CHP 13: Molecular Oncology

Molecular Basis of Cancer

Cancer is caused by nonlethal mutations in DNA. The mutations affect two types of genes: oncogenes and tumor-supressor genes. These genes control the cell division cycle and cell survival. cell cycle G1 (cell growth --> S (DNA synthesis and chromosome replication) -->G2 cell growth -->M (mitosis and cytokinesis). Oncogenes: Promote cell division. Oncogenes include cell membrane receptors that are bound by growth factors, hormones, and other extracellular signals. These receptors transduce signals through the cell membrane into the cytoplasm through a series of protein modifications that ultimately reach the nucleus and activate factors that initiate DNA synthesis (moving the cell from G1 to S phase of the cell cycle) or mitosis (moving from G2 to M). Oncogenes also support cell survival by inhibiting apoptosis, or self-directed cell death. More than 100 known oncogenes in human genome. Tumor Suppressors: Factors that control transcription, or the translation of genes required for cell division. They also participate in repairing DNA damage and promoting apoptosis. Tumor suppresors slow down or stop cell division by counteracting the movement of the cell from G1 to S or G2 to M phase. These two points are therefore referred to as the G1 check-point and G2 checkpoint in the cell division cycle. More than 30 tumor suppressor genes. (Kinda see it as cell wants to be really sure it wants to synthesize more DNA and really sure it wants to split into two cells in mitosis). In cancer cells, mutations in oncogenes are usually gain-of-function mutations resulting from amplification or translocation of DNA regions containing the genes or activating mutations that cause aberrant activity of the proteins. Mutations in tumor-supressor genes are usually loss-of-function mutations, resulting in inactivation of the tumor suppressor gene products. These mutations may occur through deletion, translocation, or mutation of the genes. Cell cycle fig 13.1 pg. 371 After mitosis M there are two haploid (one diploid) complements of chromosomes (46 chromosomes) in the G1 phase of the cell cycle. DNA is replicated during the S phase, resulting in four haploid (Two diploid complements) in the G2 phase. The chromosomes are distributed to two daughter cells at mitosis eith each receiving 46 chromosomes. Cancer results when the cell division cycle proceeds from G1 to S or G2 to M phase inappropriately.

Classification of Neoplasms (on study guide)

Cancer is divided into two broad groups: a)Solid Tumors b)Hematological malignancies a)Solid Tumors: Designated according to the tissue of origin as carcinomas (epithelial) or sarcomas (bone, cartilage, muscle, blood vessels, fat). Teratocarcinomas consist of multiple cell types. Benign tumors are named by adding the suffix -oma to the tissue of origin. For example an adenoma is a benign glandular growth. An andenocarcinoma is malignant. Metastasis is the movement of dislodged tumor cells from the original (primary) site to other locations. Only malignant tumors are metastatic. No one characteristic of the primary tumor predicts the likelihood of metatasis. Both tumor and normal cell factors are involved. Clinical analysis may be performed to detect the presence of relocated or circulating metastasized cells to aid in treatment strategy. b)Hematological malignancies: Tumors arising from white blood cells are referred to as leukemias and lymphomas. Leukemia: Neoplastic disease of blood-forming tissue in which large large numbers of white blood cells populate the bone marrow and peripheral blood. Lymphoma: Neoplasm of lymphocytes that forms discrete tissue masses. The difference between these diseases is not clear because lymphocytic leukemias and lymphomas display bone marrow and blood symptoms similar to those of leukemias. Also chronic lymphomas can progress to leukemia. Conversely, leukemias can display lymphomatuous masses without overpopulation of cells in the bone marrow. Within lymphomas, Hodgkin (or Hodgkin's) disease is histologically and clinically diff from all other types of lymphomas, termed non-Hodgkin (non-Hodgkin's) lymphoma(NHL). Plasma cell neoplasms. Which arise from terminally differentiated B cells, are also classified in a separate category. Some of the physiological symptoms of plasma cell tumors are related to the secretion of immunoglobulin fragments by these tumors. Leukemias arising from undifferentiated cells in the bone marrow are classified myeloid diseases, such as chronic (CML) and acute myeloid leukemia (AML) so 3 types of lymphomas: 1.Hodgkin 2.Non-Hodgkin 3.Plasma cell neoplasm 1 type of leukemia 1.Myeloid Myelodysplastic syndrome is a dysregulation of cells in a variety of differentiation states. google "Healthy bone marrow produces immature blood cells — called stem cells, progenitor cells, or blasts — that normally develop into mature, fully functional red blood cells, white blood cells, and platelets. In MDS, these stem cells may not mature and may accumulate in the bone marrow or they may have a shortened life span, resulting in fewer than normal mature blood cells in the circulation."

Mutation Spectra

Even with high-thoughput sequencing the allelic fraction of mutations/normal sequence and pateint characteristics (Age, gender, type and location of tumor) have to be considered. Biotech is rapidly developing standard reagent sets and sequencing panels (primers) for the most popular tests but differ from one supplier to another.

Molecular Analysis of Leukemia and Lymphoma Leukemias/lymphomas on study guide (e.g., CML, ALL, translocations, clonal rearrangements) Clonal Rearrangements:

Gene rearrangements: Gene rearrangements analyzed for hematological malignancies include V(D)J recombination, the normal intrachromosomal rearrangements in B and T lymphocytes, and the abnormal interchromsomal translocations that occur in any cell type. TLDR: During differentiation of B cells from precursor stem cells rearranegment, recombination, and mutation of the immunoglobulin V,D,J regions ultimatrly results in functional VJ results (light-chain) and VDJ (heavy-chain) genes. TLDR of the order of gene rearrangments: Immunoglobulin heavy chain gene rearrnagment preceds Ig light chain gene rearrangemnt during B cell differentiation in the bone marrow. Following succfesul IGH (heavy chain) recombination the Ig light chain loci then rearrnage first the Ig kappa locus. If the kappa rearrangment is non functional, the Ij lamba locus rearranges. With functional heavy and light chain rearrnagments the cell will develop into a mature naive B cell. V(D)J Recombination - To enhance antibody diversity, lymphocytes undergo normal genetic rearrangement of immunoglobulin (Ig) heavy- and light-chain genes and T-cell receptor genes. The gene rearrangement process is a series of intrachromosomal recombination events mediated by recombinase enzymes that recognize specific sequences flanking the gene segments. This process occurs indepdently in each lymphocyte, so that a diverse repertoire of antibodies is available to match any random invading antigen. TLDR: gene rearrangements are normal processes that occur in B and T lymphocytes as they mature from lymphoid stem cells. The genes coding for immunoglobulin heavy and light chains (IgH and IgL) begin the rearrngement process ine arly B cells and pre-B cells. The T-cell receptor genes rearrange in the order sigma, gamma, beta and alpha chains. 1)Immunoglobulin Heavy-Chain Gene Rearrangement in B cells (VDJ rearrangement I believe): Each antibody consists of two heavy chains and two light chains. The gene encoding the immunoglobulin heavy chain is located on chromosome 14. The unrearranged or germline configuration of the immunoglobulin heavy-chain locus consists of a series of gene segments or repeated exons coding for the functional parts of the antibody protein.These include 123 to 129 variable (VH) regions (38 to 46 functional gene segments) and 9 joining (JH) regions (6 functional), one of which will connect one variable region with a constant (CH) region of the antibody or receptor. There are 11 constant regions (9 functional). The immunoglobulin heavy-chain gene also contains 27 diversity (DH) regions (27 functional) one of which will connect the variable and joining regions. stopped taking detailed notes. The completion of gene-rearrangment process on only one of the two immunobloulin heavy chain gene alleles if referred to as allelic exclusion. AFTER VDJ rearrangement AID or activation-induced deaminase (AID) further changes DNA seq of the variable region. These variable regions are said to have undergone somatic hypermutation. After the gene is transcribed, one of the cosntant regions is joined to the final messenger RNA by splicing or by a secondary recombination event (Class switching). The maintenance of the constant regions in the DNA allows for antibody-type switching during the immune response. Some cells undergo class switch recombination placing the VDJ gene next to the genes encoding the IgG,IgE etc constant regions. The b cells will express a diff isotype during the secondary response. 2)Immunoglobulin Light-Chain Gene Rearrangement in B Cells: Two separate genes code for the Ig light chains the kappa locus on chromsome 2 and the lambda locus on chromosome 22. There is a kappa deleting element (KDE). This element determines the deletion of the IgK constant region in cells producing Ig lamda light chains 3)T-Cell Receptor Gene rearrangement : The T cell receptor is composed of two of four chains, alpha, Beta, gamma, delta with charatersitic structures resembling immunoglobulin V,J,C regions. The alpha and delta chains are encoded on chromsome 14 the gamma and beta are locate don chromsome 7. The four chains form pairs making two types of receptors alpha betta and gamma delta. T-cell receptor genes have fewer variable gene segmants than the immunobloulin genes. The gene sfor the gamma and alpha chains have no diveristy regions. pg. 490 advanced concepts has 3 ways diversity in antibodies is created. Rearrangment of the T-cell receptor chains proceeds in a similar manner as in the immunoglobulin genes. The V(D)J segments are joined together with addition or deletion of nts at the junctions between the gene segments. The extracellular domains of the T-cell receptor dimers are held in conformation by interchain disulfide bridges between cysteine residues in the T-cell receptor peptides.

Mutations in Hematological Malignancies a)Lymphoid Malignancies study guide 3 types!

