Various Cancers Share Common Molecular Mechanisms

Published Date: 02 Nov 2017

Chapter 14

Ajay Malik, S S Gill

‘Education never ends, Watson. It is a series of lessons with the greatest for the last.’

Sir Arthur Conan Doyle

"……future discoveries will not likely be made by morphologists ignorant of molecular biologic findings, or by biologists unaware or scornful of morphologic data, but by those willing and capable of integrating them through a team approach….."

Rosai

According to SEER cancer statistics, it is estimated that 1,596,670 men and women (822,300 men and 774,370 women) will be diagnosed with and 571,950 men and women would have died of cancer of all sites in 2011. The understanding of the central role of the genetic material of the cell has been a story of the second half of the 20th century. The diagnosis of cancer specific molecular changes in our genome has led to new approaches for diagnosing and therapeutic strategies. The unravelling of various cancers causing changes in signalling pathways has modified the classifications of tumors. The challenge of cancer treatment has been to target pathogenetically distinct tumor types, to maximize efficacy and minimize toxicity. Improvements in cancer classification have thus been central to all advances in cancer treatment. The development of miniaturized high-throughput technology for genetic analysis would permit not only the monitoring of gene sequence in an experiment, but also a comprehensive analysis of the complex networked coordinated programs and pathways that cause cancers.

140 years ago, Johannes Mueller showed that cancer was made up of cells. Thereafter, it came to be accepted that cancer was a collection of various diseases with many endogenous and exogenous influences. Kinzler and Vogelstein had said, "cancers represent tumors that have acquired the ability to invade the surrounding normal tissues." This definition highlights one of the most important distinguishing factors in the classification of neoplasms--the distinction between benign and malignant tumors.

Research has shown that various cancers share common molecular mechanisms such as uncontrolled cell proliferation, mutation, and dysregulation of genes for cell proliferation, migration, and differentiation. Tumors with similar appearance can follow significantly different clinical courses and show different responses to therapy. This kind of clinical heterogeneity can be explained by dividing morphologically similar tumors into subtypes with distinct pathogeneses e.g. subdivision of acute leukemias, non-Hodgkin’s lymphomas and childhood "small round blue cell tumors". For many more tumors, important subclasses are likely to exist but have yet to be defined by molecular markers. For example, prostate cancers of identical grade can have widely variable clinical courses ranging from indolence over decades to explosive growth causing rapid patient death.

Cancer classification has been difficult in part because it has historically relied on specific biological insights rather than systematic approaches for recognizing tumor subtypes. Cancer classification has two challenges: class discovery and class prediction. Class discovery refers to defining previously unrecognized tumor subtypes. Class prediction refers to the assignment of particular tumor samples to already-defined classes, which could reflect current states or future outcomes.

It is said that tumors grow through a process of clonal expansion driven by mutations. The first mutation leads to limited expansion from a single cell, and each subsequent mutation gives rise to new clonal growth with more proliferative potential. The idea that carcinogenesis is a multistep process is supported by morphologic observations of the transitions between premalignant (benign) cell growth and malignant tumors. In colorectal cancer, the transition from benign to malignant neoplasm can be easily documented and occurs in identifiable stages like benign adenoma, carcinoma in situ, invasive carcinoma, and eventually local and distant metastasis. The development of molecular biology has opened new territories for the molecular analysis of normal and tumour cells. The molecular features of malignant transformation of a cell are as follows:

Self sufficiency in growth signals

Insensitivity to inhibitory signals

Evasion of apoptosis

Defects in DNA repair

Limitless replication

Sustained angiogenesis

Ability to invade and metastasize

A study of these molecular features of malignant transformation has improved the understanding of many cancers, familial and sporadic. It also serves to identify recurrent, constant chromosomal abnormalities within some cancers. These genetic abnormalities are diagnostically critical and/or open new ways to their management. In hereditary cancers, childhood tumours, sarcomas and some epithelial tumours, molecular pathology is playing a decisive role.

Pathologists typically refer to anatomic tumor classifications, when they are referring to lists of primary tumors that are known to occur at a particular location. A list of tumors occurring at a body site is not a classification because it includes tumors that are biologically, clinically, and histologically unrelated. Although often referred to as World Health Organization (WHO) ‘classifications’, the WHO accurately titles its organ-based lists of neoplasms as ‘histologic typings’ for the different organs. What is needed is to develop a more systematic approach to cancer classification based on the simultaneous expression and monitoring of thousands of genes using DNA microarrays. It has been suggested that such microarrays could provide a tool for cancer classification. Microarray studies to date, however, have primarily been descriptive rather than analytical and have focused on cell culture rather than primary patient material, in which genetic noise obscures an underlying reproducible expression pattern.

