Mark Sales

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.

Chromosomal imbalances in gastric and esophageal adenocarcinoma: specific comparative genomic hybridization-detected abnormalities segregate with junctional adenocarcinomas.

The incidence of adenocarcinoma arising at the esophagogastric junction (EGJ) is increasing at a rate greater than that for any other form of solid malignancy. Commensurate with this, the incidence of histologically similar tumors arising in the gastric body and antral mucosa is declining. The increased incidence of the proximal group of tumors may reflect, in part, the higher prevalence of Barrett esophagus. These epidemiological features suggest that histologically similar tumors arising at the EGJ and from the distal stomach are different, which may be reflected in the genetic abnormalities that characterize the two groups of tumors. The purpose of this study was to screen genomic DNA from adenocarcinomas of the esophagus and stomach for regions of chromosomal imbalance, using comparative genomic hybridization to determine whether tumors at the EGJ (junctional tumors) have a different profile compared with tumors of the distal stomach. Tumor samples were derived from a series of 48 gastroesophageal adenocarcinomas (20 junctional and 28 distal) that were acquired prospectively from patients undergoing esophagogastrectomy. These tumors are characterized by several regions of chromosomal imbalance with no obvious correlation between most regions of abnormal copy number and tumor type. However, our study shows for the first time cytogenetic abnormalities (5p+ and 18q−) that identify statistically significant differences (P < 0.02 and < 0.05, respectively) between junctional and distal gastric tumors. These differences are gain of 5p (55% [11/20] of junctional tumors vs. 21% [6/28] of distal gastric tumors) and loss of 18q (25% [5/20] cases of junctional tumors vs. 4% [1/28] of distal tumors) segregating with tumors of the EGJ. These abnormalities may distinguish distinct tumor subtypes that are recognized in epidemiological and clinical studies but that are otherwise histologically identical.

Chromosomal changes in colorectal adenomas: relationship to gene mutations and potential for clinical utility.

Although the occurrence of both chromosomal aberrations and specific gene mutations in colorectal tumorigenesis is firmly established, the relationship between these different forms of genetic abnormality remains poorly understood. We have previously demonstrated, in colorectal adenocarcinomas, that mutations of APC, KRAS, and TP53 are each specifically associated with certain chromosomal aberrations. Using comparative genomic hybridization and mutational analysis of APC, KRAS, and TP53 to evaluate 78 colorectal adenomas, we have shown that several of the significant relationships between gene mutations and chromosomal abnormalities reported in colorectal adenocarcinomas also exist at the adenomatous stage. KRAS mutation correlated with 12p gain (P < 0.001) and TP53 mutation with both 20q gain and 18q loss (P = 0.03 for both). In addition, we have identified two chromosomal aberrations, gain of 13q and loss of 11q, that correlate with the presence of synchronous adenomas (P = 0.049 and P = 0.03, respectively) and several chromosomal changes (20p+, 20q+, 17p−, and 18q−) that are related to the onset of high‐grade dysplasia. These data strengthen our previous contention that the co‐occurrence of specific gene mutations and chromosomal changes is not random and significant relationships do exist. Our findings also raise the possibility that certain chromosomal aberrations may act as important clinical biomarkers.

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.

Effective monosomy or trisomy of chromosome band 2q37.3 due to the unbalanced segregation of a 2;11 translocation

We report a seven generation family in which a 2;11 chromosome translocation is segregating. Both unbalanced segregants have been found in the family, and cytogenetic analysis demonstrates that this results in effective monosomy or trisomy for chromosome band 2q37.3. Those family members who are monosomic exhibit a variable phenotype with a number of features associated with an Albrights Hereditary Osteodystrophy‐like phenotype (AHO‐like) whilst those who are trisomic have a phenotypic spectrum ranging from mild facial anomalies and growth retardation to apparent normality. The latter group of patients represent the first reported patients with pure trisomy for chromosome band 2q37.3.

