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discover cancer,cancer treatment,cancer
discover cancer,cancer treatment,cancer



Cytogenetic  analysis has become an integral part of the diagnosis and management of many malignancies. Theodor Boveri was the first to suggest that malignant tumours  could be due to an abnor- mal chromosome constitution . His hypothesis stated that the cell of a malignant  tumour has an abnormal chromosome consti- tution  and that  any event leading to an abnormal  chromosome constitution will result in a malignant tumour. He also postulated  the existence of enhancing or suppressing chromosomes, suggest- ing that malignant  growth  would result from loss of suppressing chromosomes or the predominance of enhancing  chromosomes. Thus,  prior  to  the  concept  of genes,  Boveri foreshadowed  the existence of oncogenes  and tumour suppressor genes.
The term chromosome  was first coined by Waldeyer in 1888 but it took nearly 70 years for the chromosome  complement of a normal human cell to be reliably determined. The birth of cytoge- netics is  generally dated  from Tjio and Levan’s identification  of



the true chromosome complement in human cells as it is from this time that abnormalities of chromosome  number and subsequently chromosome   structure  were reported  .  Their  discovery was made possible by a number  of advances. In his delightful  book, Hsu divided the study of human  cytogenetics  into four periods: the pre-hypotonic  period (or Dark Ages), the period from 1952 to
1959  that  included  the  discovery  of  hypotonic   solution  pre- treatment for cytological preparations, the third period (1959–1969) during  which  time  chromosome abnormalities  were  linked  to clinical syndromes, and the post-banding (modern)  period .
During  the pre-hypotonic era, chromosomes were studied in mouse and rat cancers and camera lucida drawings of metaphases suggested  the presence of many more chromosomes than normal and of structural abnormalities  within  these chromosomes. The drawings were taken from squash preparations,  a technique that was used  to  flatten  metaphase  spreads  of chromosomes  into  a two-dimensional  configuration,  but   still  resulted   in  crowded overlapping aggregations of chromosomes that were very difficult to count. Colchicine,  an extract of the autumn  crocus, was used to  arrest  the  cells in the  metaphase  stage  of the  cell cycle and increase the number  of mitoses available for analysis.
The use of a hypotonic  pre-treatment method  was an enor- mous step forward in the production of analysable chromosome preparations.   The  chromosomes  could  now  be  separated  and viewed individually. Counting chromosomes was simplified and gross structural abnormalities  could be discerned.  Hsu  describes the discovery of the utility of a hypotonic  pre-treatment as a labo- ratory accident, the perpetrator of which never owned  up to the error; thus, a major discovery in the history of cytogenetics  was apparently made by an unknown  technician.
It was in this era, in 1956, that Tjio and Levan finally answered the question  that had been plaguing investigators for more than
30 years, when they reported that there were 46 chromosomes in the human cell rather than 48. Subsequently, a number of research- ers were able to identify chromosome  abnormalities that appeared specific  for clinical syndromes.  Lejeune  and his colleagues pub- lished the chromosomal nature  of Down syndrome in 1959  . Their  observation  of an extra G group  chromosome  in patients with a specific congenital  malformation  syndrome showed for the first time  that  cytogenetic  analysis could  be used to diagnose  a human condition. From this time, there was a stream of publica- tions describing  chromosome aneuploidies  associated with other malformation syndromes. A number of the early, seminal papers in the field have been reproduced  in Peter Harper’s excellent study of the  beginnings  of human  cytogenetics  .  The study of the constitutional karyotype was aided by the discovery that phytohe- magglutinin  (PHA)  could induce peripheral  blood lymphocytes