In the past decade, molecular biologists have tried to classify tumors based on grouping of tumor samples that share similar gene expression profiles. This ability to separate tumors into groups is not equivalent to separating tumors into classes because the groups may represent expected variations of behavior within a single tumor population. For example, tumor samples of a particular type of tumor may contain groups that are separable based on proliferation rate, cell death rate, size, invasiveness, dominance of glycolytic enzyme pathways, and so on. Variant groups within a population do not qualify as classes, if it can be shown that the differences between the groups can be accounted for by transient differences in a tumor's biology. If a slow-growing tumor becomes a fast growing tumor over time, or if a single tumor has foci of slow growth and fast growth, then the tumor cannot be classed by its rate of growth. A key principle in classification is that classes are intransitive (i.e. instances of a class never change their class). Sarcomas never become lymphomas and lymphomas never become sarcomas. Grouping tumors by shared gene expression profiles may indicate that a certain tumor shares a similar profile with another tumor, but it does not guarantee an intransitive classification of neoplasms.

For over 150 years, pathologists have relied on histomorphology to classify and diagnose neoplasms. Their success has been nothing short of electrifying, permitting accurate diagnosis of thousands of different types of neoplasms with only a microscope and trained eyes. In the past two decades, cancer genomics has challenged the supremacy of histomorphology by identifying genetic alterations shared by morphologically diverse tumors and by finding genetic features that distinguish subgroups of morphologically homogeneous tumors. The sequence for most of the human genome is now publicly available and can be applied to understand, characterize, and treat complex diseases such as cancer. The biological differences between tumors that account for variations in morphology and clinical behavior can be analyzed using gene expression microarrays, comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), quantitative PCR and mutation analysis. Normal cell regulation can be disrupted by many factors, including viral infections, DNA methylation, and sequence alterations. Current molecular techniques are providing the tools needed to investigate tumor biology and to discover the genetic and epigenetic causes of cancer.

Microarrays together with clustering analysis have allowed genome-wide expression patterns in biological systems to be deciphered and compared. Hierarchical clustering of microarray data groups together genes that are coordinately expressed under different conditions. A unique signature can be found within the genetic programming of each tumor, revealing its molecular history. This allows tumor histology to be molecularly dissected based on the unique expression profile of each cell type in the sample. For example, in breast tumors, the unique gene expression of mammary tumor cells can be distinguished from other cell types within the sample, such as lymphocytes and stromal cells. In addition, molecular subtypes within a given histologic classification can often be identified. For example, there are two classes of B-cell chronic lymphocytic leukemia, two classes of diffuse large B-cell lymphoma, five classes of non-small cell lung tumors (including three types of adenocarcinoma), and at least four molecular classes of invasive ductal cell breast carcinoma. These molecular subtypes are clinically significant because they predict patient outcome and explain variability seen in the natural course of certain tumors with the same anatomic diagnosis.

The clinical practice of molecular pathology is fundamental to almost every aspect of healthcare, assisting with diagnosis of the disease, therapeutic choice, therapeutic outcome monitoring, prognosis, prediction of disease, preventive strategies and clinical epidemiology. Identification of molecular pathology of cancer cell requires the presence of specific molecular markers or changes for precise diagnosis, prognosis, as well as in determining individual treatment strategies (targeted or personalized cancer therapies).

Molecular pathology of cancer encompasses the following:

Molecular and genetic approaches to classification and the diagnosis of human neoplasms.

The practical approach in design and validation of predictive biomarkers for cancer targeted treatment, treatment response and progression of the disease.

The genetic susceptibility of individuals to develop different types of cancer.

The environmental factors implicated in carcinogenesis and tumor development.

Molecular biology techniques that are available today can be applied for the following purposes in cancer diagnosis and management –

Detection of cancer predisposition

Detection of preneoplastic changes

Cancer detection

Tumor staging

Tumor grading

Differential diagnosis

Subclassification

Prognosis of spontaneous clinical course

Prognosis of response to therapy

Traditionally, the tumor subtype is determined by its morphology, usually determined by staining of tissue sections. More recently, immunohistochemistry (e.g. for cell type-specific cytokeratins in carcinomas or CD surface proteins in leukemias) has come to play an important role in routine practice and differential diagnosis. In general, the precise identification of the tumor type already provides a great deal of information on the likely prognosis of the patient and a basis for the choice of the most appropriate treatment. In routine histopathology, additional information on the aggressiveness of a tumor is obtained by grading, which relies on subjective estimates of the degree of tissue disorganization as well as cellular and nuclear atypia.

In spite of these improvements, classification, staging and grading of many tumor types by current methods are far from perfect in predicting prognosis and the response to specific therapies. Molecular diagnostic techniques would thus be helpful in determination of tumor stage. This can be, for instance, by allowing detection of tumor cells in the blood or bone marrow. A major impact of molecular diagnostic techniques is expected to occur in the subclassification of tumors with respect to prognosis and response to therapy. In renal and breast cancer, molecular classification has corroborated previous suspicions that different subclasses of the disease exist and may need different treatments. Yet another situation is found in some cancers that are morphologically and molecularly similar, but in which progression depends on specific molecular alterations. For instance, mutation of TP53, as detected by accumulation of the mutant protein in the tumor cell, may predict a worse prognosis in Wilms tumor and urinary bladder cancer.

