Joan Cunningham

Does isochromosome 7q mandate bone marrow transplant in children with Shwachman–Diamond syndrome?

Summary. We report on nine children with Shwachman–Diamond syndrome (SDS), eight of whom had clonal abnormalities of chromosome 7. Seven children had an isochromosome 7 [i(7)(q10)] and one a derivative chromosome 7, all with an apparently identical (centromeric) breakpoint. Children with SDS are predisposed to myelodysplasia (MDS) and acute myeloid leukaemia (AML) often with chromosome 7 abnormalities. Allogeneic transplants have been used to treat these children, however, they are a high‐risk transplant group and require careful evaluation. Three of the children were transplanted but only one survived, who to our knowledge remains the longest surviving SDS transplant patient (4·5 years +). The six non‐transplanted children are well. In classic MDS, chromosome 7 abnormalities are associated with rapid progression to acute leukaemia; however, we present evidence to suggest that isochromosome 7q may represent a separate disease entity in SDS children. This is a particularly interesting finding given that the SDS gene has recently been mapped to the centromeric region of chromosome 7. Our studies indicate that i(7)(q10) is a relatively benign rearrangement and that it is not advisable to offer allogeneic transplants to SDS children with i(7)(q10) alone in the absence of other clinical signs of disease progression.

C-MYC translocation in t(14;18) positive follicular lymphoma at presentation: An adverse prognostic indicator?

Follicular lymphoma (FL) is a common subtype of low grade B-cell non-Hodgkin lymphoma (NHL). Although this form of lymphoma often pursues an indolent course, in some cases it may behave in a more aggressive manner. Clinical and histological parameters have been shown to correlate with an adverse prognosis but a number of cytogenetic abnormalities may also be associated with aggressive disease. Although, the t(14;18) in itself does not affect outcome in cases of FL, secondary abnormalities that occur in a complex polyploid karyotype may identify cases with a poor prognosis. It is unusual to find both t(14;18) and C-MYC translocation in the same tumour; those cases in which it has been described include examples of high-grade B-cell NHL (either de novo or transformed FL) or B-cell acute lymphoblastic lymphoma. In this report, three cases of FL are described in which both t(14;18) and a C-MYC translocation were identified at presentation. We also summarize four further cases from the literature. This is a small series but one which raises the possibility that the presence of a C-MYC translocations at presentation may identify a particularly aggressive subtype of FL. Further studies are required to investigate the true incidence of this aberration, the impact on C-MYC regulation, clinical course and response to treatment.

HER2 assessment on core biopsy specimens using monoclonal antibody CB11 accurately determines HER2 status in breast carcinoma

Purdie C A, Jordan L B, McCullough J B, Edwards S L, Cunningham J, Walsh M, Grant A, Pratt N & Thompson A M
(2010) Histopathology 56, 702–707
HER2 assessment on core biopsy specimens using monoclonal antibody CB11 accurately determines HER2 status in breast carcinoma

Dysregulation of autophagy in chronic lymphocytic leukemia with the small-molecule Sirtuin inhibitor Tenovin-6

Tenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways.

Molecular characterisation of a recurrent, semi-cryptic RUNX1 translocation t(7;21) in myelodysplastic syndrome and acute myeloid leukaemia

A proportion of cytogenetic abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) may escape detection by high‐resolution genomic technologies, but can be identified by conventional cytogenetic and molecular analysis. Here, we report the detection of a reciprocal translocation t(7;21)(p22;q22) in the marrow of two adults with MDS and AML, using conventional cytogenetic analysis and fluorescence‐in situ‐hybridization (FISH). Reverse‐transcription polymerase chain reaction (RT‐PCR) and sequence analysis identified a fusion between RUNX1 and the gene encoding ubiquitin specific peptidase‐42 (USP42), with splice‐variants and variable break‐points within RUNX1. Combined cytomorphology and FISH studies in MDS marrow revealed abnormal RUNX1 signals within megakaryocytes, suggesting that the acquisition of t(7;21)(p22;q22) does not confer complete differentiation arrest and may represent an early genetic event in leukaemogenesis. Single nucleotide polymorphism‐arrays failed to detect additional sub‐microscopic genomic changes predisposing to or associated with t(7;21). Molecular analysis of 100 MDS and AML marrow specimens by RT‐PCR did not reveal new cases with the RUNX1‐USP42 fusion. Thus, our studies have identified t(7;21)(p22;q22) as a rare but recurrent abnormality in MDS/AML, with the existence of alternative spliced forms of the RUNX1‐USP42 transcript in different patients. Further studies are required to identify the potential contribution of these splice‐variants to disease heterogeneity.