to divide . This method was adopted by Moorhead et al. for the study of human chromosomes and remains one of the main- stays of modern  cytogenetic analysis.
The  confirmation   of  the  correct  chromosome  number   in human  cells led in the late 1950s  and early 1960s  to a flood of publications  describing numerical and structural abnormalities of chromosomes.  The  resulting  confusion  in  the  literature  made clear that there was a need for a common nomenclature to describe these rearrangements in a manner  that  was intelligible  to other workers in the field. Thus, a small group  met in Denver,  CO,  to establish a system of describing chromosome abnormalities. They published  the results of their  deliberations  in a report entitled “A   Proposed  Standard   System  of  Nomenclature  of  Human Mitotic  Chromosomes,” also known  as the  Denver  Conference (1960),  and  this report has formed  the  basis for all subsequent nomenclature reports, now published as An International System for Human Cytogenetic  Nomenclature (ISCN).
The ability to create a banding  pattern  on human  chromo- somes understandably complicated  the  nomenclature. Meetings in Paris and Edinburgh proposed  a basic system for designating chromosome regions and bands, resulting in a report of the Paris Conference   (1971). Crucially,  this  report  provided  a  way  of describing  structural rearrangements in terms of the band  com- position and the breakpoints involved in the rearrangement. The subsequent ISCN  publications  have been updated  to encompass the  various advances in the  field, including  fluorescence  in situ hybridization (FISH)  and arrays, but their core function  remains the  description  of chromosome abnormalities  in a manner  that allows  a  cytogeneticist   to  interpret   the  report  of  a  colleague immediately from anywhere in the world. Whilst the complexity of  chromosome rearrangements in cancer  cells frequently  tests this system to the  limit, as a scientific form of Esperanto,  it has been spectacularly successful over the years.
The advent of banding  enabled chromosomes to be individu- ally identified and the normal homologues paired. Initially, band- ing patterns along the length of each chromosome were induced by  preparations  stained  with quinacrine  mustard  and  visualized via a  fluorescence microscope    or depended upon  a method whereby slides were incubated  in warm saline or buffer solutions prior to staining by Giemsa. The initial Giemsa staining method  required  3 days for completion. Seabright’s rapid banding  tech- nique was therefore  embraced  as the whole procedure could  be carried out  at  room  temperature using air-dried  slides and pro- ducing  G-banded  chromosomes  ready  for  observation   within
10  minutes  (10).  Once  banding  became  available, there  were numerous  publications  describing recurrent  chromosome abnor- malities that appeared to be found in specific tumour types.



2. Cancer
Cytogenetics

The  history of cancer cytogenetics  is not  a long  one  but  it has been eventful and much knowledge  has been accumulated  in the
50  years since Peter  Nowell  and  David  Hungerford published their finding of a small marker chromosome in the chromosome complement of cells cultured  from  seven patients  with chronic myeloid leukaemia (CML)  .  Nowell and Hungerford made their landmark discovery in 1960, only just beating another group from Edinburgh who had also noted  the same marker chromo- some in their CML patients .
Although  the  hypothesis  that  malignant  cells were  derived from normal tissue cells that had acquired an abnormal chromatin content  was first proposed by Boveri, it was not until Nowell and Hungerford’s description  of the Philadelphia chromosome  that a revolution  in our understanding of the processes underlying  the development of malignancy began. Nowell and Hungerford called the  marker  chromosome   the  Philadelphia  chromosome   1,  Ph1, after the city in which they worked and the number  1 superscript signalled that they fully expected that there were many more cyto- genetic markers of cancer to be discovered. This was an exciting finding and an exciting time that effectively launched the field of cancer  cytogenetics  but  the  following  years were  frustrating  as abnormalities were observed in various cancers but the inability to identify  specific chromosomes  by any method  other  than  their basic shape limited researchers’ abilities to link abnormalities with different   morphological  subtypes   of  haematological   or  solid tumours.  All this changed with the advent of banding techniques.
Banding allowed the chromosomes to be clearly distinguished from one another  and, most  importantly,  revealed the nature  of structural   abnormalities:   balanced   translocations    of   material between chromosomes, deletion of part of a chromosome, dupli- cation of  another  segment,  or  an  inversion  of  a chromosome segment. In 1973,  Janet Rowley reported that a reciprocal trans- location  between   chromosomes   9   and   22   resulted   in   the Philadelphia  chromosome .  Since then,  hundreds  of  rear- rangements have been identified including not only translocations  but  also deletions  and  additions  of part or all of chromosomes and also inversions of genetic material within chromosomes.
From the descriptions  of chromosome  rearrangements, long before the Human  Genome  Project shone a light on the location of genes strung along our chromosomes, the molecular biologists were able to discover critical genes at the breakpoints of transloca- tions. Banding continues to allow us to identify new chromosome abnormalities in both haematological and solid tumours and these abnormalities provide the sign posts to the critical genetic changes that underlie the transformation of normal cells into cancer cells.