Molecular changes in carcinomas and solid malignancies are more complex than those in hematological malignancies. The material harvesting is also more difficult in these. In addition, obtaining samples from solid malignancies is an invasive process. This is one of the reasons why molecular techniques have not made a significant advancement as far as the solid malignancies are concerned.

For any analytical procedure, the minimal sample obtained through noninvasive or minimally invasive procedure should be acceptable. The test being run should be fast, economical, reliable, sensitive, specific and should maintain the stability of the molecule being tested. The results should be reliable and one must be able to carry out the tests with current available expertise and laboratory equipment. After detection of cancer, the choice of therapy depends on the precise classification of the cancer. Experience and carefully collected observations are used to assess the prognosis of the cancer and select the most appropriate therapy. There are many carcinomas, in which the ‘classical’ triads of histology, staging, and grading do not consistently yield sufficient information for optimal selection of therapy. It is not at all exceptional for carcinomas with identical stage, grade, and histology to take divergent clinical courses. The most severe problem, however, is that micrometastases escape detection by current imaging methods. Therefore, staging is not really precise. The extension of the primary tumor as well as its histology and grading suggest only an estimate of the presence of micrometastases.

The decisions on how to deal with the primary tumor and whether to apply an adjuvant therapy (and which type of adjuvant, if there is a choice), are based on probabilities rather than definitive information. A large set of empirical data collected for each cancer type can be used in these decisions. In some cancers, algorithms and nomograms taking all known relevant parameters into account have been introduced as a help for patients and doctors. Nevertheless, the overall situation is far from satisfactory. In a sense, progress in therapy has aggravated this dilemma. Since a large choice of treatments is now available, definite criteria to individualize the therapy are required.. In addition, therapies differ with respect to their side-effects and their costs, which cannot be neglected. For these reasons, improvements in the classification of carcinomas are a major goal of current molecular research. Breast cancer may represent a major carcinoma, where this type of research is advanced and is translated into the clinic at a rapid pace.

In a sense, therefore, expression profiling using microarrays is a logical continuation of a development that is already underway in breast cancer diagnosis. Analysis of the expression levels of a large number of genes allows classifying breast cancers into ER+ and ER- types. It distinguishes previously unrecognized luminal cell-like and basal cell-like subtypes, with Her2/Neu+ cancers representing a distinct subclass within the basal-cell-like subtype. Cancers arising in patients with inherited mutations in the BRCA genes also exhibit characteristic profiles. Most importantly, metastatic cancers appear to show distinct expression patterns from those still growing locally. Individualization of cancer therapy helps to achieve optimal therapeutic results, to minimize suffering not only from the cancer, but also from the treatment, and to avoid unnecessary expenses. In this situation, molecular markers come in handy to continue an ongoing development in all spheres of oncology.

Nucleic acid methods are preferred over immunohistochemistry (IHC) for detection of mutations. Though IHC is a convenient method to score for molecular markers in surgical pathology, it is subject to variability from differences in antibody specificity, scoring criteria, and storage. Additional advantages of nucleic acid analysis include correlating specific mutations to treatment response and providing a marker for monitoring residual disease. The need for solid-tumor molecular markers in pathology is clear. However, how these new tests would be implemented is not clear. Specimens received in surgical pathology are routinely formalin fixed and paraffin embedded (FFPE) to preserve the architecture of the tissues. This processing makes recovery of mRNA unreliable and thus, expression analysis by microarray or reverse transcription-PCR is not feasible unless there is a novel method available for sample procurement in solid tumors. Markers in the form of DNA and protein are typically more stable than RNA, allowing them to be used within the current framework of surgical pathology for determining mutation status and gene expression.

Real-time quantitative PCR can analyze multiple genes simultaneously within a single reaction. The main advantages of multiplexing over single-target analysis are the ability to provide internal controls, lower reagent costs, and preservation of precious samples. Multiplexing can be particularly important when there is a need to analyze several targets from microdissected tissue. Microdissection of solid tumor samples is usually necessary when determining gene copy numbers because the presence of DNA from normal diploid cells interferes. Protocols for obtaining nucleic acids from microdissected tissue are well established. Two genes in which quantitative DNA analysis is important for prognosis and treatment of breast cancer are HER-2/Neu and topoisomerase II alpha. In about 20% of breast cancers, the HER-2/Neu gene becomes amplified at the DNA level, leading to an increase in an over expression of the protein. The topoisomerase-II alpha gene is physically located near HER-2/Neu gene within the chromosome band region 17q12-q21, an area that is frequently mutated in breast tumors. DNA amplification of HER-2 gene can occur concomitantly with topo-II alpha gene alterations (amplification/deletion), and changes in topo II alpha copy number may dictate response to chemotherapy with topo II alpha inhibitor drugs.