Immunoglobulin and T-cell receptor gene rearrnagments are normal and take place regardless of malignancy. The unique nature of gene rearrangement system with cell-specific antibody and antibody receptor formation ios exploited in finding abnormal cell pop clonally derived from these cell specific features. Targeting these gene rearrangements can only be applied to tumors arising from lymphocytes (B and T cells). Other types of malignancies such as myeloid tumors cannot be analyzed using these targets. Cant do this target analysis with myeloid tumor malignancy. Translocations involved in tumors --> t(14;18)(q32;q21) The reciprocal translocation between the long arms of chromosomes 14 and 18 moves and dysregulates the BCL2 gene located on chromosome 18q21.3. BCL2 (B-cell leukemia and lymphoma 2 or B-cell CLL/lymphoma 2) is an oncogene. The gene product of BCL2 is a member of a group of related proteins that control apoptosis. The Bcl2 protein inhibits apoptosis in B lymphocytes, that is, enhances survival of cells that normally would die. Survival of genetically damaged cells may contribute to the development of tumors. One of the most frequent hematological malignancies follicular lymphoma is associated with t(14;18). The translocation partner of chrom 18 is chrom 14 in the vicinty of the Ig heavy chain gene. The trasnlocation may occur through cryptic recognition sites for the gene-rearrnagment recombinase enzymes on chromsome 18. There are several breakpoints in the chromsome 18 region 3' to the BCL2 most of which occur in the major breakpoint region (MBR). ABout 10 to 20% of the breakpoints fall into a cluster closer to the BCL2 gene thousands of bases from the MBR in the minor cluster region (MCR). An intermediate cluster region (ICR) and other breakpoints outside of MR and MCR have also been reported. Molecular detection of t(14;18): Southern blot with a probe to MBR region of chromosome 18 will reveal the translocation by the presnce of bands diff from those expected from a normal chromsome 18. (positive specimen has extra bands). On the results with positive sample you would see extra bands in the positive specimen because the positive specimen introduced a restriction site so is cut by the enzyme. Using PCR or qPCR. Forward primers to chromosome 18 and reverse primers complementary to the Ig heavy chain joining region will yield a product only if the two chromosomes have been joined by translocation. The amplicons are visualized by gel electrophoresis or with a qPCR probe. The t(14;18) can be used to quantify tumor load by qPCR. most methods using this include a standard curve for regression analysis of the test sample measurements. A std curve is established using cultured cells with the t(14;18) counted and mixed at different proportions with cultured cells that do not have the translocation. The illustration pg. 399 represents DNA isolated from each mixture of cells analyzed by qPCR with a Taqman probe. Alternatively, internal controls such as known amounts of plasmid DNA may be added to the test specimens. In this method, the amount of translocated cells (or translocated chromosomes) is determined relative to the internal control. Most frequently the results are reported as the percentage of translocated cells in the specimen type. For instance, the raw number of translocated cells is determined using the standard curve of threshold cycles versus the known cell count. The raw number of cells is then divided by the number of total cells represented in the PCR reaction. For example: A dliution series ranging from 50 to 10,000 translocation cells in 1 million untrasnlocated cells, the strand curved generates the formula y=1.5631[Ln(x)] + 42.396 where y is the threshold cycle number and x is the number of translocated cells (50 ng of DNA was used per sample). So if a sample crosses the fluorescence threshold at y=36 cycles (average of duplicate measurements) the number of cells x is approximately 60 cells. Assuming that 50 ng of DNA represents 7,500 cells (1 ng of DNA = approximately 150 cells). 60/7,500 = .008. .008 X 100 = 0.8% translocated cells in the specimen. The main limitation of any PCR procedure targeting the t(14;18) is the inability of the primers to detect all of the possible breakpoints on chromosome 18. Primer pairs, sets of primer pairs and novel probe methods have been designed. Test reports should include an estimation of false-negative results expected due to breakpoints that remove binding sites for the primers used. Unless a trasnlocation has been previously obervsed by a given PCR method a negative result should acknowledge that a trasnlocation may be present but undetecbakle with the primers used. t(11;14)(q13;q32): This translocation joings the Ig heavy chain gene rion on chromsome 14 with part of the long arm of chromsome 11. The cyclin D1 (CCND1) gene also called the parathyroid andeomatosis 1 gene (PRAD1) or BCL1 on chromsome 11 is attached to the long arm of chromsome 14 in the intron between the Ig heavy chain gene joining and constant regions. The trasnlocation increases expression of CCNDI, resulting in passage of the cell cycle from the G1 to the S phase. This is primarly found in mantle cell lymphoma (MCL) but may also be present in chromic lymphocytic leukemia CLL, B-prolymphocytic leukemia, plasma cell leukemia, multiple myeloma, and splenic lymphoma. Mantle cell lymphoma without the t(11;14) is likely to show expression of the transcription factor SOX11. SOX11 therefore represents an inrpotant performed by IHC or western blot. Methods for detectiuon similar to 11;18. Southern blot methods have been replaced with mopstly PCR and RT-PCR. If primers miss some translocations due to variations in break points then then use FISH or flow cytometry analysis. Of the breakpoints on chromsome 11 80% are in the major trasnlocation cluster 5' to the CCDN1 gene. PCR analysis detects 40-60% of the trasnlocation breakpoints. The PCR product can be detected by qPCR fluorescence or by gel or capillary electrophoresis. Primers are complementary to the joining region of Ig heavy chain gene and the CCND1 gene. advanced concept: many molecular events can result in overexpression of c-myc. The DNA virus Epstein-Barr virus (EBV) is associated with Burkitt lymphoma. EBV induces cell prolieferation and thus more oppotunity for translocation events. Certain types of another DNA virus human papillomavirus inserted in the vicinity of c-myc cause overexpression of the gene. t(8;14)(q24;q11) Burkitt lymphoma The avian myelocytomatosis viral oncogene homolog (c-myc) gene on chromosome 8 is one member of a gene family including n-myc and l-myc. The c-myc gene codes for a helix-loop-helix/leucine zipper transcription factor that binds to another protein, Max, and activates trasncription of other genes. The translocations at 2;8 and 8;22 are found in about 10% of burkitt lymphomas. In t(8;14) the breakpoints on chromosome 8 are spread over a 190kbp region 5' to and within the c-myc gene. As a result of translocation the c-myc gene is separated from its normal promoter and regulatory region and moved into the switch recombination region of the Ig heavy chain gene on chromosme 14. In the t(2;8) and t(8;22) translocations the chromsome 8 breakpoints are 3' to the gene and c-myc is moved into the Ig kappa or lambda locus. Also c-myc can translocate into T-cell recpetor alpha gene also. Detection: Labs used southern blot analysis with a probe complementary to exon 3 of the c-myc gene such as a 32P or digoxigenin labeled 1.4 kb ClaI-EcoRI restriction fragment. Interphase FISH and CISH are now used with seprate probes to chromsomes 8 and 14 or with chromsome 14 probe pairs. Amplification of the gene can be detected by FISH using a 120kb c-myc (8q24.12-q24.13) fluorescent probe. The figure on pg 400 says: CISH. Two probes labeled with biotin or digoxigenin are complementary to sequences flanking the chromosome 14 breakpoint. In the abensce of the translocarion the probes will appear next to one another in the nucleus. The translocation will move one probe to chromosme 14, leaving the other behind on chromosome 8 resulting in sepraration of signals in the nucleus. So will see one probe another probe color and then one chromosome where the probe color are next to each other (The normal non translocated chromosome 8). Invitrogen website: CISH, or chromogenic in situ hybridization, is a process in which a labeled complementary DNA or RNA strand is used to localize a specific DNA or RNA sequence in a tissue specimen. CISH methodology may be used to evaluate gene amplification, gene deletion, chromosome translocation, and chromosome number. CISH utilizes conventional peroxidase or alkaline phosphatase reactions visualized under a standard bright-field microscope, and is applicable to formalin-fixed, paraffin-embedded (FFPE) tissues, blood or bone marrow smears, metaphase chromosome spreads, and fixed Myc protein expression is measured by IHC

Banding Patterns

Interpretation of clonality by PCR depends on gel banding patterns. False negative results may arise from primers that do not match the gene rearrangement in the tumor. On the other hand, artifactual single bands will produce false-positive results. Patterns of multiple single bands may arise from specimens with low cell numbers, such as cerebrospinal fluid or paraffin sections. Do not indicate monoclonality just that only a few cells are present. These cells may or may not be maligant. False single-band patterns may also occur within polyclonal smears detected on non-denaturing polyacrylamide gels (PAGEs). Heteroduplex analysis is recommended if amplicons are resolved by nondenaturing PAGEs. Similar problems occur with resolution by capillary electrophoresis where peak heights and widths are compared to distinguish wide polyclonal peaks from narrow monoclonal spikes. Pretretaments such as eosin staining may yield false single bands by capillary electrophoresis.

