R.A. de Weger

Current technologies for HER2 testing in breast cancer

BACKGROUND Molecular techniques play an increasingly important role in breast cancer detection and help in the prediction of prognosis and treatment response. HER-2/neu predicts the sensitivity of breast tumors to trastuzumab and lapatinib. Presently there are several ways to assess HER2 status at the protein level (e.g. ELISA), at the RNA level (RT-PCR, microarray) and at the DNA level (fluorescence in situ hybridization, chromogenic in situ hybridization (ISH), silver in situ hybridization or multiplex ligation-dependent probe amplification). DESIGN This paper provides an overview of new developments in HER2 testing. RESULTS Although these techniques correlate well in comparative studies, discrepancies remain. Each technique has its own (dis)advantages and thus there is no real gold standard. Not surprisingly, there is no consensus at present on which of the protein- or gene-based techniques is superior, on the use of mono- or duo-probe ISH systems, nor on the use of manual or fully-automated staining- and scoring systems. CONCLUSION Until large clinical trials clearly point out one strategy as the best predictive one for trastuzumab response, the choice for a testing strategy will probably be based on local preferences which consider both practical and economic issues. Standardization, proper internal and external quality control assessment, laboratory accreditation and automation of tissue processing (autostainers) and interpretation methods (image analysis) will play an increasingly important role in HER2 testing.

Donor leukocyte infusions for recurrent hematologic malignancies after allogeneic bone marrow transplantation: impact of infused and residual donor T cells

We evaluated the efficacy and toxicity of different doses of donor T cells given with donor leukocyte infusions (DLI) as treatment for relapse of various hematologic malignancies after allogeneic bone marrow transplantation (BMT). We also studied whether DLI treatment was more effective if circulating T cells were exclusively of donor origin (complete donor T cell chimeras) as compared with T cells originating from both donor and recipient (mixed T cell chimeras). Twenty-eight patients were studied of whom 24 had a complete donor T cell chimerism. The malignancies were as follows: chronic myeloid leukemia (CML) in chronic phase (CP) (n = 9); more advanced CML (n = 5); multiple myeloma (MM) (n = 5); acute leukemia (AL) (n = 9). T cell doses varied from 0.1 × 107 to 33 × 107 T cells/kg. Eight patients received two to four DLI courses because they failed to respond to one course. Thirteen of 14 patients with CML, including four patients with more advanced CML, achieved complete remission (CR). All five patients with MM responded, including three CRs. Six patients (three with CML, three with MM) responded only after two to four DLI courses. Patients with CML-CP were likely to respond to as few as 1 × 107 T cells/kg whereas patients with MM generally responded when they received ⩾10 × 107 T cells/kg. However, despite the infusion of high T cell doses (up to 32 × 107 T cells/kg), practically all patients with AL failed to respond. The likelihood of response was strongly related to the occurrence of graft-versus-host disease (GVHD) in patients with CML and MM (P = 0.0002), although GVHD was not helpful for patients with AL. Higher T cell doses (⩾10 × 107/kg) induced serious GVHD (n = 17) and marrow aplasia (n = 5), and GVHD was directly or indirectly the cause of death for six patients. Finally, there were no obvious differences in responses between complete donor T cell chimeras and mixed T cell chimeras.

Localization in situ of costimulatory molecules and cytokines in B-cell non-Hodgkin's lymphoma.

Costimulatory molecules are essential in cognate interactions between T and B lymphocytes. To study the prerequisites of functional interactions between malignant B cells and intermingled T cells in B‐cell non‐Hodgkin’s lymphomas (B‐NHL), we examined the expression of CD40, CD80 and CD86 and their ligands CD40 ligand (CD40L, CD154), CD28 and CTLA4 (CD152) using immunohistochemistry and confocal laser scanning microscopy. Almost all mucosa‐associated lymphoid tissue (MALT) NHL were positive for CD40 and CD80 and in nine out of 14 cases were positive for CD86. The majority of follicle centre cell lymphomas (FCCL) expressed CD40, but were heterogeneous in their expression of CD80 and CD86. Most diffuse large cell lymphomas (DLCL) were CD80+, but lacked expression of CD86. These patterns reflect the differences in phenotype of normal marginal‐zone B cells (as counterparts of MALT NHL) and germinal centre cells (as counterparts of FCCL and DLCL). Counter‐receptors on T cells were detectable in 13 of 14 MALT NHL, 12 of 16 FCCL but only occasionally in DLCL (three of 12 cases). A subgroup of FCCL was identified with T‐cell expression of CD40L, CD28 and CTLA4 simultaneously with strong expression of CD40 and CD86 on the tumour B cells. These results indicate that MALT NHL and a subset of FCCL are most optimally equipped for functional interactions with T cells. This may be supported by the demonstration of cytokine production – mainly in T cells – in MALT NHL [interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ), IL‐10] and FCCL (IL‐2, IFN‐γ) and to a lesser extent in DLCL.

