Diagnosis, risk stratification and management of monoclonal gammopathy of undetermined significance and smoldering multiple myeloma.

Monoclonal gammopathy of undetermined significance (MGUS) is one of the most common premalignant disorders. IgG and IgA MGUS are precursor conditions of multiple myeloma (MM), whereas light-chain MGUS is a precursor condition of light-chain MM. Smoldering MM (SMM) is a precursor condition with higher tumor burden and higher risk of progression to symptomatic MM compared to MGUS. Assessment of the risk of progression of patients with asymptomatic monoclonal gammopathies is based on various factors including clonal burden, as well as biological characteristics, such as cytogenetic abnormalities and light-chain production. Several models have been constructed that are useful in daily practice for predicting risk of progression of MGUS or SMM. Importantly, the plasma cell clone may occasionally be responsible for severe organ damage through the production of a M-protein which deposits in tissues or has autoantibody activity. These disorders are rare and often require therapy directed at eradication of the underlying clone. Importantly, recent studies have shown that asymptomatic patients with a bone marrow plasma cell percentage ≥60%, free light-chain ratio ≥100, or >1 focal lesion on MRI (myeloma-defining events) have a 80% risk of developing symptomatic MM within 2 years. These patients are now considered to have MM requiring therapy, similar to patients with symptomatic disease. In this review, we provide an overview of the new diagnostic criteria of the monoclonal gammopathies and discuss risk of progression to active MM. We also provide recommendations for the management of patients with MGUS and SMM including risk-adapted follow-up.

Diffuse large B-cell lymphoma with MYC gene rearrangements: Current perspective on treatment of diffuse large B-cell lymphoma with MYC gene rearrangements; case series and review of the literature.

In the past decade, patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) were treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. Standard treatment is now changing as a result of deeper understanding of underlying biologic differences of such lymphomas. One of the most powerful predictors of an adverse outcome on R-CHOP therapy is the presence of a MYC gene rearrangement (MYC+ lymphoma). Determination of MYC gene rearrangement by FISH (fluorescent in situ hybridisation) has recently become a standard diagnostic procedure. In this paper, an overview of current literature on MYC function and MYC+ lymphoma patient outcome is presented. Furthermore, we present 26 patients from our tertiary referral centre who were diagnosed with MYC+ lymphoma between 2009 and 2014. In our patient series, we confirm the dismal prognosis of MYC+ lymphoma patients. Intensification of classical chemotherapy does not lead to better overall survival, justifying new treatment modalities. First line therapy should be more specifically targeted against MYC and the genes and proteins that are deregulated by MYC. To this end, the first clinical trial in which MYC+ patients will be offered targeted treatment has recently been launched.

Distinctive Phenotypic Abnormalities Associated with Submicroscopic 21q22 Deletion Including DYRK1A.

Partial monosomy 21 has been reported, but the phenotypes described are variable with location and size of the deletion. We present 2 patients with a partially overlapping microdeletion of 21q22 and a striking phenotypic resemblance. They both presented with severe psychomotor delay, behavioral problems, no speech, microcephaly, feeding problems with frequent regurgitation, idiopathic thrombocytopenia, obesity, deep set eyes, down turned corners of the mouth, dysplastic ears, and small chin. Brain MRI showed cerebral atrophy mostly evident in frontal and temporal lobes, widened ventricles and thin corpus callosum in both cases, and in one patient evidence of a migration disorder. The first patient also presented with epilepsy and a ventricular septum defect. The second patient had a unilateral Peters anomaly. Microarray analysis showed a partially overlapping microdeletion spanning about 2.5 Mb in the 21q22.1-q22.2 region including the DYRK1A gene and excluding RUNX1. These patients present with a recognizable phenotype specific for this 21q22.1-q22.2 locus. We searched the literature for patients with overlapping deletions including the DYRK1A gene, in order to define other genes responsible for this presentation.

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome.