Microsatellite Instability (unsure if type of solid tumor mutation)

Lynch syndrome, or hereditary nonpolyposis colorectal cancer HNPCC is an inherited cancer predispotion syndrome, accounting for 3 to 5% of colon cancers and 3% of endometrial cancers. There is also an increased risdk for dveloping other types of cancers, including cancer of the stomach, breast, ovary, pancreas, prostate, urinary tract, liver, kidney or bile duct. Predisposition to cancer in this syndrome is caused by mutations in the mismatch repair (MMR) protein complexes, including mutS homologs, MSH2, MSH6, mutL, homolog MLH1, and the human postmeiotic segregation hPMS2. These complexes are responsible for correcting replicative errors and mismatched bases in DNA. The MMR system originally discovered in bacteria and further in yeast. Similar (homologous) genes were subsequently identified in humans and named after the bacterial and yeast genes. The protein complex binds to mismatched bases in the DNA double helix or loops formed by replicative errors. The repair is methyl-directed. At the end of S phase (DNA replcation) the system recognizes errors in the newly synthesized (un methyalted) daughter strand and uses the template strand which is methaylted as a guide for reapir. Included in the types of DNA lesions that are repaired by this system are replication errors (RERs) caused by slippage between the replication apparatus and the DNA template. RERs occur espeically in microsatlletes where one to three nt are repeated in the DNA sequence. If the errors remain in the DNA until the end of the next round of replication, new alleles will arise, generating increasing numbers of alleles for the locus, or microsatellite instabilities (MSI). (so if error is not repaired the next round of replication will create a new allele of the original locus. Additional uncorrected errors will produce more alleles). In contrast, a stable locus will retain the same alleles through many rounds of replication. Microsateliite slippage occurs every 1,000 to 10,000 nromal cell divisions most of which are repaired in normal cells. Dysfunction of one or more components of the MMR system will result in MSI an increase in the number of alleles due to loss of repair. The majority of MMR mutations in Lynch syndrome are found in the MSH2 and MLH1 genes. Mutations in hPMS2 account for 9% of MMR variants compared with 39% MLH1 and 33% MSH2 and 19% MSH6. Mutations in hPMS1 and MSH3 are rare. Although direct sequencing of the affected genes is definitive and identifies the specific mutation ina. family, the test may miss mutations outside of the structural gene sequences or in other genes. Screening for MSI normal MMR proteins by IHC is often the first step in MSI analysis. Lack of staining of the normal protein is evidence of loss of MMR function.Bevcause this loss of MMR gene function causes MSI, MSI can be used to screen indirectly for mutations in the MMR genes. With an inherited mutation in one copy of an MMR gene, somatic mutation of the remaining copy will result in the MSI phenotype in tumor cells. MSI, therefore will be apparent in the tumor where both functional copies of the gene have been lost but not in normal tissue that retains one normal copy of the gene. To perform MSI analysis, therefore, normal and tumor tissue from the patient must be compared. MSI is apparent from the increased number of alleles in the tumor tissue compared with that in the normal tissue after PCR amplification of microsatellite loci and gel electrophoresis or capillary electrophoresis. The detection of instability (more bands or peaks in the tumor tissue compared with normal tissue) is strong evidence for lynch syndrome. Increased alleles in the tumor scans reveals instability at those loci. For cap electrophoresis the stable loci look the same in normal and tumor tissue. Screening two mononucleotide-repeat (more sensitive than di) loci, BAT25 and BAT26 and three dinucleotoide-repeat loci, D5S346, D2S123, and D17S250 is sufficient for determination of MSI. If at least two of the five or if more than 30% of loci show instability, the specimen is classified as having high instability (MSI-H). Tumors showing MSI in one or a minority if loci tested are classified as low instability (MSI-L). If no instability is detected the tumor is stable (MSS). Initially MSI-L and MSI-H tumors are interpreted as microsatellite stable, and MSI-H tumors are considered microstaelitte unstable. More recent clinical correlations suggest that MSL-L may have specific implications. MSI-H is reported as MSH unstable with an increased likelihood of Lynch syndrome. IF MSI is discovered, the inherited mutation can be confirmed by direct sequencing of the MMR genes. Sequencing will not detect epigenetic silencing of the MLH1 gene, which can result in MSI. A separate test for MLH1 promoter methylation bu bisulfite sequencing or methylation specific PCR may also be performed. Testing for deletion of the EPCAM gene by sequencing or multiplex dependent probe amplification (MLPA) is another approach to assess linked deletion of MMR loci. The cost of testing increases with IHC,PCR, and sequencing respectively.

Detection of Clonality

Polyclonal means normal and good monoclonal gene rearrangement means bad and positive and could be cancer. Gene rearrangements occur independently in each lymphocyte so that a normal population of lymphocytes is polyclonal (polytypic) with respect to their rearranged immunglobuin or T-cell receptor genes. Overrepresentation of a single rearrangment in a specimen cell population can be an indication and characteristic of a lymphoma or leukemia. When over 1% of cells make the same gene rearrangment, the cell population is referred to as a monoclonal (monotypic) with respect to the rearranged genes. Differentiate large monotypic population of tumor cells from a reactive clone or oligoclone or reactive clone of cells responding to an antigen. Oligoclones are not only smaller but transient in nature so shouldnt be consistenly present. 1)Molecular Analysis of Immunoglobulin Heavy-Chain Gene Clonality: TLDR: southern blot with RE and probes to detect gene rearrangements or PCR or cytogenetics. Clonality can be detected by protein and nucleic acid analyses.First tests for this were southern blot for nucleic acids. With regard to heavy chain gene, restriction sites were mapped in the germline confirguation. BamH1, EcoR1, and HindIII were RE commonly used. When the germline sequence is rearranged the RE sites are moved, created, or deleted, reuslting in a unquie restriction pattern for every gene rearrangment. For the southern blot, DNA cut with the restriction enzymes is transferred to a nitrocellulose membrane and probed to the joining region of the gene. Normal results should reveal the expected fragments generated from the germline DNA, in the example, an 18kbp EcoRI fragment, an 18-kbp BamHi fragment and an 11-kbp HindIII fragment. The normal fragments are visible for two reasons. First, a normal patient specimen contains cells other than lymphocytes that do not undergo gene rearrangement. Second reacon for the germline bands is that only one chromosome in a lymphocyte undergoes gene rearrangement, leaving the homologous chromosome in the germline ststate. In a normal specimen, there will be millions of immunoglobulin gene rarrangements, but no one rearrnagment is present in high enough amounts to be visible as nongermline bands on the membrane or autoradiogram; thus, only the germline bands will be visible. If however 2-5% of the cells in the specimen consist of a clone of cells all with the same gene rearrangement that clone will be detected by the presence of additional bands diff from the germline bands. Interpretation of the results is positive if extra bands are present and negative if only the germline bands are present. Analysis of clonality by southern blot affords the advantage of detecting all gene rearrnagments, including incomplete rearrnagments involivng only the D and J regions of the gene. Cons of southern blot: The southern blot method is limited by the requirement for at least 20 to 30ug of quality DNA from the specimen. Artifcats such as cross-hybridizations may complicate the interpretation of southern blot results. PCR is the most frequent approach for the detection of gene rearrangements. For this method forward and primers complementary to the variable region and reverse primers to the joining or constant regions of the b-cell-rearranged genes are used. The resulting ampilicons are resolved by agarose, polyacrylamide, or capillary electrophoresis. If 1% at least of the sample is monoclonal gene rearrangement product will be amplified preferentially and revealed as a sharp band on gel. In polyclonal specimen amplification products in a range of sizes will result. A normal cell population will yield amplicons consisting of fragments of diff lengths. Yielding a cloud or smear of bands or series of peaks. A monoclonal cell population will yield a predominat or single band or peak. Presence of which is interpreetd as a positive result. Con of PCR: a)With regard to gene rearrangement studies - Inability to amplify all possible gene rearrangements by PCR as primer binding sites are lost during recombination and somatic mutation. ex:Limitation to detection of clonality by PCR is the loss of the FR3 primer binding site due to the rearrangement process which may destroy or remove the sequences bound by the FR3 primer. So some labs use FR2 and FR1 framework 1 and 2. other methods utilize primers to the leader region in addition to the FR primers. These primers increase the number of gene rearrangements that can be amplified and therefore detected by this assay. Deletion of the IgH locus can occur in some lymphomas precluding amplification with any primers. If the gene rearrangment cannot be amplified, cytogenetics may be used to assess clonality at the immunoglobulin heavy chain gene locus. For immunoglobulin heavy-chain gene rearrangement, diff approaches to the variable region primer binding sites have been taken. The immunoglobulin heavy chain gene variable region is divided into two types of domains. The complementarity determining regions (CDRs) code for the a.a that will contact the antigen. The CDRs, are the most variable therefore or unstable in sequence. The framework regions FRs code for a.a. that have more of a structural role in the antibody protein and are more stable in sequence. Standard methods for clonality utilize a forward consensus primer to the innermost framework region FR3 and the reverse primer complementary to the joining region. The consensus primer has sequences that match the most frequently occurring sequences in the FR3 region and may not be identical to any one sequence. Enough nt will hydrogen bond however to natch the Most rearrnaged variable regions. Primers directed at the diversity region are useful for amplification of the germline configuration of the immunoglobulin heavy-chain genes as well as DH-JH rearrangements. Primers complementary to the seven family spefici sequences of the diversity region and a primer to the 5' most joining region (JH1) are use to amplifiy the DH -JH junction. The primer complementary to the DH7-27 segment, the sequences cloeset to the joining region will yield a defined product from the Ig HEAVY CHAIN GENE IN THE GERMLINE CONFIGUARITON. The DH7 primer will yield a specific 350 bp product from the unrearranged germ line gene due to the short distance between the DH7 gene segment and the joining region primer. Diversity region primers are also useful for targeting incomplete rearrangements where the variable region primer binding sites are lost. Another approach to the detection of B-cel clonality is to make patient-specific primers. For this method, a consensus primer is used to amplify the rearranged gene from a positive specimen. The amplification product is then purified from the gel or from the PCR reaction mix and sequenced. Primers exactly matching the variable region sequence of that specimen are then manufactured for use on subsequent samples. Advantage is that it is more sensitive because fewer or none of the gene rearrangements from normal cells are amplified. Furthermore, the tumor load can be measured quantitatively by real-time PCR (RT-PCR). Con: more time consuming to perform and the primers used are patient specific. Moreover, in a patient with a chronic condition and for whom monitoring is likely to be done tumor cells may undergo futher mutation in the variable region and inactiivate the specific primer binding requriing the manufacture of new primers. 2)Molecular Analysis of Immunoglobulin Light-Chain Gene Rearrangement: The immunglobulin light chain genes are also targets for clonality detection. In addition to protein analysis, southern blot, PCR, and RT-PCR have been used to detect light gene clonality. Targeting light chain gene is useful for tumors arising from terminally differentiated B cells (plasma cells) that have undergone extensive somatic hypermutation at the heavy chain gene locus. In these tumors the rearranged heavy chain genes are frequently unamplfiable, yielding false negative results because of accumulation of base changes in the variable region primer bidning sites. Most of these tumors are amplifiable however at the light chain genes. Gene rearrangements in the kappa light chain locus are amplfiable using primers complemntary to the sequence families of the Vk region or to the intron between the Jk regions and Ck. Opposing primers are complementary to Ck and to one or more of the five joining regions. KDE can also be used for a primer binding sites. Using KDE allows for detection of lambda expressing cells that have deleted Jk or Ck. Immunoglobulin heavy chain gene rearrnagment preceds Ig light chain gene rearrangemnt during B cell differentiation in the bone marrow. Following succfesul IGH (heavy chain) recombination the Ig light chain loci then rearrnage first the Ig kappa locus. If the kappa rearrangment is non functional, the Ij lamba locus rearranges. With functional heavy and light chain rearrnagments the cell will develop into a mature naive B cell. Therefore detection of clonality at the Iglamba locus on chromsome 22 is also useful for confirming or monitoring the diagnosis of b-cel leukemias and lymphomas. Forward primers complementary to the Vlambda gene segments and reverse primers to the Jlambda and C lambda gene segments are frequently used for these assays. RT PCR is used with the constant region primers to eliminate the intron between the constant and joining regions. 3)Molecular Analysis of T-cell receptor Gene Rearrangement Like B-cell clonality and B-cell malignancies, clonality of T-cell receptor gene rearrnagments may demonstrate that a high white cell count is of T cell origin or the presence of a clonal T-cell infiltrate accompanycing another malignancy. Clonality may also be used to moniotr treatment efficacy. Cloncal T cel receptor gene rearrangements are detected in a manner simialr to the Ig gene reaarngements by southern blot, PCR and sequencing. For southern blot studies, probes complementary to the variable, joining, and diversity regions of the Tcell receptor genes are used to detect monoclonal populations. Just as with Ig rearrnagments no one gene rearrnagment should be visible in a normal specimen. The presence of a large monoclonal population is revealerd by the abnds dectable in addition to the germline bands seen in the normal control. The T-cell receptor gene rearrnagment assays done by PCR most often target the TCRgamma gene. Assays are also performed on the TCR beta and TCR gamma genes. Dectection of gene rearrnagments in TCR alpha is difficult due to the 85kb lenght of the Jalpha gene. The TCR alpha gene rearrangements may be inferred from the TCR gamma gene DELETIONS. Primers are designed complementary to rearranged gene segments. Multiple primer sets are often used to ensure detection of the maximum gene rearrangement. PCR will yield a product consistent with a single-gene rearrangement in a positive sample, whereas a normal sample will yield a polyclonal pattern. Due to the limited range of lengths of the population of rearranged T-cell receptor genes heteroduplex analysis may be used to improve the resolution of poly and monoclonal patterns. T cell receptors do not undergo somatic hypermutation when a given T cell is exposed to antigen. Therefore, the specificty of a given T-cell clone maintains a common antigen specificty which is the clonotype of that population. Massive parallel sequencing of T-cell receptors and alignment of the data to the germline gene sequences can reveal a rearrnagment. By taking a tally of the reads a full pic of T CELL receptor clonotype can be generated.

