester of 6-(4'-(4"-carboxyphenoxy)butyl)-2, 10-dimercapto-2,10-dimethyl-4,8-diazaundecane (NHS-BAT ester) to monoclonal antibodies (MAbs) was investigated. The functionalities of the resulting BAT conjugated and 99mTc-labeled MAbs BW 431/26, MAb 425 and bispecific MDX210 (fragment construct) were tested by immunoreactivity and immunoscintigraphy. METHODS: The kinetics and chemistry of the conjugation reaction were monitored by high-performance liquid chromatography, size-exclusion chromatography and positive fast-atom-bombardment mass spectra (FAB-MS). The 99mTc BAT-MAbs were tested with various immunoreactivity assays. The biodistribution of 99mTc-BAT-BW 431/26 in rats was compared with directly labeled BW 431/26. RESULTS: At pH 8.5 and 25 degrees C, the reactivity of the NHS-BAT ester was high with 90% completion after 30 min. The conjugation yield of 19 microM MAb and 228 microM NHS-BAT ester amounted to 30%. Higher NHS-BAT ester concentrations afforded higher BAT-to-MAb ratios. According to FAB-MS, the conjugation competing hydrolysis surprisingly occurred at the NHS ring. Almost quantitative 99mTc labeling was achieved after 5 min at 25 degrees C. Immunoreactivity of the 99mTc-BAT antibodies showed > 90% recovery and proved to be insensitive to BAT-to-MAb ratios of up to 10. The 99mTc-BAT-BW 431/26 showed similar organ distribution but revealed less urinary excretion compared with the directly labeled BW 431/26. Immunoscintigraphy with 99mTc-labeled and BAT-BW 431/26 and BAT-MAb 425 showed the respective biological function in vivo. CONCLUSION: According to straightforward conjugation chemistry, the ease of 99mTc labeling and the application of a simple ultrafiltration technique, the NHS-BAT ester represents a nondestructive, universally applicable biofunctional ligand to introduce stable 99mTc protein binding sites. Kit formulated conjugation/labeling can be performed with little time requirements and laboratory experience.