BACKGROUND: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. AIM: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. RESULTS: We estimate the prevalence of the syndrome to be 1 in 16,000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10(-5)). CONCLUSION: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

Comparative genomic hybridization for cytogenetic evaluation of stillbirth.

OBJECTIVE: To ascertain the feasibility and reliability of comparative genomic hybridization for cytogenetic evaluation of macerated stillbirths. MATERIALS: We examined ten stillborn fetuses above 15 weeks' gestation whose karyotypes were unknown because of tissue culture failure. Sixteen fetuses that were successfully karyotyped using prenatal or postnatal tissues were also examined as controls, including five pregnancy terminations with autosomal aneuploidy, one with sex chromosome aneuploidy, one with a chromosomal deletion; five macerated fetuses with normal karyotypes, three with autosomal aneuploidy, and one with sex chromosome aneuploidy and discrepancy between chorionic villi and fetus. RESULTS: All comparative genomic hybridization analyses in fresh and macerated tissues were successful except for one. All normal karyotypes and aneuploidies were confirmed. Comparative genomic hybridization failed in one fetus with a deletion of the short arm of chromosome 18. In the stillborn fetuses without known karyotypes, one aberrant profile was found; however, the results were not confirmed with interphase fluorescence in situ hybridization. In one fetus triploidy was diagnosed with DNA flow cytometry. CONCLUSION: Comparative genomic hybridization is a valuable backup technique for aneuploidy screening in tissues from macerated stillborn fetuses when tissue culture fails. Gains or losses can subsequently be confirmed by fluorescence in situ hybridization, using DNA probes that focus on specific loci of a chromosome.

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

Decreased expression of Ki-67 in atrophic cervical epithelium of post-menopausal women.

Papanicolaou-stained cervical smears (Pap smears) of post-menopausal women often present difficulties in distinguishing atrophic cervical epithelium from high-grade cervical intraepithelial neoplasia (CIN2-3). The aim of this study was to disclose differences in proliferative activity in normal cervical epithelium, cervical atrophy, and high-grade CIN lesions, in order to develop specific and sensitive classifiers to discriminate between cervical atrophy and high-grade CIN, both in cervical smears and in tissue sections. A case-control study was done on 83 patients. Proliferative activity was assessed in histological sections using the monoclonal antibody MIB1. An image analysis system was used to characterize different proliferation-associated features. Preceding Pap smears were restained with MIB1 and proliferative activity was measured by a point-counting procedure, carried out on a training set of 32 cases and a test set of 51 cases. In cervical atrophy, proliferative activity was significantly lower than in normal epithelium (p<0.001). Proliferative activity measured in both biopsies and cervical smears was considerably higher in high-grade CIN than in normal epithelium (p<0.001). Discriminant analyses resulted in four classifiers, based on proliferation parameters, to discriminate between cervical atrophy and high-grade CIN, and between CIN2 and CIN3, in biopsy specimens and cervical smears, respectively. The two classifiers for biopsy specimens resulted in 100% correct classification. Application of the classifier obtained from the training set of Pap smears resulted in 100% correct classification of the Pap smears in the test set. The classifier to discriminate between CIN2 and CIN3 in Pap smears, obtained from 36 patients, resulted in 87% and 90% correct classification, respectively.

Interphase cytogenetic analysis of cervical intraepithelial neoplasia.

The aim of this study was to detect numerical chromosomal aberrations that may be involved in the progression of cervical intraepithelial neoplasia (CIN) toward cervical carcinoma. Therefore, cervical lesions (five CIN 1, seven CIN 2, six CIN 3, six invasive carcinomas, and six normal samples) were studied by in situ hybridization (ISH) on serial 3-microm-thick paraffin tissue sections, using a panel of eight centromeric DNA probes for chromosomes 1, 3, 6, 7, 8, 11, 17, and X. An estimation of the percentage of dysplastic epithelium with abnormal ISH signals per nucleus was made. Chromosome aneusomy could be detected in all persisting and high-grade CIN lesions and invasive carcinomas. In most cases, when one of the chromosomes showed aneusomy then all studied chromosomes showed numerical changes. Interestingly, the abnormal ISH signals were found only in a varying part of the morphologically dysplastic epithelium, the remainder showing no such changes. In aneuploid regions of the CIN 1 lesions the mean chromosome index for all chromosomes was 1.97+/-0.03 with a range of 1.92 to 2.00. The chromosome index ratios of chromosomes 1, 7, and X showed a significant positive correlation with CIN grade (r > or = 0.74; P < or = 0.006). It is concluded that chromosome aneusomy of chromosomes 1, 7, and X may be involved in the progression of CIN lesions.