Liquid Biopsy

SOlid tumors release cells and nucleic acids into circulation. This may have a role in metastatic cancers. Liquid biopsy is usually from blood plasma or serum or urine but may also be done on other body fluids. Two approaches: Cell free or circulating free nucleic acids (cfDNA or cfRNA), exosomes (Cell-derived vesicles carrying nucleic acid or other moelcules) and circulating tumor cells (CTC). Circulating nucleic acid analysis can be classified as targeted or untargeted. Targeted procedures are directed at specific mutations using sequencing or PCR methods specifically designed for those mutations or rearrangements. These methods can reach high sensitivity (1% to 0.001%) and provide an estimation of mutant allele frequency. Untargeted methods assess whole exomes or more frequently gene panel by sequencing and comparative genomic hybdrization (CGH) array technology (digital karyotyping), with sensitivties of 0.2% to 2%. They can detect copy number variations and structural abnormalities Plasma DNA is the most frequent source material. Plasma RNA has seen less clinical application but has the potential to provide additional info on tumor gene expression and tissue specific gene expression. The latter might be used to determine the tissue of original of tumors as well. Urine has also been a source of genetic information from cfDNA. Urine analysis has been applied to the detection of acquired mutations in lung cancer after treatment with targeted therapies. Circulating tumor cells CTCs were considered to be a mechanism of tumor metastases from one tissue site to another. Although low in number they can travel in clusters of 2 to more than 50 cells. There are several technologies for the isolation of CTCs. Antibody capture, using immobilized antibodies to epithelial cell adhesion molecules (EpCAM), most frequently used ( i beleiev the antibodies grab onto the antigens on tumor cells). Alternatively, negative depletion of leukocytes can also be performed. Cell morphology and physical characteristics such as size (epithelial cancer cells are larger than leukocytes) or density may be used for selection. Once the tumor cells are enriched they may be stained and other characteristics measured. Extracted DNA is tested using allele-specific PCR or other sensitive methods. Gene-expression profiling and translocations may be found in extracted cDNA made from extracted RNA. cfDNA and CTC are also sources of epigenetic info. Methylation patterns specific to tissues or tumor state can be assessed using sequencing, methylation-specific PCR, and methylation arrays. miRNA can be isolated from plasma as well. Not in clinical routine yet but the presence or absence or expression levels of particular miRNA species may provide info on prognosis or tumor state.

Oncology (on study guide)

Study of tumors

Loss of Hetereozygosity (seen in solid tumors I believe)

TLDR: LOH is detected by PCR and cap electrophoresis of heterozygous STR loci linked to disease genes. A deletion or loss of the normal allele uncovering a recessive mutant allele is identified by the loss of the STR linked to the normal gene. Increased risk of cancer is a concern for members of families carrying cancer-predisposition mutations. These mutations are recessive regarding the cancer or malignant cell phenotype but dominant regarding the cancer risk. Therefore, relatives of patients with suspected inherited conditions are tested for purposes of cancer surveillance and family planning. Once the familial gene mutation is identified in the patient or proband (like patient zero of family of the tested subject), targeted tests for the mutation in other family members is performed. In an inherited condition such as Lynch syndrome or a history of breast and ovarian cancer (HBOC), blood samples are sufficient for mutation analysis. In tumor cells, further testing for loss of heterozygisty (LOH) may be performed. LOH reveals the loss of the "good allele" at a locus, uncovering the homologous locus with with a recessive mutation. LOH can be detected by PCR amplification of heterozygous STR or variable-number tandem repeat (VNTR) closely linked to the disease gene followed by capillary electrophoresis. Amplification of loci in tumor cells with LOH will reveal a loss of the allele linked to the normal allele of the gene when comared with the mutant allele.Comparing peak heights in normal (N) and tumor (T) tissues the formula for LOH is see pg. 385 peak height of normal allele in N / peak height of normal allele in T all divided by peak height of mutant allele in T over peak height of mutant allele in N. A ratio of less than 0.5 or more than 2 indicates LOH. Alternately, LOH is assumed if the peak height of the normal allele in the tumor is less than 40% of the height of the normal allele in the normal DNA. Cons: Technical artifacts such as allelic dropout or PCR bias complicaye interpretation. When analyzing tumor tissue may be necessary to test more than one area of a tumor to confim LOH.