The first cytogenetic  abnormality  to have its genetic  secrets unlocked  was the 8;14  translocation which characterizes Burkitt lymphoma/leukaemia. Researchers identified  that  the transloca- tion caused two genes, MYC  on 8q24  and the immunoglobulin heavy chain gene, 1GH on 14q32, to come together . It is  now  known  that  two  classes of  translocations   are found  in malignancies. The first type is epitomized by the t(8;14) in Burkitt lymphoma,  one gene which is already actively transcribed  in the cell type, such as IGH  in B lymphocytes, is juxtaposed  to a gene such as MYC which is, by virtue of its resulting proximity to IGH, up-regulated. Other  translocations  such as the  t(8;21) in acute myeloid  leukaemia  (AML),  also described  by Janet  Rowley  in
1973  ,  and  the  t(9;22) in CML,  form fusion genes with a “new” gene product  which incorporates  part of the normal genes broken  at the  sites of translocation. In  the  case of t(8;21), the genes involved are the RUNX1T1 (originally named  ETO  after “Eight  Twenty-One”) gene on 8q22  and the RUNX1 (AML1) gene on 21q22  ; the t(9;22) causes a fusion of the BCR gene on 22q11.2 and ABL1  on 9q34  .  It is the altered func- tion  of these “new”  fusion genes that  appears to transform  the cell, as shown by the development of CML-like disorders in mice into which BCR–ABL1 constructs  have been inserted .
It took  time for the  medical and scientific worlds to realize the importance  of the  cytogenetic  discoveries of the  1960s  and
1970s.  It was necessary to convince clinicians that  the chromo- some  changes  being  described  in  the  marrow  and  peripheral blood  of their patients  with a variety of malignancies could pro- vide valuable information  about  the type and prognosis of these disorders. Many important clinical correlations were either identi- fied or confirmed in the International Workshops on Chromosomes in Leukaemia. These constituted gatherings  of physicians and sci- entists from around  the world who brought together case studies of  chromosome analyses together with  clinical and  laboratory data relating to each case.
The  first of these  was held  in Helsinki,  Finland  in August
1977  .   Laboratories   participated  from  Belgium,  Finland, Sweden,  England,  Germany,  and  the  USA and  the  participants reviewed the data of 223 patients with Ph1-positive CML and 279 patients with acute non-lymphocytic leukaemia. A number  of fur- ther workshops  were held  and  information  regarding  the  inci- dence  and  prognostic  significance of rearrangements in CML, AML, acute lymphoblastic leukaemia (ALL), and myelodysplastic syndromes (MDS) provided by these workshops formed the basis for all future studies. Subsequently, national and multinational clini- cal trial groups  have incorporated cytogenetic  studies into  their prospective trials and provided a wealth of data to show that cyto- genetic analysis is of diagnostic and prognostic importance in most haematological  malignancies and a number  of solid tumours.