Human papilloma virus detection by in situ hybridisation signal amplification based on biotinylated tyramine deposition.

Aim-To describe a method for amplifying human papilloma virus (HPV) in situ hybridisation (ISH) signals.Methods-Three human cervical cell lines, namely CaSKi, HeLa and SiHa, containing different copy numbers of integrated HPV DNA were studied. Following ISH, catalysed reporter deposition (CARD), based on the deposition of biotinylated tyramine at the location of the DNA probe, was used to amplify the ISH signal.Results-Using CARD-ISH, one to three HPV type 16 copies were detected in situ both in cell suspensions and paraffin wax sections of SiHa cells. CARD-ISH can also be used to detect oncogenic HPV DNA sequences, such as HPV types 16 and 18, in routinely processed formalin fixed, paraffin wax embedded cervical specimens.Conclusions-CARD-ISH is a fast and highly sensitive ISH method for the routine detection of low copy number HPV DNA sequences in cervical cell lines and routinely processed tissue sections. Application of this technology also enables the routine detection and cellular localisation of other viral DNA sequences present at copy numbers below the detection limit of conventional ISH methods.

Loss of chromosome 9 in tissue sections of transitional cell carcinomas as detected by interphase cytogenetics. A comparison with RFLP analysis.

Interphase cytogenetics by in situ hybridization (ISH) using a panel of centromere-associated DNA probes for chromosomes 1, 7, 9, 10, 11, 16, 17, and 18 was performed on 5 microns thick frozen tissue sections of transitional cell carcinomas (TCCs) of the urinary bladder. By this approach, chromosome ploidy, numerical chromosome aberrations, imbalance between chromosomes, and heterogeneity of aberrations within individual tumours were determined. In 15 of 24 TCCs, loss or underrepresentation of chromosome 9, compared with the ISH copy numbers of at least five other chromosomes, was demonstrated. Independently, RFLP analysis were performed on the same cases to detect loss of heterozygosity (LOH) of chromosome loci 9q34, 11p15, 16q22-24, 17p13, and 18q21. LOH was found in 9 of 19 informative cases for chromosome locus 9q34. Comparison of the ISH and RFLP results showed no correlation between numerical aberration and LOH for the loci on chromosomes 11, 16, 17, and 18. However, numerical loss of chromosome 9 was found in 89 per cent (eight of nine cases) with LOH for 9q34. Conversely, LOH at 9q34 was observed in only 67 per cent (eight of 12 cases) with underrepresentation of chromosome 9. Moreover, in 60 per cent of the non-informative cases (three of five cases), underrepresentation for chromosome 9 was observed. These results indicate that the heterochromatin probe for chromosome 9 can be reliably used in TCC tissue sections for the detection of chromosomal loss. In aneuploid TCCs, this DNA probe can be used for the detection of chromosomal underrepresentation only in combination with other centromere-associated DNA probes.

Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretion receptor superfamily with an unusual extracellular domain.

CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and a single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).

A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.

For amplification of in situ hybridization (ISH) signals, we describe a method using catalyzed reporter deposition (CARD). This amplification method is based on the deposition of biotinylated tyramine (BT) at the location of the DNA probe. The BT precipitate can then visualized with fluorochrome- or enzyme-labeled avidin. Both for bright-field ISH (BRISH) and for fluorescence ISH (FISH), the detection limit was highly increased. This method is especially suitable for visualization of very weak ISH signals, such as those obtained by ISH using locus-specific DNA probes. Furthermore, CARD amplification of ISH signals (CARD-ISH) is highly sensitive, rapid, flexible, and easy to implement. Successful application of CARD-ISH with locus-specific DNA probes on histological and cytological samples may improve the determination of structural chromosomal aberrations in archival material.