Gene and Chromosomal Mutations in Solid Tumors (on study guide)

TLDR:Remember locations of gene rearrnagements on chromsomes. Detection by IHC is used to detect increased protein, and sequencing is used for detection of missense mutations. FISH or sequencing and qPCR and RT-qPCR to measure expression levels. Also SSCP, and southern blot. "a)Human Epidermal Growth Factor Receptor 2, HER2/neu/erb-b2 1(17q21.1) What it does: Found in glioblastoma in rats. Gene product is a 185 kd cell membrane protein that adds phosphate groups to tyrosine on itself and other proteins (tyrosine kinase activity). The receptor is one of several transmembrane proteins with tyrosine-kinase activity. It is very similar to a family of epidermal-growth-factor receptors that is overexpressed in some cancers. These molecules share similarities in that they include a kinase domain, transmembrane domain, cysteine-rich domain, and immunoglobulin-like domain. In normal cells, this protein is required for cells to grow and divide. HER2/neu is overexpressed in 25% to 30% of human breast cancers, in which overexpression of HER2/neu is a predictor of a more aggressive growth and metastasis of the tumor cells. Therapy: It is also an indication for the use of anti-HER2/neu antibody drug therapy, which works best on tumors overexpressing HER2/neu. Herceptin therapy is indicated presently for women with NER2/neu-positive(HER2/neu overexpressed) breast cancer. note: remember herceptin as HER is what it targets! google"Herceptin is a monoclonal antibody specifically designed to target HER2 receptors, which are transmembrane receptors on both normal cells and HER2+ tumor cells." To detect: 1) IHC. Overexpression of the HER2/neu oncogene is performed by immunohistochemistry (IHC) using monoclonal and polycloncal antibodies to detect the HER2/neu protein. IHC test works best on fresh or frozen tissue. google" A Polyclonal Antibody represents a collection of antibodies from different B cells that recognize multiple epitopes on the same antigen. Each of these individual antibodies recognizes a unique epitope that is located on that antigen. ... In general, ability to detect multiple epitopes gives more robust detection." 2)FISH, which measures DNA and RNA of Her2/neu is more reliable than IHC, especially in older fixed tissue but is less readily available. 3)Southern, northern, and western studies have shown that HER2/neu gene amplification is highly correlated with the presence of increased HER2/neu RNA and protein. In contrast to IHC, which measures increased amounts of HER2/neu protein, FISH directly detects increased copy numbers of the HER2/neu gene in DNA likely responsible for the increased protein. (so not just overexpressed is also that there is a change in copy number this is a genome mutation i believe). HER2/neu gene amplification occurs as a result of tandem duplication of the gene or other genetic events as the tumor cells continue to divide. FISH testing for HER2/neu gene amplification requires a labeled probe for the HER2/neu gene and a differently labeled probe for the centromere of chromosome 17. Ex: A probe that spans the entire HER2/neu gene labeled in orange and a probe that binds the centromere of chromosome 17 labeled in green should yield two green signals, each associated with an orange signal per nucleus. The copy number of HER2/neu relative to centromere 17 indicates whether HER2/neu is amplified (present in multiple copies on the same chromosome). Data are reported as a ratio of the number of HER2 signals to chromosome 17 centromere signals. A ratio of more than 2 is considered positive or amplified. The number of signals is enumerated in 50 to 100 cells. 4)Chromogenic in situ hybridization (CISH) is another method that has been used to detect HER2/neu gene amplification. Using a standard bright-field micrcope, CISH technology also detects deletions, translocations, or a change in the number of chromosomes. An attarctive feature of CISH is that the slide images are permanent, facilitating dopcumentation and consultations. 5)Another bright field imaging method Silver-enhanced in situ hybridization (SISH), introduced for the determination of HER2 status, has a high correlation with FISH. Initially, in situ hybridization with chromogenic detection by gene-specific probes was limited by high background and low signal intesnity. Probes with reptitive sequences removed were disgned to increase specificty. Although FISH, SISH, and CISH are more accurate and less subjective methods than IHC, IHC is faster, less expensive, and allows the pathologist to asses target gene expression along with other visible landmarks on the slide. Furthermore, protein overepxression (Detectable by IHC) can occur without gene amplification (Detectable by ISH). (So IHC can catch the HER/neu in some cases while ISH can't if no gene amplification). Some labs use IHC as an initial screening method and then confirm the results with ISH. ERBB family of growth factor receptors includes the HER2 receptor and EGFR. Factors bind them such as EGF, TGF-a, heregulins, neuregulins. EGF, NRGs, and heregulins are small peptides that are active in the development of various cell types, such as gastric mucosa, the heart, and the nervous system. b)Epidermal Growth Factor Receptor, EGFR (7p12) Like HER2/neu the epidermal growth factor receptor gene (EGFR, ERBB1) is a member of the ERBB family of growth factor receptors that also includes ERB3/HER3 and ERB4/HER4. All of these proteins are located in the cell membrane and form dimers with one another upon binding of growth factor from outside the cell. Binding of growth factors evokes tyrosine-kinase activity EGFR is frequently overexpressed in solid tumors. Overexpression has been observed in a variety of tumor types. For this reason, the EGF receptor has been an attractive target for the design of therapeutic drugs. Treatment: a)Monoclonal antibodies were developed to block ligand (Growth factor) binding to the receptor. b)Agents were also designed to inhibit kinase activity of the receptor. These are called tyrosine-kinase inhibiotrs (TKIs) Detection: 1)IHC analysis of EGFR protein expression, similar to the testing for HER2/neu protein overexpression was assessed for correlation with response to monoclonal antibody drugs and methods approved by the U.S. FDA for this application. Con: Interpretation of the results of EGFR expression testing and the predictive value of the test are not always straightforward, however. 2)Quantitative polymerase chain reaction qPCR has also been proposed to assess EGFR gene copy number through increased gene expression. Particular mutations in the kinase domain of the EGFR protein have proven better predictors of response to tyrosine-kinase inhibiting agents. 3)These mutations can be detected by a number of methods, including sequence-specific PCR, single-strand conformational polymoprhism (SSCP), and direct sequencing (such as NGS). Testing for predictors of response or prognosis, even with comprehensive sequencing, is complex because multiple clinical and genetic factors contribute to the response to targeted therapies and the natural course of the tumor. These include intronic polymoprhisms in the EGFR gene, expression of other components of the signal transduction pathway, or other tumor suppressors such as p53. Multiple molecular and morphological characteristics can be found in single tumors as a result of tumor heterogeneity. c)Kirsten Rat Sarcoma Viral Oncogene Homolog, k-ras (12p12); Neuroblastoma ras, N-ras (1p13); and Harvey Rat Sarcoma Viral Oncogene Homolog, H-ras (11p15) Kras,N-ras,H-ras Singals from extracellular stimuli, such as growth factors ad hormones are transmitted through the cell cytoplasm to the nucleus, resulting in cell proliferation or differentiation. The mitogen-activated protein kinase (MAPK) pathway is a cascade of phosphorylation events that transduces growth signals from from the cell membrane to the nucleus. Critical components of this pathway are small proteins that bind to GTP in order to become active. These small GTP-binding proteins include the ras genes that receive signal from the cell surface proteins and activate the initial steps of the MAPK cascade. Gain of function mutations in ras proto-oncogenes occur in many types of cancers. Mitogens: Normally cells grow in the presence of nutrients and factors that stimulate cell divison (mitogens). Lack of mitogen stimulation results in cell arrest or apoptosis. If oncogene or suppressor gene mutations stimulate aberrant growth signals, cells grow in the abensce of controlled stimulation. Mammals have three diff ras genes that produce four similar proteins K-ras, N-ras, and H-has. The Ras proteins differ only in their carboxy termini, the end of the proteins that anchor them to the inner surface of the membrane. Because they bind and hydrolze GTP for energy, the ras genes are members of a family of G-proteins. The GTP hydrolysis is regulated by GTPase-activating proteins GAPs. Activation of membrane-bound K-ras is initiated by activated receptors bound to mitogens (growth factors or hormones). Active K-ras bound to GTP then initiates a cascade of phosphorylation events that ends in the nucleus where transcription factors modulate gene expression. GDP/GTP exchange on K-ras is modulated by GTPase-activating proteins (GAPS), guanosine nculeotide exhcnage facotrs (GEFs), and guanosine nucleotide dissociation inhibitors (GDIs). Ras proteins are anchored to the cell membrane through farnesyl groups and palmitoyl groups on the ras proteins. K-ras has only farnesyl groups where as N-ras and H-ras have both farnesyl and palmitoyl groups. (Think kentucky fried). google" GTPase-activating proteins or GTPase-accelerating proteins (GAPs) are a family of regulatory proteins whose members can bind to activated G proteins and stimulate their GTPase activity, with the result of terminating the signaling event." advanced: regulation of ras GTPase activity is controlled by rasGAP. Several other GTPase-activating proteins (GAPs) besides rasGAP are important in signal transduction. Two clinically important proteins of the GAP family of proteins are the gene product of the neruofibromatosis type-1 (NF1) locus and the gene prodcut of the breakpoint cluster region (BCR) gene. The NF1 gene is a tumor suppresor gene and the protein encoded is called neruofibromin. The BCR locus is rearranged in the philadelphia+chromsome (Ph+) oberseved in chronic myelogenous leukemais and acute lymphocytic leukemias. How it works: Mutations in K-ras are the most common oncogene mutations in human cancers. The most frequently occurring mutations are located in codons 12,13,22 and 61 in exons 2 and 3 of KRAS gene. Clinically significant mutations may also be found in exon 4. These mutations affect sequences coding for the GTP-binding domain of the protein and throw the KRAS protein into a permanently active state that does not require stimulation from GTP hydrolysis. As a result, the RAS proteins harboring these single-nucleotide substitutions remain constitutively active in the GTP-bound form. Detection: KRAS mutations are highly correlated with tumor histology and may predict the progress of tumorigenesis in early-stage tumors. Furthermore the presence of KRAS mutations may affect treatment strategy, especially with targeted therapies such as kinase inhibitors and farnesyl-transferase inhibitors. KRAS mutations are also a target of 1)liquid biopsies that is analysis of tumor-specific mutations in blood. 2)KRAS mutations are detected and identified by direct sequencing. 3)Site specific methods such as pyrosequencing have also been developed. 4)Sequencing of selected exons of the KRAS and NRAS and downstream BRAF genes have become a common method for mutation analysis in lung, colon, thyroid, and skin cancer. Cons:Certain characteristics of tumor tissue, such as poor cellularity, tumor heterogenity, and previous chemotherapy treatments affecting tumor cells, confound the molecular diagnosis. d) Ewing Sarcoma, EWS (22q12) A group of tumors arising from primitive neuroectodermal tissue (PNET), Ewing sarcomas comprise a family of childhood neoplasms referred to as the Ewing family. Although immunohistochemical staining for the cell surface protein HBAy1 (p30/p32MIC2), a neuronspecific enzyme, is helpful in the diagnosis of these tumors, no unquie characteristics disntinguish the diff types of tumors that make up this group. Detection of specific translocations by cytogenetic or molecular methods is useful for diagnostic and prognostic accuracy. Translocations involving the EWS gene at 22q12 (Also called EWSR1 for EWS break-point region 1) with the FLI-1 gene at 11q24, t(11;22) (q24;q12) are present in 85% of Ewing sarcomas. Another translocation between EWS and the ERG gene at 21q22 is present in 5% to 10% of Ewing sarcomas. Other partners for the EWS gene such as ETV1 at 7p22, E1AF at 17q12, and FEV at 2q33 are present in fewer than 1% of cases. Detection:Lab testing at the molecular level includes detection of the tumor-specific translocations by reverse transcriptase polymerase chain reaction (RT-PCR). Positive results are revealed by the presnece of a PCR product. Negative specimens will not yeild a product. As with all assays of this type an amplification control such as GAPDH or 18S RNA must accompany all samples to avoid false-negative results. These tests can be performed on tissue or liquid biopsies. e)Synovial Sarcoma Translocation Chromosome 18 - Synovial Sarcoma Breakpoint 1 and 2, SYT =-SSX1,SYT-SSX2 T(x;18)(p11.2q11.2) A recurrent reciprocal translocation between chrosome 18 and the X chromosome is found in synovial sarcoma, a rare type of cancer of the muscle, fat, fibrous tissue, blood vessels, or other supporting tissue of the body. Synovial sarcoma accounts for 8% to 10% of all sarcomas and occurs mostly in young adults. google "Sarcoma is a type of cancer that can occur in various locations in your body. Sarcoma is the general term for a broad group of cancers that begin in the bones and in the soft (also called connective) tissues (soft tissue sarcoma)." The t(x;18) translocation fuses the synovial sarcoma translocation, chromosome 18 gene (SS18 or SYT) with either of two related genes, synovial sarcoma translocated to X (SSX1 and SSX2), on the X chromosome. There are 5 SSX variants, SSX1, SSX2,SSX3,SSX4,and