Recurrent chromosomal translocations have traditionally been associated with leukemias/lymphomas and sarcomas. In contrast, carcinomas generally have complex karyotypes with no recurrent translocations like leukemias. Instead, carcinomas typically have activating mutations in oncogenes (e.g. RAS) or inactivation of tumor suppressor genes (e.g. p53). Nowadays an increasing number of carcinomas are being recognized as having recurrent translocations. Notable examples are pediatric renal cell carcinoma and thyroid carcinoma. Translocations are traditionally identified by conventional cytogenetics (karyotyping/chromosomal banding analysis), although FISH and PCR-based assays are increasingly been utilized for identification of translocations. In contrast, small deletions and point mutations typical of carcinomas generally cannot be visualized by cytogenetics and require nucleic acid-based methods. In some instances IHC can be used to identify aberrant protein expression resulting from a translocation or mutation (such as Bcl2 in follicular lymphoma). Instances where IHC can be applied as a surrogate to a molecular test are of particular relevance to anatomic pathologists.

An interesting rule of thumb is that sarcomas with characteristic translocations are morphologically uniform and lack atypical mitoses, whereas truly pleomorphic MFH-like sarcomas typically have complex cytogenetics with multiple non-recurrent changes analogous to carcinomas. There is a trend for some genes (EWS, FUS) to appear with multiple translocation partners in different tumors. Even more curiously, there are rare examples of an identical translocation (ETV6-NTRK3) in histogenetically disparate tumors (infantile fibrosarcoma, congenital mesoblastic nephroma, secretory breast carcinoma, and mammary analogue secretory carcinoma of salivary glands). By convention, translocations are designated in numerical order for chromosomes [e.g. t(11;22)], and in 5’-3’ order for chimeric gene products [EWSWT1], which confusingly may not necessarily be in the same order [EWS gene is on chromosome 22, and WT1 on 11].

Some of the molecular associations in solid tumors are tabulated in table 14.1 for easy reference and learning.

Table 14.1:

Gene

Chromosome

Tumor Association

n-myc

2

neuroblastoma

p53

17p

many sporadic tumors ( associated with over expression) and in Li-Fraumeni syndrome

WT1

11p13

Wilms tumor 11p13 mutation / deletion; DSRCT t(11;22) / EWS-WT1

EWS

22

ES t(11;22) / EWS-FLI1; DSRCT t(11;22) / EWS-WT1; Clear cell sarcoma; Angiomatoid Fibrous Histiocytoma: identical translocation t(12;22) / EWS-ATF1; Myxoid liposarcoma t(12:22) / EWS-CHOP; Extraskeletal myxoid chondrosarcoma t(9;22) / EWS-CHN/TEC

ALK

2

IMT - various translocations of 2p23; Lung adenocarcinoma EML4-ALK

ETV6TEL

12

Infantile fibrosarcoma, Congenital mesoblastic nephroma, Secretory carcinoma of breast, Mammary analogue secretory carcinoma of salivary glands: identical translocation t(12;15) / ETV6-NTRK3

TFE3

Xp11.2

ASPS, RCC with Xp11.2: identical translocation t(X;17) / ASPL-TFE3

PEComas

INI1

22q11

Rhabdoid tumors (renal and extra-renal rhabdoid tumors, AT/RT of the brain); epithelioid sarcoma, myoepithelial carcinoma of soft tissue, medullary carcinoma of kidney

FUS(TLS)

16p11

Myxoid liposarcoma t(12;16) / FUS-CHOP; Low grade fibromyxoid sarcoma (Evans tumor) t(7;16) / FUS-CREB3L2; Angiomatoid Fibrous Histiocytoma t(12;16) / FUS-ATF1

VHL

3p

Sporadic and hereditary clear cell RCC, vHL syndrome

RET

10

Activating mutations: Thyroid (papillary and medullary carcinoma), MEN2a, MEN2b

MET

7

Papillary RCC (hereditary and occasionally sporadic)

kRAS

12

Pancreas, colon, lung, ovary (mucinous)

BRAF

7

Papillary thyroid carcinoma, melanoma, colorectal carcinoma – targeted therapy under development

c-kit

4

GIST (targeted therapy with imatinib available), melanoma, Mastocytosis

PDGFR

4

GIST

EGFR

7

Lung cancer – targeted therapy with EGFR inhibitors available

HER2

17

Amplified in breast carcinoma – trastuzumab target therapy available

Some of the solid tumors with recurrent genetic changes can be subtyped / classified and remembered as given in table 14.2

Table 14.2:

Tumor

Chromosomal abnormality

Genes/proteins

ASPS

t(X;17)(p11;q25)

ASPL-TFE3

Xp11.2 – same present in renal carcinomas. TFE + by IHC

CCS of tendon sheath

t(12;22)(q13;q12)

t(2;22)(q34;q12)

EWS-ATF1

EWS-CREB1

DFSP

t(17;22)(q22;q13)

ring chromosome 17

COL1A (collagen gene) - PDGFβ

Endometrial stromal sarcoma

t(7;17)(p15;q21)

JAZF1-JJAZ1

Extraskeletal myxoid chondrosarcoma

t(9; 22)(q22;q12)

t(9;17)(q11;q11)

RBP56-CHN (TEC)

ES/PNET

t(11;22)(q24;q12)

t(21;22)(q22;q12)

t(7;22)(p22;q12)

t(17;22)(q12;q12)