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry.

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome 1-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using beta-galactosidase/ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. The described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.

p53 mutations have no additional prognostic value over stage in bladder cancer.

Evidence is accumulating that the tumour-suppressor gene p53 is involved in the development of bladder cancer. Therefore we studied p53 mutations in 47 bladder cancers obtained from 45 patients using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. Eight out of 24 invasive tumours appeared to have a p53 mutation, while no p53 mutations were found in the superficial tumours. All the p53 mutations were found in grade 3 tumours. The tumours with altered p53 showed a higher frequency of allelic loss (FAL) than the tumours without a mutation (55.8% vs 21.1%, P < 0.05, chi 2 test). This increase in FAL suggests a correlation between p53 mutations and genetic instability. A significant correlation between mutated p53 and poor survival in the whole group studied was found (P < 0.001, log-rank test). However, within the group of muscle-invasive tumours the occurrence of p53 mutations had no additional prognostic value. Therefore, even though p53 mutations were found in aggressive tumours, the clinical usefulness of its detection seems limited. Nevertheless, these results imply that p53 is involved in the clinical behaviour of bladder cancer; its role in the progression of superficial cancer to invasive disease merits further attention.

Double-target in situ hybridization in brightfield microscopy.

For brightfield detection of two different DNA target sequences in one sample, we developed a double-target in situ hybridization (ISH) technique, using biotin- and digoxigenin-labeled chromosome-specific DNA probes. First, several immunochemical detection systems were optimized and compared for sensitivity and simultaneous applicability. Two non-interfering immunochemical systems were chosen for simultaneous detection of the DNA probe labels. This resulted in combination of an alkaline phosphatase (AP)-conjugated avidin-biotin system with a horseradish peroxidase (HRP)-conjugated antibody system for detection of biotin- and digoxigenin-labeled DNA probes, respectively. Development of AP with New Fuchsin-naphthol phosphate and HRP with diaminobenzidine-H2O2 resulted in stable, well-contrasting (red and black, respectively) color precipitates visible by conventional light microscopy. The double-target ISH technique was successfully applied on a wide variety of biological materials, such as metaphase spreads, cytospin, and Thin-prep samples of cytological specimens, frozen tissue sections, and formalin-fixed, paraffin-embedded tissue sections. In particular, on tissue sections, where quantitative interpretation of ISH data can be hampered by truncation of nuclei, the double-target ISH technique appeared to be a valuable tool for demonstration of chromosome aberrations and chromosome imbalances.

DNA in situ hybridization as a diagnostic tool in the discrimination of melanoma and Spitz naevus.

As the clinical and histological differential diagnosis between Spitz naevus and cutaneous melanoma may be very difficult, we have investigated whether DNA in situ hybridization maybe helpful in resolving this problem. To this end, routinely-processed paraffin sections of 15 typical Spitz naevi, 15 typical nodular melanomas, and five cases originally misdiagnosed as Spitz naevi but which later metastasized and were reclassified as melanoma were analysed using a method previously described (De Wit et al., J Invest Dermatol 1992; 98: 450-458). Microscopical semi-quantitative evaluation revealed that the number of nuclei with supernumerary aberrations of the centromere region of chromosome 1, suggestive of aneuploidy, was significantly different in Spitz naevi and nodular melanoma. The mean number of aberrant nuclei per high power field was 0.41 and 4.01, respectively (P = 0.0001). On applying the results of the typical lesions to the equivocal, originally misdiagnosed lesions, three out of five could be identified as melanoma. These results suggest that the application of DNA in situ hybridization may contribute to the positive identification of histologically equivocal pigmented lesions. The advantages of this technique are that it is cheap, requires little tissue, and can be applied on routinely-processed paraffin sections.

