NEOCENT: a randomised feasibility and translational study comparing neoadjuvant endocrine therapy with chemotherapy in ER-rich postmenopausal primary breast cancer.

Neoadjuvant endocrine therapy is an alternative to chemotherapy for women with oestrogen receptor (ER)-positive early breast cancer (BC). We aimed to assess feasibility of recruiting patients to a study comparing chemotherapy versus endocrine therapy in postmenopausal women with ER-rich primary BC, and response as well as translational endpoints were assessed. Patients requiring neoadjuvant therapy were randomised to chemotherapy: 6 × 3-weekly cycles FE100C or endocrine therapy: letrozole 2.5 mg, daily for 18-23 weeks. Primary endpoints were recruitment feasibility and tissue collection. Secondary endpoints included clinical, radiological and pathological response rates, quality of life and translational endpoints. 63/80 patients approached were eligible, of those 44 (70, 95% CI 57-81) were randomised. 12 (54.5, 95% CI 32.2-75.6) chemotherapy patients showed radiological objective response compared with 13 (59.1, 95% CI 36.4-79.3) letrozole patients. Compared with baseline, mean Ki-67 levels fell in both groups at days 2-4 and at surgery [fold change: 0.24 (95% CI 0.12-0.51) and 0.24; (95% CI 0.15-0.37), respectively]. Plasma total cfDNA levels rose from baseline to week 8 [fold change: chemotherapy 2.10 (95% CI 1.47-3.00), letrozole 1.47(95% CI 0.98-2.20)], and were maintained at surgery in the chemotherapy group [chemotherapy 2.63; 95% CI 1.56-4.41), letrozole 0.95 (95% CI 0.71-1.26)]. An increase in plasma let-7a miRNA was seen at surgery for patients with objective radiological response to chemotherapy. Recruitment and tissue collection endpoints were met; however, a larger trial was deemed unfeasible due to slow accrual. Both regimens were equally efficacious. Dynamic changes were seen in Ki-67 and circulating biomarkers in both groups with increases in cfDNA and let-7a miRNA persisting until surgery for chemotherapy patients.

Microsatellite alterations plasma DNA of primary breast cancer patients.

The aim of this study was to analyze plasma DNA from primary and metastatic breast cancer cases for tumor-specific alterations and to compare these findings with immunocytochemistry and estimation of cytokeratin 19 (CK19) mRNA for detection of micrometastases. DNA was extracted from plasma, lymphocytes, and microdissected tumor tissue sections obtained from 71 patients with breast cancer and 9 controls. DNA samples were analyzed for loss of heterozygosity (LOH) and/or microsatellite instability (MI) by PCR with two polymorphic markers (DM-1 and D16S400). Reverse transcription-quantitative PCR (QPCR) and immunocytochemistry were used for detection of CK19 mRNA and protein. Breast cancer plasma DNA displayed frequent LOH (31.3%) and MI (11.6%) supported by the same alteration in microdissected tumor DNA. Most notably, 10 of the 39 patients with primary breast cancer showed LOH (n = 6) or MI (n = 4). We compared plasma tumor DNA, plasma and bone marrow QPCR, and blood and bone marrow immunocytochemistry in 32 of the patients with primary cancer. Of these, only one patient had immunocytochemically detectable carcinoma cells in the blood, and three showed abnormally high levels of plasma CK19 mRNA. All four of these patients had plasma DNA alterations. We then compared bone marrow findings: of the 10 primary breast cancers that showed LOH or MI, 6 had elevated CK19 mRNA and 5 had immunocytochemically positive cells. Tumor DNA is readily detectable in plasma of primary and metastatic breast cancer patients, and plasma DNA alterations (LOH and MI) reflect those seen in the tumor. The application of microsatellite analyses to plasma DNA may be useful in assessing tumor burden in breast cancer patients, particularly when combined with QPCR, and is preferable for patients with breast cancer, for whom sequential bone marrow aspiration is undesirable.

Reproducibility in the quantification of mRNA levels by RT-PCR-ELISA and RT competitive-PCR-ELISA.

The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibility of RT, PCR, RT-cPCR and measurement, amplifying the glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene from isolated renal glomeruli. We used an enzyme-linked immunosorbent assay (ELISA) to quantify PCR products. We also report our PCR-based method for constructing a competitor DNA identifiable independently of the native product. Our results show that the entire RT-PCR and ELISA process had a standard deviation (SD) of less than 10% (n = 10). This compared to an SD of less than 13% (n = 10) in PCR and ELISA. The SD for ELISA alone was less than 11% (n = 10). RT-cPCR quantitation gave an SD of approximately 15% (n = 10). These results support the use of standard RT-PCR for the relative quantitation of mRNA. RT-cPCR is also suited to relative quantitation, but it is also independent of the amplification saturation curve and permits the identification of differences in cellularity between samples.

The presence of Epstein-Barr virus in multiple organs in a fatal case of virus-associated haemophagocytic syndrome.