EWS-FLI1

EWS-ERG

EWS-ETV1

EWS-E1AF

(FLI1 & EWS have various breakpoints – type 1 is favorable prognosis. FLI 1 detectable by IHC but is nonspecific)

IMT

t2p23 – t(2;5)

Alk1 fusion

DSRCT

t(11;22)(p13;q12)

EWS-WT1

Infantile fibrosarcoma

t(12;15)(p13;q25)

ETV6(TEL)-NTRK3

Liposarcoma – round cell & myxoid variant

t(12;16)(q13;p11)

t(12;22)(q13;q12)

FUS/TLS-CHOP

EWS-CHOP

Well differentiated Liposarcoma

Ring chromosome 12

HMGA2, MDM2

Amplification

Neuroblastoma

–1p or del 1p32-36

Double minutes

+17q

Gene unknown

N-myc amplification – bad prognosis

Hyperdiploidy

Good prognosis

Rhabdomyosarcoma

Alveolar

t(2;13)(q35;q14)

t(1;13)(p36;q14)

PAX3-FKHR (unfavorable)

PAX7-FKHR (favorable)

Embryonal rhabdomyosarcoma

loss of 11p15

"fusion-negative" ARMS are

Clinically / molecularly indistinguishable from ERMS

Rhabdoid tumors

22q11.2 del or mutation

INI1 (also seen in AT/RT)

Synovial sarcoma

t(X;18)(p11;q11)

SYT-SSX1

t(X;18)(p11;q11)

SYT-SSX2 (monophasic SS)

WT

11p13 del or mutation

WT1

11p15 mutation

WT2

Trisomy 12

Fresh and frozen tissues are optimal source of DNA and RNA to serve as template for targeted molecular analysis. However, archival FFPE tissue is a frequently used alternative source of DNA for clinical testing. FFPE tissue is especially critical as a source of nucleic acid when pathological evaluation renders an unexpected diagnosis. It also provides the advantage of allowing archival analysis with correlation to outcome. Molecular analysis can be performed at different levels of resolution, from the whole chromosome down to the specific nucleotide sequence. At the chromosomal level, classical cytogenetics helps in finding out the chromosomal structure, numbers, translocations and deletions. In situ hybridization (chromogenic/fluorescent – CISH/FISH) can be carried out on cells or paraffin embedded sections. FISH is a powerful tool for molecular analysis as it is difficult to get DNA extracted from archival material. In surgical pathology laboratories, PCR has changed the outlook for diagnosing malignancies. Quantitation of the molecular changes is now possible with pure cancer tissue microdissected with the help of laser capture microdissection (LCM) methods (Fig. 14.1).

Fig. 14.1:

Tissue Microdissected

Manually / LCM

Proteins – Western Blot Analysis, IHC

RNA – Northern Blotting, cDNA analysis

DNA – LOH, SNP analysis, Southern Blot Mutational Analysis, DNA Arrays

Many studies of potential diagnostic and therapeutic usage in various tumors like breast carcinomas, lung and colon carcinomas, GIST, childhood tumors, soft tissue sarcomas and many other carcinomas have resulted in discovery of some biomarkers of value. These have both prognostic and predictive importance. As per the current knowledge of tumorigenesis, majority of the cancers have multiple genetic and epigenetic changes occurring in multistep pathways. These changes can be correlated morphologically like in colon cancer.

Mutations can be in the form of point mutations, deletions or amplifications. These mutations can be detected by using sequencing for point mutations, translocations and amplifications detection by using FISH/CISH in paraffin embedded tissues. IHC can also aid in detection but is not so sensitive or specific. PCR, RT-PCR and ISH are utilized for detection of translocations. Many of the known oncogenes involve specific point mutations e.g. K-RAS mutations in lung carcinomas involve one of the following three codons – exon 12,13 and 61. K-RAS mutations can also occur in pancreatic, colonic and thyroid carcinomas. In colon carcinomas, K-RAS mutation testing is a must by the reporting laboratories before anti-EGFR therapies can be instituted. This can be accomplished in rapid PCR based analysis. In breast carcinomas, EGFR testing is done by IHC but in a more sensitive way by analysing gene copy number by FISH. The tests that are run in parallel show good correlation. The same is seen in glioblastoma multiforme and lung carcinomas. Tumor suppressor genes have two active copies normally and both get mutated before tumorigenesis occurs. The best way to analyse the TSG is by LOH, CGH and FISH.

Soft Tissue and Bone Tumours

Ewing’s sarcoma/ primitive neuroectodermal tumor (EWS/PNET)

On cytogenetic examination, over 95% cases of EWS/PNET show reciprocal translocation 11; 22 (q24; q12), which results in fusion of the EWS gene with the FLI1 or ERG genes. The most common fusion is the one that results in frame linking of EWS exon 7 with FLI exon 6 (type I). The EWS-FLI1 fusion gene results in formation of a highly expressed protein with strongly driven transcription activity. These translocations are detected by FISH and/or RT-PCR, can be used for primary diagnosis, detection of metastatic or residual disease. Sensitivity is significantly higher with fresh tissue than with FFPE tissue and wherever possible, fresh tissue should be preferred over FFPE tissue. Clinical relevance of detection of type I transcripts in stem cells harvested from bone marrow of patients has not been established. IHC detection of the protein is sensitive but not specific.