SSX5. With rare exceptions only SSX1 and SSX2 are fused to SYT in the t(X;18) translocation. The fusion gene acts as an aberrant transcription factor with both activation and repression functions from the SYT and SSX portions respectively. Detection: The t(X;18) translocation is detected by FISH or RT-PCR. In the latter method, total RNA reverse-transcribed to cDNA is amplified with primers specific for SSX and SYT genes. In a seminested PCR version of this procedure, the SSX primer used in the first round is a consensus primer for both SYT-SSX1 and SYT-SSX2 After the first amplfiication, SSX1- and SSX2-specific primers discriminate between the two translocation types. The PCRO products are detected by agarose gel electrophoresis and ethidium bromide staining. This method can be performed on fresh, frozen, or fixed tissue, depending on the condition of the specimen RNA. f)Paired Box-Forkhead in Rhabdomycosarcoma, PAX3-FKHR, PAX7-FKHR, t(2;3) Rhabdomycosarcoma (RMS) is the most common soft tissue sarcoma of childhood accounting for 10% of all solid tumors in children. In addition to alveolar rhabdomyosarcoma (ARMS), there are two additional histological forms of RMS: embryonal (RMS-E) and primitive (RMS-P).Although histological classification of ARMS is sometimes difficult, accurate diagnosis is important for the management and treatment of this malignancy because ARMS has a worse prognosis than other subtypes. Translocations involving the Forkhead in the Rhabdomyosarcoma gene (FKHR, also called FOXO1A) and the paired box genes (PAX3 and PAX7). The chimeric genes resulting from the translocations encode transcriptional activators with DNA-binding motifs homologous to the forkhead transcription factor. PAX-FKHR translocations have been observed in all subtypes of RMS but are more characteristic of ARMS. PAX7-FKHR t(2;13) is associated with better outcomes than PAX3-FKHR t(1;13). Mutations in the PAX3 gene are also found in Waardenburg syndrome, a congenital auditory pigmentary syndrome. The majority of ARMS displays the t(2;13) translocation with the t(1;13) variant present with one third the frequency as t(2;13). Detection: Both translocations above are detected by FISH, RT-PCR, quantitative polymerase chain reaction (qPCR), and RNA sequencing. g) Tumor protein 53, TP53 (17p13) Mutations in TP53 are found in all types of cancer, and about 50% of all cancers have TP53 mutations. The gene product of TP53, p53, is a 53,000-dalton DNA-binding protein that controls the expression of other genes. Normally p53 participates in the arrest of cell division in the event of DNA damage. The arrest in G1 phase of the cell cycle allows repair enzymes to correct the DNA damage before DNA synthesis begins. Once the damage is repaired, p53 protein is removed by binding to another protein MDM2 and through degradation. When p53 (tumor supressor gene) is not functional, replication proceeds on damaged templates, resulting in further genetic abnormalities. Also, the mutant product does not degrade properly and accumulates in the cell nucleus and cytoplasm. Due to the frequency and ubiquity of its mutations, mutated p53 protein is a potential therapeutic target. Small molecules that can reactivate mutant p53 protein have been tested in clinical trials. Several studies have shown that mutated TP53 in tumor tissue is also an indicator of poor prognosis in breast cancer, lung cancer, colon cancer, leukemia, and other types of cancer. Controversy on this tho. Detection: Common method was the detection of the stabilized mutant protein by IHC. Several monoclonal antibodies directed at diff epitopes in the p53 protein were used for this purpose. Because normal p53 protein is transient, significant staining (2+ or above on a scale of 0 to +4) of p53 is considered positive for the mutation. Use of microarrays or panel sequencing to screen expression of multiple genes with TP53 was proposed as a more accurate method for predicting survival than either IHC or mutation analysis of TP53 alone. Methods that include sequencing of the entire IHC, is another accurate approach. SSCP and direct sequencing of microdissected tumor tissue are other methods often used to detect TP53 mutations. SSCP methods cover selected exons 5 to 8 or 4 to 9 of the TP53 gene because these exons encode the regions involved in DNA binding and protein-protein interactive functions. Sequencing methods can include the entire gene. In early studies results from IHC didn't always match direct DNA analysis. In addition for screening for somatic alterations, mutation analysis of TP53 is also performed to aid in the diagnosis of Li-Fraumeni syndrome, a cancer-prone condition caused by inherited mutations in the p53 gene. In this case, normal tissue will be heterozygous for the mutation, removing the challenge of isolating pure samples of tumor tissue. Once an inherited mutation is detected, further analysis of relatives requires targeting only that mutation. h) Ataxia Telangiectasia Mutated Gene, ATM (11q22) Predisposition to cancer is one symptom of the neurological disease ataxia telangiectasia (AT). AT occurs in at least 1/40,000 live births. This disease is caused by mutations in the ATM (stands for A-T mutated) gene on chromsome 11. ATM mutations are also present in some types of leukemias and lymphomas. Carriers of the autosomal-recessive mutations in ATM are at increased risk for developing leukemia, lymphoma, or other types of cancers. The ATM gene product is a member of the phosphatidylinositol-3 kinase family of proteins that repsond to DNA damage by pshoprylating other proteins involved in DNA repair and/or control of the cell cycle. The ATM protein participates in pausing the cell cycle at the G1 or G2 phase to allow completion of DNA repair. Detection: Direct DNA sequencing is the method of choice for detection of ATM mutations, especially in family members of carriers of previously identified mutations. Other methods such as SSCP have been used. A functional test for the repair of double-strand breaks induced by irradiation was developed for ATM. For this assay, exponentially growing cells were irradiated followed by the addition of colcemid to inhibit spindle formation. The cells were harvested for giesma staining and the karyotypes examined. The ratio of aberrations/cell was calculated from the number of chromatid and chromosome breaks (Counted as one breakage event) in addition to dicentric chromsomes, trasnlocations, ring chromsomes, and chromatid exchange figures (counted as two breakage events). More recent measures of DNA damage utilize chromogenic reporters. Heteryzygous carriers of an ATM mutation may also be at increased risk for mantle cell lymphoma, B-cell lymphocytic leukemia, or T-cell prolymphocytic leukemia. i) Breast Cancer 1 Gene, BRCA1 (17q21), and Breast Cancer 2 Gene, BRCA2 (13q12) Approx 5% of breast cancers result from inherited gene mutations, mostly in the breast cancer genes BRCA1 and BRCA2. Women who carry a mutation in BRCA1 have a 60% to 80% lifetime risk of breast or ovarian cancer. Men carrying a mutation especially BRCA2, have a 100 fold increased risk of breast cancer compared with men without a mutation, as well as an increased risk of colon and prostate cancer. Both men and women can transmit the mutation to subsequent generations. The BRCA1 gene product has a role in embryonic development, and both BRCA2 and BRCA2 gene products are involved in DNA double strand break repair by homologous recombination. Defects in homologous recombination repair (HRR) are associated with mutations in repair genes including BRCA/NRCA2, ATM, ATR, PALB2, RAD51, CHEK1, and CHEK2, as well as loss of BRCA2 expression through promoter methylation or overexpression of the BRCA2 transcriptional repressor EMSY. Poly(ADP-ribosyl)transferase or PARP is also part of the repair process, and therapeutic agents inhibiting its activity in combination with other gene inhibitors have shown efficacy against BRCA associated cancers. Detection: SSCP, protein truncation tests, chromosome breakage tests, and other procedures have been used to screen for mutations in the BRCA genes. The method used for clinical applications however is direct sequencing. for study guide i would know this (Three mutations 187delAG and 5382insC in BRCA1 and 6174delT in BRCA2 occur frequently in particular ethnic populations. Can be detected by several targeted assays, including sequence-specific PCR and allele-specific oligomer hybridization (dot blotting). The significance of a BRCA mutation test will depend on several factors including penetrance of the gene mutations. If mutation is not detected in coding region of genes the possibility of mutations in the non coding regions cannot be rules out. j)Von Hippel-Lindau Gene, VHL (3p26) Benign blood vessel tumors in the retina found in 1895. Another pathologist further noted that these retinal tumors were linked to tumors in the blood vessels in other parts of the central nervous system, sometimes accompanied by cysts in the kidneys and other internal organs and that the condition was heritable. The Von Hippel-Lindau syndrome (VHL) is now recnogized as a genetic condition involving the abnormal growth of blood vessels in organs, especially those that are particularly rich in blood vessels. It is caused by mutations in the VHL gene which is located on thr short arm of chromoisome 3. Normally, VHL functions as a tumor-suppressor gene, promoting cell differentiation. VHL syndrome is a predisposition for renal cell carcinoma and other cancers. Deletions, point mutations, and splice-site mutations have been described in patients with VHL. In addition, cases of renal cell carcinoma and tumors of the adrenal gland are accompanied by somatic mutations in the VHL gene. Direct sequencing is the preferred method of testing for VHL gene mutations. k)V-myc Avian Myelocytomatosis Viral Related Oncogene, Neuroblastoma-derived, MYCN or n-myc (2p24) Myc family proteins, activated by mitogen signals, increase the expression of several genes through interactions with conserved cis elements (Myc-boxes) and transcriptional coactivaors. The Myc family includes the genes c-myc, n-myc, and l-myc. They are basic helix-hoop-helix transcription factors. The c-myc oncogene (8q24.21) is the most frequently expressed myc. The c-myc gene is amplified in breast and ovarian cancer, lymphomas, and leukemias. Myc expression is regulated by transcription factors specific to each type of cancer. Increase Myc expression is a negative prognostic marker. Expression of l-myc (1p34.2) can induce differentiation in cultured cells. Amplification of l-myc is associated with oral cancer. The n-myc gene on the short arm of chromosome 2 (2p24) is amplified in cases of neuroblastoma and retinoblastoma. n-myc is a oncogene that is counteracted by the tumor supressor gene neurofibromatosis type 1 (NF1). Detection: Myc gene amplification (or protein exoression) is detectable by IHC, FISH, sequencing, or array analysis. Transcription of n-myc may also be measured by using qPCR. l)V-ROS Avian UR2 Sarcoma Virus Oncogene Homolog 1 (ROS1) Proto-Oncogene (6q22.1) and Rearranged During Transfection (RET) Proto-Oncogene (10q11) The ROS1 oncogene, coding for a membrane receptor tyronsine kinase, is rearranged in a variety of human cancers. The resulting fusion protein contains a constitutively active ROS1 kinase domain and drives cellular proliferation. ROS1 is rearranged in 1% to 3% of lung adenocarincomas. The RET proto-oncogene is located on the long arm of chromosome 10 (10q11). Its gene product is a membrane tyrosine kinase that participates in sending cell growth and proliferation signals to the nucleus. The general structure of a large first intron with small exons is characteristic of tyrosine kinase recpetors such as receptors for KIT,EGFR, and platlet derived growth factor PDGF genes. The RET gene is an ex of how diff mutations in the same gene result in diff diseases. Translocations that result in overexpression of RET are found in thyroid papillary carcinomas. Point mutations that activate RET (also called MEN2A) are found in inherited multiple endocrine neoplasia (MEN) syndromes a group of diseases resulting in abnormal growth and function of the pituitary, thyroid, parathyroid and adrenal glands. In contrast, loss of function mutations in the RET gene are found in Hirschsprung disaese, a rare congenital lack of devleopment of nerve cells in that colon that results in colonic obstruction. mutations have been reported in about 50% of congenital cases and 20% of sporadic cases of this disorder. Because about 16% of children with congenital central hypoventilation syndrome CCHS have Hirschsprung disease, RET mutations were also sought in CCHS, but most mutations detected were determined as polymorphic variants. Detection of RET gene mutations can aid in the diagnosis of MEN diseases. Clinical testing targets, mainly exons 10,11,and 16 are where most reported mutations have been found. RET and ROS1 rearrangements are detected by IHC, FISH or sequencing. RET alterations occur in about 1% of lung cancers and both RET and ROS1 are therapeutic targets. m)Anaplastic Lymphoma Receptor Tyrosine Kinase (ALK) Proto-oncogene, 2p23.1 ALK is a receptor tyrosine kinase classfied in the insulin receptor superfamily. The ALK gene has been rearrnaged, mutated, or amplified in tumors of various types incluidng lymphomas, neuroblastoma, and non-small-cell lung cancer. Chromosomal rearrangements are the most common genetic alterations in this gene, with multiple fusion gene partners, including the Echinoderm Microtuble-Associated Protein like 4 gene (EML4; 2p21) and others. ALK gene rearrangements are detected by FISH analysis and sequencing. n)V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog, KIT, c-KIT (4q12) KIT protein is a transmembrane receptor with tyorinse kinase activity. Mutations in this gene are associated with GI stromal tumors (GISTs), mast cell disease, and AML. KIT activation has oncogenic activity. Targeted therapeutics have been used to treat patients with melanoma and gists. Increased KIT activity can result from amplification, overexpression or missense mutations. IHC is used to detect increased KIT protein, and sequencing is used for detection of missense mutations. Other molecular Abnormailities For potential diagnostic targets see table 13.4 pg. 383