We describe a case report of a 21-year-old male with fatal Epstein-Barr virus-associated haemophagocytic syndrome. Virus is detected in multiple organs by polymerase chain reaction and in the tissue-specific cells of those organs by in situ hybridisation. It is suggested that organ failure may be a direct response to infection.

Thrombocytopenia following autologous bone marrow transplantation: evidence for autoimmune aetiology and B cell clonal involvement.

Thrombocytopenia outlasting anaemia and neutropenia is a well recognised sequel of autologous bone marrow transplantation (BMT) but the pathogenesis remains unclear. Autoimmune destruction of platelets has been suggested as a possible mechanism. We studied 5 patients who had undergone autologous BMT and were found to have persistent thrombocytopenia (< 150 x 10(9)/l) 6 months from transplantation with a normal haemoglobin level and granulocyte count. Apart from a mild reduction in the megakaryocyte numbers in one case, no other quantitative or qualitative defects of the megakaryocyte lineage were present to explain the peripheral thrombocytopenia. Two cases had positive anti-platelet autoantibodies. Immunoglobulin heavy chain gene rearrangement studies of peripheral blood and bone marrow mononuclear cells using the polymerase chain reaction showed evidence of clonal rearrangement in one of the two cases with positive anti-platelet autoantibodies. Our results support the previous reports that anti-platelet antibody-mediated destruction of platelets may play a role in the pathogenesis of post-autologous BMT thrombocytopenia. Furthermore, the demonstration of a clonal B cell expansion in one of the cases with anti-platelet antibodies suggests an aetiological link between clonal B cells, autoantibody production and thrombocytopenia.

Detection of immunoglobulin heavy chain gene rearrangements in Hodgkin's disease using PCR.

AIM: To detect clonal rearrangements of the immunoglobulin (Ig) heavy chain gene in Hodgkin's disease tissue using the polymerase chain reaction (PCR). METHODS: DNA extracted from 36 samples of Hodgkin's disease was analysed using PCR and primers from conserved sequences in the variable (VH) and joining (JH) regions. RESULTS: Clonal rearrangement was detected only in one case. Evidence of clonal immunoglobulin gene rearrangement had been detected previously in this case using conventional Southern blot analysis. CONCLUSIONS: The sensitivity of the two techniques is equivalent and clonal Ig heavy chain gene rearrangements are rare in Hodgkin's disease.

In situ and slot hybridization analysis of RNA in colorectal tumours and normal colon shows distinct distributions of mitochondrial sequences.

cDNA clones of mRNAs with an abnormal abundance in familial adenomatous polyposis were used to examine the levels and distribution of the mRNAs in tissues from 15 patients with colorectal cancer. Of 12 cloned sequences studied by slot hybridization, one was substantially reduced in tumours compared with normal tissue. Sequence analysis showed this to code for IgA. In situ hybridization was consistent with slot hybridization and immunohistochemistry. Two mitochondrially encoded sequences had distinct distributions detected by in situ hybridization but did not have detectable quantitative differences in whole tumour or mucosa extracts.

In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes.

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.

Identification of a human ribosomal protein mRNA with increased expression in colorectal tumours.

A human ribosomal protein cDNA was selected from a normal colon cDNA library on the basis of overexpression in familial adenomatous polyposis. Nucleotide sequence analysis was used to identify this cDNA as corresponding to the human equivalent of the rat ribosomal protein L31 (HL31). We have quantified the expression of HL31 mRNA in colorectal tumours and found overexpression in 23 out of 23 cases. Our results indicate that HL31 is associated with a malfunction of normal growth regulatory mechanisms in these tumours, and suggest a role for HL31 in proliferation and neoplasia.

Functional activity of intestinal epithelium demonstrated by mRNA in situ hybridization.

To elucidate the synthetic activity in relation to the morphology of the epithelial cells of the small and large intestine, in situ hybridization with a poly-deoxyribothymidine (poly d(T)) probe was applied to paraffin sections of formalin-fixed blocks. This effectively displays poly-adenylated RNA and, by implication, messenger RNA (mRNA). By minimizing proteinase K pretreatment, the relative concentrations of cellular mRNA were visualized. This revealed minimal mRNA in crypt columnar cells, and maximal mRNA in proliferating cells and in cells showing terminal differentiation. The latter include surface epithelial cells, endocrine cells, Paneth cells, and maturing, but not mature, goblet cells. The goblet cells showing positive hybridization can be regarded as active cells and show characteristic morphology. Such cells are particularly evident in untreated coeliac disease, remitting ulcerative colitis, and transitional mucosa. The proliferating cells showing increased hybridization include normal mitotically active crypt epithelium, regenerating epithelium in ulcerative colitis, adenomatous epithelium, and adenocarcinomatous epithelium. The similarity of hybridization between metaplastic polyp epithelium and surface colonocytes is consistent with the concept that metaplastic polyps are formed of cells showing premature terminal differentiation.

In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.

An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.