Desmoplastic small round cell tumour

The characteristic genetic abnormality in DSRCT is the t(11;22) (p13;q12) translocation producing a fusion gene between the EWS and the Wilm’s tumour gene (WT1). This leads to formation of a chimeric protein whose function is not yet known. The EWS-WT1 fusion is specific for DSRCT. This has broadened the clinical spectrum of a young male with widespread involvement of the peritoneum to a number of sites outside the peritoneum including pleura, hand, parotid, pancreas, ovary, testes, bone and brain. Occasional reports of DSRCT being associated with EWS-ERG gene has now somewhat limited the specificity of the EWS-WT1 fusion gene in the diagnosis of DSRCT. The fusion gene detection is by RT-PCR, multiplex PCR or FISH. The presence of a highly specific monoclonal antibody to the carboxy-terminal region of the WT1 gene by IHC reliably differentiates DSRCT from EWS/PNET.

Alveolar rhabdomyosarcoma (ARMS)

75% cases of ARMS harbour t(2;13)(q35;q14) translocation resulting in a fusion of PAX3 gene (chromosome 2) with 3’ end of the FKHR gene (chromosome 13). 10% cases harbour t(1;13)(p36;q14) translocation of PAX7-FKHR. The chimeric protein acts as an aberrant transcription factor that drives the PAX gene binding site. Some ARMS associated with other amplifications including the MYCN oncogene. PAX3-FKHR has a poorer prognosis and a higher propensity for bone marrow involvement. Detection of the fusion gene is by RT-PCR and FISH. These methods can be used for detection of submicroscopic bone marrow metastasis which carries a poor prognosis. Both PAX3 and PAX7 are expressed in developing myotomes. Interestingly bi-phenotypic small cell tumours with both neuroectodermal (NE) and muscle differentiation simultaneously express EWS-FL1 and PAX3-FKHR fusion transcripts.

Undifferentiated soft tissue sarcoma (USTS)

These small cell tumours of indeterminate type show a diffuse hyper-cellular pattern consisting of sheets of medium sized cells that have a variable nuclear morphology. They lack identifying histochemical, IHC and ultra-structural features. In up to 40% cases, RT-PCR detects fusion transcripts of a specific sarcoma type. Detection of such fusion transcripts has specific prognostic and therapeutic implications.

Spindle cell tumours

Within the group of spindle cell tumours, high prevalence of fusion transcripts are found in synovial sarcoma, dermatofibrosarcoma protuberance (DFSP), giant cell fibroblastoma, congenital/infantile fibrosarcoma, extragastrointestinal (soft tisuue) stromal tumours and inflammatory myofibroblastic tumour. To a lesser degree, chromosomal abnormalities may also be seen in malignant peripheral nerve sheath tumour (MPNST), angiomatoid fibrous histiocytoma and low grade fibromyxoid sarcoma.

Synovial sarcoma (SS)

80% cases of SS arise in deep soft tissues. Characterization of genetic abnormalities has markedly expanded the clinicopathological spectrum of SS. It is now known to occur in all ages, involving a wide variety of sites such as head and neck, kidney, prostate, skin, vulva, penis, intrathoracic, intraneural and intraosseus sites. The genetic hallmark of SS is t(X;18)(p11;q11) translocation. This creates a fusion gene of SYT group (chromosome 18) and the SSX gene (Xp11). In about one third of cases of SS, more complex translocations and aneuploidy are also present. The SYT-SSX gene encodes a chimeric protein that is oncogenic as a result of aberrant transcriptional regulation. Detection is by RT-PCR, FISH and Southern blot. RT-PCR is equally sensitive in fresh and FFPE tissue. IHC is of little utility as SYT and SSX are expressed in a wide variety of tissue and tumours. Diagnostically, SYT-SSX is rarely and weakly detected in other tumours such as MPNST and EWS/PNET group. Nevertheless, SYT-SSX fusion transcripts strongly suggest the diagnosis of SS. Prognostically, SYT-SSX2 has a better clinical outcome than SYT-SSX1. This may guide treatment stratification in future. The significance of detection of minimal residual disease (MRD) or of positive margins by RT-PCR has not been established.

Congenital/infantile fibrosarcoma (CIF)

The translocation t(12;15)(p13;q25) producing ETV6-NTRK3 fusion gene is the commonest cytogenetic abnormality in CIF with other less common events such as trisomies 8,11,17 and 20. It is postulated that the translocation is the initial event with the polysomies being follow-up events in more mitotically active phenotypes. The fusion gene is created with the ETV6 transcription factor fusing to the tyrosine kinase domain of NTRK3 gene; the chimeric protein so produced has a constitutively active tyrosine kinase (TK) activity through a number of different cell activation pathways. Detection of the fusion gene is best done by FISH and RT-PCR on fresh tissue. Cytogenetic detection of translocation by banding techniques is difficult because of the similar size and banding pattern of the exchanged fragments. IHC based upon the NTRK3 protein is not specific because NTRK3 is expressed in several tumours. The ETV6-NTRK3 fusion gene is present only in mesoblastic nephroma within pediatric tumours. However, it is common in AML and secretory carcinoma of breast in adults.