Cancer

Term that includes all malignant tumors.

Analytical targets of molecular testing

There are tissue-specific and tumor-specific targets. Tissue-specific targets are molecular characteristics of the tissue from which a tumor arose. The presence of RNA,DNA, protein, or other molecules from these targets in abnormal amounts or locations is used to detect and monitor the presence of tumor. For example molecular tests are designed to detect DNA or RNA from cytokeratin genes in gastric cancer, carcinoembryonic antigen in breast cancer, and rearranged immunoglobulin or T-cell receptor genes in lymphoma. Although tissue-specific markers are useful, they are also expressed by normal cells. and their presence does not always prove the presence of cancer. In contrast, tumor-specific targets are not present in normal cells and are, therefore, more definitive with respect to the detection of a tumor. Tumor-specific genetic structures result from genome, chromosomal, or gene abnormalities in oncogenes and tumor-supressor genes that are associated with the development of the tumor. Gene mutations and chromsomal translocations are found in solid tumors, leukemias, and lymphomas. Cell free nucleic acid or circulating tumor cells carrying oncogenic mutations can be detected in blood and other body fluids. Genome mutations, or aneuploidy, result in part from the loss of coordinated DNA synthesis and cell division that occurs when tumor suppressors or oncogenes are dysfunctional.

Mutations in Hematological Malignancies b)Myeloid Malignancies study guide 4 types! (you are analyzing DNA from cancerous WBC in blood or bone marrow here) all kinase receptors except t(15;17)