Chimerism analysis within 6 months of allogeneic stem cell transplantation predicts relapse in acute myeloid leukemia

The role of chimerism analysis as a prognostic indicator of relapse after hematopoietic stem cell transplantation (SCT) is controversial. We monitored chimerism status by short tandem repeat-based polymerase chain reaction (PCR) in T- and non-T-cell subsets and retrospectively evaluated clinical outcome in 96 patients with acute myeloid leukemia after myeloablative (MA) or reduced-intensity conditioning SCT. Fifty-six percent of 80 patients in the MA group demonstrated complete donor chimerism (CC) at all time points, whereas 6% had decreasing mixed chimerism (MC), 8% stable MC, 25% increasing MC and 3% increasing and decreasing MC. In 16 RIC patients, these percentages were 12, 50, 6, 6 and 19, respectively, together with 6% nonengraftment. Forty-three out of 96 patients experienced relapse. The last chimerism evaluation before relapse revealed increasing MC in only eight patients. In samples taken between 1 and 6 months post SCT, CC/decreasing MC was significantly related with a lower risk of relapse (31 versus 83%, P<0.000) and mortality (38 versus 83%, P<0.000) than with MC/increasing MC. However, the development of relapse was very rapid. Only very frequent monitoring of chimerism status by highly sensitive methods might identify impending relapse and allow early immunological intervention.

Identification of human ccn2 (connective tissue growth factor) promoter polymorphisms

Background—Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor β1 (TGF-β1), interleukin 10 (IL-10), and tumour necrosis factor α (TNF-α), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions. Aim—To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression. Materials/Methods—Seventy seven human DNA samples were sequenced—45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)—using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit. Results—A C to G substitution was identified at position −132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position −447. These substitutions at −132 and −447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position −43 (G), −47 (C), −71 (G) and a C to T substitution at position −198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites. Conclusion—Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level ⩾1 × 10−4 were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1–4). The intervention was associated with graft versus host disease ⩾grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0–30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.

Sentinel Node Biopsies in Melanoma Patients: A Protocol for Accurate, Efficient, and Cost-Effective Analysis by Preselection for Immunohistochemistry on the Basis of Tyr-PCR

Background: Immunohistochemistry (IHC) of serial sectioning is considered the gold standard for detection of melanoma activity in sentinel node (SN) biopsies. However, this is cost and labor intensive. In contrast, tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) is simple and quick, but it is hampered by its extreme sensitivity. This study was performed to test whether a strategy that combines the two methods, using tyrosinase RT-PCR to preselect nodes for IHC, could be accurate and cost effective.Methods: In 36 patients, SNs were identified by scintigraphy and patent blue uptake. Of each SN, one cross section was analyzed first by hematoxylin and eosin staining. Next, all nodes were examined by serial sectioning and IHC of one-half and tyrosinase RT-PCR of the other. Before comparison, all results were documented in a blinded manner. Material costs and workload estimates were noted per SN.Results: Fifty-five SNs were retrieved from the 36 patients. Hematoxylin and eosin staining of the first cross section revealed tumor positivity in 3 patients (6 SN). Tyrosinase RT-PCR was positive in 11 of the remaining 33 patients (19 of 49 SN). Of these same 11 patients, only 5 were shown to have tumor-positive SNs by using IHC on serial sections (7 SN). All these nodes had been positive for tyrosinase on PCR. For IHC, an average of 40 sections were prepared and examined per SN at a cost of

Cutaneous Malignant Melanomas in 57 Cats: Identification of (Amelanotic) Signet-ring and Balloon Cell Types and Verification of Their Origin by Immunohistochemistry, Electron Microscopy, and In Situ Hybridization

200(U.S.)/SN. In contrast, routine tyrosinase RT-PCR costs

Detection of calcitonin-encoding mRNA by radioactive and non-radioactive in situ hybridization: improved colorimetric detection and cellular localization of mRNA in thyroid sections.

37(U.S.)/SN, and takes 5% of the time necessary for IHC. A strategy including hematoxylin and eosin staining on the first cross section, followed by tyrosinase RT-PCR on half of each negative (half) node, could preselect nodes to be taken through serial sectioning. In these series, such a strategy would have prevented serial sectioning and IHC of 30 SN from 22 patients. Apart from a considerable gain in efficiency, this would have reduced material costs by a minimum of

Macrophages and antitumor reactions

6000 (U.S.). This iscrepancy would be even higher if work intensity of analysts and pathologists were considered.Conclusions: In routine analysis of SN biopsies in melanoma patients, tyrosinase RT-PCR can be used effectively to preselect nodes for further IHC of serial sections. This method seems both time and cost effective.