Interphase cytogenetics on agar cultures: a novel approach to determine chromosomal aberrations in hematopoietic progenitor cells.

We describe a novel approach to determine the presence of chromosomal aberrations in progenitor cells by in situ hybridization (ISH) on agar cultures. Bone marrow cells of 3 patients suffering from acute myeloid leukemia (AML) were selected to develop the method. In all 3 cases, numerical aberrations for chromosome 1 and/or 8 were detected by karyotyping and ISH using chromosome-specific centromeric-associated DNA probes. These aberrations were used as markers in this study. After in vitro culture of the bone marrow samples in agar, the cells were pretreated to perform ISH. This approach retains the cytological architecture of the agar assay, allowing discrimination between chromosomal aberrations detected in the clonogenic and non-clonogenic cells in culture. With this new technique, the presence of the cytogenetic aberration in clonogenic cells can be shown at the interphase level.

Structural chromosome 1 aberrations in transitional cell carcinoma of the bladder: interphase cytogenetics combining a centromeric, telomeric, and library DNA probe.

Fluorescence in situ hybridization (FISH) was used to study numerical and structural chromosome 1 aberrations in interphase nuclei of transitional cell carcinomas (TCCs) of the urinary bladder. One of the characteristic numerical aberrations, as detected previously in low-grade noninvasive TCCs, included trisomy for chromosome 1 (A. H. N. Hopman et al., Cancer Res., 51: 644-651, 1991). We examined in more detail 22 cases with a centromeric (1q12) and a telomeric associated (1p36) DNA probe and with a library DNA probe from sorted human chromosome 1 in single- and double-target FISH procedures. All flow cytometrically determined DNA diploid TCCs (13 cases), which showed three spots for 1q12 (6 cases), had two spots for 1p36. Since the library DNA probe showed three separate domains in the nuclei of these cases, the additional copy for 1q12 could be explained as an extra chromosome 1p-, containing the 1q12 target. In the flow cytometrically determined DNA tetraploid/aneuploid tumors, the results were more complex. In 6 of 9 cases, we observed an overrepresentation of 1q12 as compared to 1p36, also suggesting the presence of extra copies of 1p- chromosomes. The results of the present study demonstrate the utility of the FISH method to assess structural chromosome aberrations in interphase nuclei of solid tumors.

Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes.

Numerical chromosome aberrations were detected in hematological cancers by nonradioactive in situ hybridization (ISH) procedures, using centromere specific probes for chromosomes 1, 7, 8, 9, 10, 11, 16, 17, 18, X, and Y. All 15 cases could be evaluated by ISH for these 11 probes. Our experiments show that in seven of these randomly selected leukemia bone marrow cell suspensions numerical aberrations for one or two chromosomes could be detected by this method. The results of ISH on interphase nuclei and in some cases on metaphase preparations were compared with karyotyping data. Seven cases of chromosomal aberrations observed with ISH (three for monosomy and four for trisomy) were confirmed by this classical cytogenetic technique, whereas in five instances an aberration was found only with ISH (twice for monosomy, twice for trisomy, and one disomy for the Y-probe). One case of a trisomy for chromosome 1 observed by ISH on interphase nuclei could be explained by a marker chromosome, a finding that was further substantiated by ISH on metaphase spreads. In this case double-target ISH on interphase cells with the probes for chromosomes 1 and 16 strongly suggested a translocation between these chromosomes. Also, in one case a marker chromosome could be characterized as a translocation between chromosomes 7 and 17. In this latter case the cytogenetic examinations revealed only monosomy for chromosomes 7 and 17 in addition to noncharacterized marker chromosomes. Our results indicate that the nonradioactive ISH procedure in combination with chromosome specific repetitive centromeric probes is a powerful tool for studying both numerical and structural chromosomal aberrations in interphase nuclei of leukemias. It may therefore become a valuable and routine diagnostic tool in addition to the existing karyotyping procedures.