It has only been by the careful observation of chromosome abnormalities and their correlations with clinical features that true insights have been obtained  as to the underlying  genetic basis of malignancy.
Whilst the basic cytogenetic methods used in laboratories around the  world  today  are very similar to those  first described  in the
1960s and 1970s,  there are areas where improvements have been made. Mitogens  were introduced  into cultures to induce chronic lymphoid malignancy  cells to divide in the late 1970s    but further refinements  and combinations  of mitogens  are still being discovered. For example, Chapter 9 describes a recently discovered method  that  enables chromosome  abnormalities  to be identified in the majority of cases of chronic lymphocytic leukaemias .



3. Introduction of FISH Testing

The identification  of the genes involved in chromosome  translo- cations paralleled the development  of in situ hybridization  (ISH)  and so allowed the most  significant advancement  in the field of cytogenetics to come into being. Early ISH studies used tritiated thymidine  to label the DNA fragments that  were used as probes . Slides were prepared by dropping  fixed cytogenetic suspen- sion onto the slide. After the subsequent  application of probe, the slide  was  immersed  in photographic emulsion,  wrapped  in foil, and  stored away in a light  proof  box for up to 2 months.  The localization of the probe was identified by “developing” the slide so that  silver granules were deposited  at the site of the tritiated thymidine  emissions. In expert hands,  this method  worked  well and  many of the  early gene  localizations  were made  using  this method. However,  the necessity of performing  most of the steps in total darkness and the time required for hybridization made this a most frustrating method  as, after 2 months,  it was entirely likely that the test had been unsuccessful and determining  the reasons for failure after such a time period was extremely difficult.
The  development  of  FISH   was  therefore   very  welcome indeed.  FISH  did not  require  total  darkness for successful com- pletion of testing and a result could be obtained  in 24 h. The ease with which routine  diagnostic laboratories  could establish FISH techniques now enabled them to be used in the routine diagnostic setting for the first time. Moreover,  FISH probes were developed that  allowed  the  cytogeneticist   to  determine   the  presence  or absence of extra copies of chromosomes, translocations,  and dele- tions in non-dividing cells. Initially, the probes were home-grown with single colour fluorescent signals for gains and losses of chro- mosomes  and  the  translocation probes  produced only  a single fusion signal. However,  the  increasing use of commercial  FISH


discover cancer,cancer treatment,cancer
discover cancer,cancer treatment,cancer

probes  in  the  clinical  setting  ensured  that  the  probe  designs evolved. False-positive and false-negative results were reduced  by designing  probes with built-in  controls or with a resulting signal pattern  that  could  not  be  readily duplicated  by accidental  co- localization in a normal patient control slide.
The ability to identify chromosome rearrangements in non- dividing cells has proved particularly useful in the  chronic  lym- phoid malignancies. FISH has been used with panels of probes to identify prognostic  subgroups  within  CLL    and plasma cell myeloma  .  FISH  has  also identified  cryptic  translocations such  as  the  t(12;21)  in  paediatric  ALL    and  t(4;14)  in myeloma   and  cryptic deletions  such as the  4q12  deletion that results in a PDGFRA–FIP1L1 fusion gene .  Both trans- locations  and  deletions  are invisible microscopically  and  could only be found by molecular methods.
Further  refinements  of FISH  methods  enabled  the effective painting  of each chromosome a different colour so that complex karyotypes could be elucidated  (30,  31) and the combination of FISH probes with fluorescent-labelled  antibodies to identify indi- vidual cell types has proved invaluable in identifying chromosome abnormalities  in disorders with variable marrow infiltration  such as myeloma (32). Such strides have been made in the last 20 years in the use of FISH  in cancer diagnosis that  it now seems incon- ceivable for cytogenetics laboratories  not to use FISH routinely.