Factors influencing a second myeloid malignancy in patients with Philadelphia-negative -7 or del(7q) clones during tyrosine kinase inhibitor therapy for chronic myeloid leukemia

The detection of Philadelphia-negative (Ph(neg)) cells with non-random karyotypic abnormalities after tyrosine kinase inhibitor (TKI) therapy of chronic myeloid leukaemia (CML) can be associated with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). To our knowledge, however, there have been no studies on variables influencing the risk of MDS/AML in patients with specific Ph(neg) karyotypes. We systematically examined studies reporting -7 or del(7q) within Ph(neg) cells in TKI-treated CML patients, and abstracted clinical and cytogenetic data from individual reports into a standardized format for further analysis. Of 53 patients, 43 had Ph(neg) -7 clones [as the sole abnormality (-7(sole)) in 29, or with other clones (-7(dual)) in 14], and del(7q) was present in 10. A total of 16/51 evaluable patients, all with -7, transformed to MDS/AML. Transformation was more frequent (15/16 patients) within 6 months of Ph(neg) -7 detection rather than subsequently (P < 0.0001). At first detection after TKI therapy, Ph(neg) abnormal clones comprised ≥50% of Ph(neg) cells in a greater proportion of patients with -7 than del(7q) (P = 0.035). Upon comparing -7(sole) and -7(dual), the latter was likely to be transient (P = 0.004), and AML was frequently observed with persistent -7 clones (P = 0.03). By logistic regression analysis (n = 36), clone size (P = 0.017), time-to-detection longer than 15 months (P = 0.02), and CML response (P = 0.085) were associated with MDS/AML. Validation of these novel associations in registry-based studies will help develop predictive criteria that define the MDS/AML risk in individual patients.

Multiparameter DNA content analysis identifies distinct groups in primary breast cancer.

Background:Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique.Methods:The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox’s Regression (CR) analysis.Results:Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76–1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18–1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI⩾1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and ‘multiploid tumours’ (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (⩾10%) independently associated with poor overall survival (P=0.027, CR).Conclusion:Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.

Application of combined immunofluorescence and fluorescence in situ hybridization on paraffin‐embedded sections to characterize T‐cell lymphoma with EBV‐infected B‐cell blasts

Combined immunofluorescence (IF) and fluorescence in situ hybridization (FISH) on formalin‐fixed, paraffin‐embedded tissue sections were used to examine lymph node tissue from two patients diagnosed with T‐cell lymphoma with Epstein–Barr virus (EBV)–infected B‐cell blasts. The majority of cells within the samples comprised T‐cells staining positively for CD3. In addition, both patients had a population of large pleiomorphic cells that were positive for the B‐cell marker CD20 and for EBV LMP‐1. Standard PCR clonality testing of the nodes revealed both immunoglobulin heavy chain (IGH) and T‐cell receptor (TCR) clonal rearrangements in one patient, although in the other case monoclonality was demonstrated only for TCRG. Cytogenetics of cultured lymphocytes from nodal tissue revealed two apparently unrelated abnormal clones in both patients. Combined IF and FISH revealed that these phenomena reflected two abnormal populations of B‐ and T‐cells rather than reactive B‐cell hyperplasia or biphenotypic evolution from a common ancestral lymphoma. True B‐cell malignancy probably emerged within a preexisting but unrelated T‐cell lymphoma. This is the first study to relate the phenotype of the abnormal cells in such cases to specific clonal populations of cells, and it demonstrates a method that may easily be introduced into a diagnostic cytogenetics laboratory with access to standard pathology laboratory resources.

Autoimmune pancytopenia following combination chemotherapy for chronic lymphocytic leukaemia

Autoimmune haemolysis or thrombocytopenia can complicate purine nucleoside monotherapy for chronic lymphocytic leukaemia (CLL), but Evans syndrome is rare. This is a report of the occurrence of pancytopenia secondary to a unique combination of red cell aplasia with autoimmune thrombocytopenia and neutropenia in a patient with CLL following treatment with fludarabine and cyclophosphamide. This case is unusual for the simultaneous targeting of three haemopoietic lineages by immune dysfunction following fludarabine and cyclophosphamide, which is a treatment regimen believed to reduce autoimmune haematological toxicity in CLL.