Inflammatory Myofibroblastic tumour (IMT)

In children and young adults, ALK receptor tyrosine kinase gene at 2p23 is characteristic of IMT. It is much less common in adults over 40 years. The ALK gene has been shown to be fused to a variety of other genes paralleling the genetics of anaplastic large cell lymphoma (ALCL). Importantly, the characteristic rearrangements are seen only in the myofibroblasts. The fusion genes encode proteins through cytoplasmic activation of tyrosine kinase activity. Detection is best done by FISH. ALK immunoreactivity of IHC has inconsistent sensitivity and specificity. For instance, it is immunoreactive in MPNST, rhabdomyosarcoma, leiomyosarcoma, malignant fibrous histiocytoma and ARMS. Diagnostically, demonstration of the ALK fusion gene provides enough evidence to say that IMT is neoplastic. However as ALCL and IMT share the same fusion gene, it has diagnostic uncertainty especially as ALCL has wide morphological spectrum.

Tumours of adipose tissue

Conventional cytogenetic analysis of lipomas has demonstrated karyotypic abnormalities in 55%-75% of cases. However, these are heterogeneous and clustered into three defined subgroups related to the high mobility group (HMG) protein encoding genes. These rearrangements are also found in a wide variety of mesenchymal tumours that do not enter into a morphological differential diagnosis with lipomas. High prevalence of genetic abnormalities is seen in lipoblastoma, atypical lipomatous tumor and dedifferentiated liposarcoma.

Importantly 95% of myxoid liposarcomas contain t(12;16)(q13;p11) translocation that fuses the TLS gene at 16p11 with the CHOP gene, thus encoding a chimeric protein that strongly promotes transcription activity. The normal function of the CHOP gene is in adipocyte differentiation. TLS-CHOP gene fusions are characteristic of both myxoid and round cell liposarcomas indicating an etiological relatedness and a high degree of specificity. Detection is by FISH and RT-PCR. The high specificity permits application in MRD detection but implications are still not clear.

Clear cell sarcoma (CCS)

The t(12;22)(q13;q12) translocation is the hallmark of CCS and results in the fusion of the EWS (chromosome 22) gene with the ATF1 (chromosome 12) gene. The EWS-ATF1 chimeric protein is thought to lead to dysregulated expression of gene normally controlled by cAMP although the in vivo targets remain uncertain. Besides the characteristic translocation, other recurrent cytogenetic changes described in CCS include trisomy 7, trisomy 8, and structural and numerical aberrations of chromosome 22. This translocation is specific to CCS and sets it apart from melanoma. This translocation is also seen in the osteoclast rich GIT tumour and suggests a link with CCS. Prognostic significance is to be established. Characterization of the translocation found in CCS has broadened the clinicopathological spectrum of the tumour. It is now known to involve a wide range of anatomic sites including dermis, ear, penis, kidney, bone and GIT other than its earlier association with tendons and aponeuroses.

Alveolar soft part sarcoma (ASPS)

The ASPL-TFE3 fusion gene resulting from the del(17) t(x;17)(p11;q25) translocation is seen in almost all ASPSs. It is postulated that the loss/gain of copy number of genes in these regions may also contribute to the pathogenesis of ASPS. The fusion gene results in formation of a chimeric protein that causes transcriptional deregulation and tumour development. ASPL-TFE3 fusion gene is also characteristic of a distinctive subgroup of childhood renal neoplasms. However, in children the fusion gene is a result of a balanced translocation; in ASPS, it is a non-reciprocal translocation. The fusion gene can be detected by karyotyping, SKY, FISH, RT- PCR its chimeric protein by IHC with high specificity and sensitivity. RT-PCR has 100% sensitivity in fresh tissue.

Extraskeletal myxoid chondrosarcoma (EMC)

There is rearrangement of the EWS and CHN genes in 75% cases of EMC as a result of t(9;22)(q22;q12) translocation. This has far more potent transcriptional activity than the native CHN. Both EWS-CHN and TAF2N-CHN act through abnormal transcriptional factors. TAF2N-CHN is seen in 10%-20% of EMC and is as a result of t(9;17)(q22;q11) translocation. The chimeric protein resulting from the translocations is thought to act via abnormal transcription factor activity. These translocations are not seen in chondrosarcomas or extraskeletal mesenchymal chondrosarcoma indicating that EMC is a distinct neoplasm and is the gold standard in the diagnosis of EMC. Detection is by karyotyping, FISH and RT-PCR, which is 100% sensitive in fresh tissue.