4. Array-Based Karyotyping Methods





One of the innovative uses for FISH testing that has evolved has been  the  development  of  comparative   genomic   hybridization (CGH). CGH  involves the labelling of patient  DNA and normal control  DNA  with  green  and  red  fluorochromes,   respectively. The two DNAs are then allowed to compete for hybridization on a  slide containing  normal  chromosome preparations.  The  con- cept relies on a computer “reading” each chromosome and assess- ing  the  proportion  of  green-  and  red-labelled  DNA  that  has hybridized along the length of each chromosome. If there are no gains or losses of DNA in the patient  sample there should  be an equal proportion of patient and control DNA hybridized to each chromosome and an equal mixture of green and red fluorescence rendering  each chromosome yellow. In the event that there is loss of part of a chromosome in the patient genome,  there is a dispro- portionate amount  of control  DNA hybridizing  to that chromo- some and so it appears red. In contrast, an extra segment of DNA in the patient sample causes that segment of the normal chromo- some to appear green. CGH  has been used largely in the research setting  and achieved only limited  use in diagnostic  laboratories.



It produced a picture of genetic abnormalities across the genome without  the  need  to  produce metaphase  spreads  but  its major drawback was the resolution  only allowed the detection  of very large gains and losses of DNA.
However, the application of CGH  to arrays of bacterial artifi- cial chromosomes (BACs) or oligonucleotides dotted  onto  slides or  “chips”  has overcome  the  problem  of resolution  and  made CGH an enormously  powerful tool in cytogenetics.  Array CGH  is  capable  of mapping  deletions  or  amplifications  measured  in kilobases rather than megabases. It is also possible to design arrays that target  specific areas of interest  or cover the entire genome. To date, these arrays are not capable of detecting  balanced trans- locations  but  their  ability to  detect  changes  in copy number  is extraordinary. For the cancer cytogeneticist,  the challenge will be how to  interpret  the  vast amount  of information  generated  by these arrays. A leukaemia karyotype may appear to contain a simple chromosome abnormality but the array CGH  applied to the same genome  may uncover  hundreds  of sub-microscopic  rearrange- ments. Only large clinical trials incorporating array data collection will  enable  us to  determine  what  is important and  what  is not from these vast repositories of information.
Another  refinement  has been added to the use of arrays. It is now possible to detect uniparental disomy or, as those who work in the field of acquired abnormalities in cancer prefer, copy num- ber neutral  loss of heterozygosity  (LOH) or acquired isodisomy. LOH without loss of one copy of a DNA segment refers to regions of cancer  genomes  where  it appears that  one  chromosome has lost a region but has replaced it with a duplication  of the identical segment from the other homologue. The regions of LOH  can be identified  by the  use of single nucleotide  polymorphism  (SNP) arrays. SNP arrays detect  the presence of thousands  of polymor- phisms along the length of each chromosome, and so a stretch of DNA  without  any  variation  observed  between  the  two  homo- logues indicates either that only one copy of the region is present or, if it is clear that  there is no deletion,  that  duplication  of one copy has occurred. The biological impetus for this action appears to be, in many instances, to achieve a doubling  of a gene muta- tion such as TET2 mutations in chronic myelomonocytic  leukae- mia (CMML). CMML  usually has a normal karyotype but  SNP arrays have shown copy number neutral LOH  involving a region of the long arm of chromosome 4 in up to 35% of cases and most of these have been shown to carry homozygous TET2 mutations (33).  The  power  of these  arrays  appears  likely to  reveal many more genetic rearrangements in different cancers.
Ultimately, the challenge for cytogeneticists and for clinicians will be how to  use the  current  and  future  technologies to  best serve the needs of our patients. Conventional cytogenetics remains a powerful and affordable test that is integral to the management



of patients  with a wide variety of malignancies.  FISH,  too,  has become an important tool both  for diagnosis and to predict out- come  in many cancers. The  potential  of the  array technologies cannot  be under-estimated but  their  role in the  care of cancer patients  remains to be defined.  And thus,  just as the 1960s  and
1970s were exciting decades for cytogeneticists, so too will be the coming  years as we cope with integrating existing and emerging technologies.
discover cancer,cancer treatment,cancer
discover cancer,cancer treatment,cancer

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