Non‐invasive RNA‐based determination of fetal Rhesus D type: a prospective study based on 96 pregnancies

Objectives To develop a non–invasive method for determining fetal RhD status in order to provide improved care for women most at risk.

p53 and cell cycle independent dysregulation of autophagy in chronic lymphocytic leukaemia

Background:Activation of wild-type p53 with the small molecule sirtuin inhibitor Tenovin-6 (Tnv-6) induces p53-dependent apoptosis in many malignant cells. In contrast, Tnv-6 reduces chronic lymphocytic leukaemia (CLL) cell viability with dysregulation of autophagy, without increasing p53-pathway activity.Methods:Here, we have investigated whether a quiescent phenotype (unique to CLL) determines the Tnv-6 response, by comparing the effects of Tnv-6 on activated and proliferating CLL. We further studied if these responses are p53-dependent.Results:Unlike quiescent cells, cell death in activated cultures treated with Tnv-6 was consistently associated with p53 upregulation. However, p53 acetylation remained unchanged, without caspase-3 cleavage or apoptosis on electron microscopy. Instead, cellular ultrastructure and protein profiles indicated autophagy inhibition, with reduced ubiquitin–proteasome activity. In specimens with mutant TP53 cultured with Tnv-6, changes in the autophagy-associated protein LC3 occurred independently of p53. Cells treated with Tnv-6 analogues lacking sirtuin inhibitory activity had attenuated LC3 lipidation compared with Tnv-6 (P⩽0.01), suggesting that autophagy dysregulation occurs predominantly through an effect on sirtuins.Conclusion:These cell cycle and p53-independent anti-leukaemic mechanisms potentially offer novel therapeutic approaches to target leukaemia-sustaining cells in CLL, including in disease with p53-pathway dysfunction. Whether targets in addition to sirtuins contribute to autophagy dysregulation by Tnv-6, requires further investigation.

Increased ALK1 copy number and renal cell carcinoma-a case report.

There have been recent reports of a rare variant of renal cell carcinoma associated with upregulation of the anaplastic lymphoma kinase gene (ALK) arising as a consequence of chromosomal translocations. The tumours were described as having a characteristic morphology. Here, we describe a case with similar morphology characterised by eosinophilic cells, abundant intracytoplasmic lumina and scattered large ganglion-like tumour cells. There was focal staining for ALK demonstrated by immunohistochemistry. However, rather than exhibiting a chromosomal translocation involving ALK, the use of FISH and a break-apart probe demonstrated that there was increased copy number of intact 2p23, the chromosomal region containing the ALK gene. Furthermore, the use of comparative genomic hybridisation showed increase of the whole of chromosome 2 along with chromosomes 6 and 17. There was no evidence of loss of 3p nor of trisomy of 7 associated with clear cell and papillary carcinoma, respectively. We suggest that this demonstrates a novel mechanism of upregulation of ALK activity by increased copy number occurring during the development of a renal carcinoma with the characteristic ALK-associated morphology.

Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study.

The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r2=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.

Significance of Chromosome 9p Status in Renal Cell Carcinoma: A Systematic Review and Quality of the Reported Studies

Defining the prognosis of renal cell carcinoma (RCC) using genetic tests is an evolving area. The prognostic significance of 9p status in RCC, although described in the literature, remains underutilised in clinical practice. The study explored the causes of this translational gap. A systematic review on the significance of 9p status in RCC was performed to assess its clinical applicability and impact on clinical decision-making. Medline, Embase, and other electronic searches were made for studies reporting on 9p status in RCC. We collected data on: genetic techniques, pathological parameters, clinical outcomes, and completeness of follow-up assessment. Eleven studies reporting on 1,431 patients using different genetic techniques were included. The most commonly used genetic technique for the assessment of 9p status in RCC was fluorescence in situ hybridization. Combined genomic hybridisation (CGH), microsatellite analysis, karyotyping, and sequencing were other reported techniques. Various thresholds and cut-off values were used for the diagnosis of 9p deletion in different studies. Standardization, interobserver agreement, and consensus on the interpretation of test remained poor. The studies lacked validation and had high risk of bias and poor clinical applicability as assessed by two independent reviewers using a modified quality assessment tool. Further protocol driven studies with standardised methodology including use of appropriate positive and negative controls, assessment of interobserver variations, and evidenced based follow-up protocols are needed to clarify the role of 9p status in predicting oncological outcomes in renal cell cancer.