t(9;22)(q34;q11) know per annette The t(9;22) is a reciprocal exchange between the long arms of chromsome 9 and 22. The translocation generates the Philadelphia chromosomes (Ph1), which is present in 95% of cases of chronic myelogenous leukemia CML, 25% to 30% of adult acute lymphoblastic leukemia ALL and 2-10% pf pediatric ALL. study question answer: The presence of the translocation is a negative prognostic factor in adult ALL. The t(1;19) translocation may be present in pre-B-ALL. Monoclonal Ig heavy chain and light chain gene rearrangements may be used to monitor B-ALL (by like PCR and gel and look at bands for polyclonal or smear and monoclonal which would be defined 1 or 2 bands). Monocloncal T-cell receptor gene rearrnagments monitor T-ALL. The breakpoints occur within 2 genes, the breakpoint cluster region (BCR) gene on chromosome 22 and the cellular counterpart of the Abelson leukemia virus tyrosine kinase (c-abl) on chromsome 9. The result of the trasnlocation is a chimeric or fusion gene with the head of the BCR gene and the tail of the c-abl gene. Both genes are tyrosine kinases; thay is they pohsphorylate other genes at tyrosine residues. The fusion gene is also a kinase but has aberant kinase activty. There are two major forms of the BCR/abl fusion gene, joining either exon 13 or 14 (b2 or b3) of the BCR to c-abl exon 2 (a2). The b2a2 or b3a2 fusion genes code for a 210 kd protein, p210. A third form of the fusion gene joins exon 1 of BCR with exon a2 of c-abl resulting in expression of an e12a transcript which codes for p190 protein. Another less common junction occurs at exon 19 of the BCR gene (c3). The c2a2 transcript encodes a p230 protein. All of the fusion proteins have been observed in CML HOWEVER P190 mostly occurs in ALL. Detection: a)Uses common PCR with forward primers deisgned to hybridze to the BCR gene on chromosome 22 and reverse primers to chromosome 9 in the c-abl gene. (you want forward primer on the 5' located gene and reverse primer on the 3' located gene of the fusion gene) Also in temrs of the order of naming the translocation t(9;22) means the revrse primer is on 9 and 9 is on the 3' end of the fusion gene is seems but not always. A product will result only if the two genes are joined by a translocation. b)Due to the length of the introns that separate the primer binding sites nested RT-PCR was intiallity utilized. To min risk of false positives may require confirmatory testing expecially for adult ALL. Primers capable of dectecting major AND minor breakooints in BCR gene are used. c)An internal RNA integrity control (or amplification control) is included for each sample to avoid false-negative results from poor RNA quality or inadequate cDNA synthesis. Transcripts from the abl or BCR genes are used most frequently for the RNA integrity control for the t(9;22) but other unique genes with constitutive expression have been used. d)Target amplicons are detected by agarose gel electrophoresis with ethidium bromide staining or capillary electrophoresis. Flourescent dye-labeled primers are required for the latter detection method. f)Quantitative PCR provides an estimation of treatment response espeically with targeted therapies for CML and ALL. g)Although cytogenetic methods espeically FISH and standard RT-PCR are most practical for diagnosis and in early stages of treatment, quantitative PCR provides a valuable estimation tumor load over the course of treatment. Because transcript levels are being measured using RT-PCR, it is important to stablize the specimen RNA on receipt by resuspending the WBC in protective buffers.Another recommendation is to collect sufficent peripheral blood for analysis to avoid false negative results. The primers used for this method are similar to those used for standard PCR. A fluorescent probe provides the signal. A standard curve or a high positive, low positive (Sensitivty) control and negative control should accompany each run. Frequently used methods reported measurements as a ratio of the BCR-abl transcript level to the RNA intefrity control usually the abl transcript, the BCR transcript or the trasncript of a housekeeping gene such as G6PDH. For ex: a standard curve for transcript number generates the formula y=-1.7328[Ln(x)]+48,627 where y is the threshold cycle number, and x is the number (or dilution) of transcripts. If quantitative PCR analysis of the patient specimen RNA yielded a threshold cycle number of 39 (average iof duplicate samples) for BCR-abl trasncripts and a threshold cycle number of 30 for the able trasncripts then solving for x yields 300 BCR-abl transcripts and 50,000 abl transcripts in the sample thus (300/50,000) = 0.6%. this formula is BCR-abl/abl X 100 A three log drop in transcript levels and a BCR-abl/abl X 100 level below 0.05% have been proposed as indicators of good prognosis. advanced: diff formula. [BCR-abl/(abl-BCR-abl]X 100 for the trasncript ratio is used if the abl primers also amplify the BCR-abl translocation. Therapeutic tyrosine-kinase inhibitors that target the chimeric BCR-abl protein have changed CML from deadly disease to chronic condition treatable by a pill. Reference standards were establiushed to be used to normalize measurements from diverse lab methods. These reference standards are protected RNA molecules or "Armored RNA" . Armored RNA is an enginerred MS2 phage protein complex encapsulating an RNA fragment of the BCR-abl target gene designed for use as a qPCR calibration standard. The protected RNA is resistant to RNase digestion and shows high stability in plasma and other fluids. Sets of known concentrations of these reference standards are distributed to labs where they are included in the measurmenet. The results are sent to a test center where the testing lab results are adjusted to the actual numbers for the standards with a correction parameter (CP) or conversion factor (CF). The CP or CF is then applied to standadize the BCR-abl test results. ALtenraqtiveluy commerical reagent sets containing internal calibrators and controls may be used. these system will generate results on the International Scale without requirement for adjustments. t(15;17)(q22;q11.2-q12) Reciprocal trasnlocation between the long arms of chromosomes 15 and 17 results in fusion of the retinoic acid receptor alpha (RARA) gene on chromosome 15 with the myelocytic leukemia (MYL or PML) gene on chromosome 17. Both genes contain zinc finger-binding motifs and therefore bind DNA as transcription factors.The PML/RARA fusion is found specifically ion promyelocytic leukemia. The presence of this trasnlocation is also a predictor of the response to retinoic acid therapy that is used as treatment for this disease. The trasnlocation forms a fusion gene with the first three (type A or S translocation) or six (type B or L trasnlocation) exons of the PML gene joining to exons 2 to 6 of the RARA gene. Detection: Test methods similar to past translocations are used. No current standardization other than standard curves generated from refernece standards. RT-PCR and RT-qPCR are most frequently used. Primers complementary to sequences in exon 3 or 6 of the PML gene and exon 2 of the RARA generate products only if translocation occurred. The presence of translocation product is interpreted as a positive result. As with all these type of tests a amplification control is required to avoid false-negative results. For quantitative PCR, results normalized to an internal control using calculations described previously yield the most consistent day to day results (lowest coeffienct of variance). Adanvced: The reciprocal fusion gene RARA/PML is also expressed in 70% to 80% of cases of APL. Non reciprocal events can produce either fusion alone as well. Although the reciprocal fusion may participate in the tumorgenesis process, it does not predict repsonse to all-trans retinoic acid therapy. The presence of the reverse transcript may be used to confirm the translocation and could be useful in cases where PML/RARA transcript is poorly expressed. FMS-Related Tyrosine Kinase 3 (FLT3), 13q12 Third class of growth factor receptor tyrosine kinases include FMS, PDGFR, FLT1 and KIT. They display 5 IG like domains in the extracellular region and the catalytic doamin is interupted by a hydrophili "interkinase" sequence of variable length. The fibroblast growth factor receptors (FGFR) represent the fourth class which differ from the third class by having on 3 Ig like domains in the extracellular region and a short kinase insert in the intracellular doamin. FLT3 is a member of the third class of tyrosine kinase receptors. Particular mutations in the FLT3 gene aberrantly activate the FLT3 kinase and predict prognosis in AML. These mutations include internal tandem duplications (ITDs) close to the trasnmembrane domain or point mutations affecting an aspartic acid residue in the kinase domain (D835). The ITD can easily be detected by PCR with primers flanking the potentially duplicated region. The size of the amplicon observed by agarose or capillary eelctrophoresis will increase in the event of an ITD. D835 mutations can be detected by PCR-RFLP, where an EcoRV restriction site is destroyed by the presence of the mutation or by RT-PCR with FRET probes. AML pg. 404. Prognosis: For AML prognosis it is recommended to interpret FLT3 mutation results with karyotyping and tests for other gene mutations, including nucleophosmin (NPM) insertion mutations and point mutations in the CCAAT/enhancer-binding protein alpha (CEBPA) and the isocitrate dehydrogenase genes (IDH1 and IDH2. If the karyotype is normal with an NPM1 mutation but no flt3 itd, or with a CEBPA mutation, the prognosis is favorable similar to that of patients with chromoskme 16 inversions or a t(8;21). With an FLT3 ITD mutation and normal karyotype the prognosis is less favorable. IDH muytations are potential therapeutic targets. Janus Kinase 2 (JAK2), 9p24 The JAK2 gene codes for a kinase enzyme that phosphorylates several Signal Transducer and Activator of Transcription (STAT) gene products, bringing about cellular repsonses. A high prop of patients with polycthemia vera, essential thrombocythemia, or idopathic myelofibrosis carry a dominant mutation causing a valine-to-phenylaline amino acid substitution at position 617 of the Jak2 protein (V617F). Detection: Dteetcion of this mutation aids in the diagnosis of these myeloproliferative disorders. a)Multiplex-sequence-specific PCR. Four primers are used one forward and one reverse primer flanking the region of the mutation, one forward ending at the mutation site complementary to the normal base, and fourth reverse primer also ending at the site of the mutation but complementary to the mutant base. The mutation is revealed as the product of the fourth reverse primer and the outer forward primer. pg. 214 fig 8.18. In some labs white blood cells are fractionated in order to perform JAK2 testing specially on the granulocyte fraction. The mutant/normal ratio is important based on data suggesting that the heterozygous/homozygous state of the V617F mutation has implications regarding which myleoproliferatibve disorder is present. Also very low levels of V617F may be found in normal individuals. Four mutations affecting exon 12 of JAK2 have been identified in some V617F-negative patients: F537...pg. 404 too long. These mutations are not located in the kinase domain of the Jak2 protein, bu they do promote higher signaling in the cell than does the V617F mutation. Although the exon 12 mutations are not found in essential thrombocythemia, mutations in the myeloproliferative leukemia (MPL) gene may be present, suggesting a molecular differentiation between polycythemia and thrombocythemia. These mutations are detected using direct sequencing although can also use nonisotopic Rnase cleave assay (NIRCA) for JAK2.

Tumor or neoplasm

growth of tissue that exceeds that of normal tissue and is not coordinated with it. Tumors are either benign (not recurrent) or malignant (invasive and tending to recur at multiple sites).

Molecular oncology

the study of cancer at the molecular level, using techniques that allow the direct detection of genetic alterations down to single bp changes.