Aneurysmal bone cyst (ABC)

The characteristic genetic abnormality of ABC involves the rearrangement of CDH11 gene at 16q22 or the USP6 gene at 17p13 and 70% cases of ABC harbour these rearrangements. Only 10%-19% cases of ABC harbour a CDH11-UPS6 fusion gene. CDH11 encodes osteoblast cadherin that is highly expressed in osteoblasts and osteoblast precursors. USP6 encodes a ubiquitin specific protease that contains the domain involved in GTPase signalling. These rearrangements are restricted to the spindle cells in ABC and do not involve giant cells or any other cellular elements in ABC, showing that the spindle cells are the neoplastic cells. The partner gene has not been identified and the mechanism of tumorigenesis is not known. The presence of recurring genetic aberrations has convincingly demonstrated that it is a neoplastic lesion. Also it has helped to define the rare soft tissue ABC. Secondary ABC associated with GCT, chondroblastoma, osteoblastoma and fibrous dysplasia do not show the rearrangements. These rearrangements are also not present is other osseous and soft tissue malignancies. At present clinicopathologic and prognostic impact of the aberrations is not known.

Nuggets

Soft tissue and bone tumours have a heterogenous set of mutations.

IN Ewings sarcoma/ PNET, most cases show a reciprocal transcloation of 11; 22 (q24; q12) which results in the fusion of the EWS gene with the FLI1 or ERG genes.

The characteristic abnormality in the desmoplastic small round cell tumour is t(11;22)(p13;q12) producing a fusion gene between the EWS and WT1.

In alveolar RMS, 75% cases show the t(2;13)(q35;q14) translocation resulting in fusion of PAX3 gene (chr 2) with 3’ end of the FKHR gene.

In synovial sarcoma, T(X;18)(p11;q11) translocation is the genetic hallmark. In one third cases of synovial sarcoma, more complex translocations and aneuploidy are also present.

In congenital/infantile fibrosarcomas, the translocation t(12;15)(p13;q25) producing ETV6-NTRK3 fusion gene is the commonest cytogenetic abnormality. It is likely that the translocation is the initial event followed by other cytogenetic abnormalities like polysomies.

In inflammatory myofibroblastic tumours, the ALK receptor tyrosine kinase gene is the characteristic abnormality.

95% of myxoid liposarcomas contain t(12;16)(q13;p11) translocation that fuses the TLS gene at 16p11 with the CHOP gene encoding a chimeric protein that strongly promotes transcription activity.

The t(12;22)(q13;q12) translocation is the hallmark of CCS and results in the fusion of the EWS (chr 22) gene with the ATF1 (12) gene. Trisomy 7, trisomy 8 and numerical aberration of chromosome 22 may also be seen.

In alveolar soft parts sarcoma, the ASPL-TFE3 fusion gene resulting from the der(17)t(x;17)(p11;q25) translocation is seen.

In extraskeletal myxoid chondrosarcoma, a t(9;22)(q22;q12) translocation producing a rearrangement of the EWS and CHN genes is seen in 75% of cases.

The characteristic genetic abnormality of ABC involves the rearrangement of CDH11 gene at 16q22 or the USP6 gene at 17p13 and 70% harbour these rearrangements.

Central Nervous System

Brain tumours are not characterized by signature translocations and fusion products as seen in hematologic and soft tissue tumours. The alterations of interest consist predominantly of gain in oncogene function and tumour suppressor losses through a variety of genetic and epigenetic mechanisms. The most common utilization of molecular diagnostics is testing for co-deletion of 1p and 19q as an ancillary diagnostic, prognostic and predictive aid in oligodendroglioma.

Alteration of genes involved in cell-cycle control:

It is known that the progression of the cell cycle is controlled by positive and negative regulators. The p16 gene and the p15 gene are located in 9p21; a region commonly deleted in astrocytomas. The expression of p16 gene is frequently altered in these tumors: in 33%-68% of primary glioblastomas and 25% of anaplastic astrocytomas. The Rb gene located on13q chromosome plays an important role in the malignant progression of gliomas. The p53 gene is a tumor suppressor gene located on chromosome 17p13.1; loss or mutation of p53 gene has been detected in many types of gliomas and represents an early genetic event in these tumors.

Loss or inactivation of tumor suppressor genes: In addition to p53 gene, other tumor suppression genes also play an important role in astrocytomas. The loss of chromosome 10 is the most frequent abnormality associated with progression of malignant astrocytic tumors; more than 70% of glioblastomas show LOH on chromosome 10; amplification of EGFR is always associated with loss of chromosome 10. The PTEN gene located at the 10q23 locus is implicated more frequently in glioblastomas than in anaplastic astrocytomas. Another suppressor gene, the DMBT1 (10q25.3-26.1) gene has also been located on the distal portion of chromosome 10. More recently annexin VII (ANX7;10q21) as detected by IHC in glioblastomas has shown to be a strong predictor of prolonged survival independent of other clinical variables. The allelic loss of chromosome 22q which contains the neurofibromatosis type 2, tumor suppressor gene NF2 is observed in 20-30% of astrocytomas. Most of these genes participate in the progression of astrocytomas (fig 14.)

1p/19q co-deletions: These co-deletions have been identified in all classical morphology oligodendroglioma. They indicate susceptibility of the tumour to procarbazine, CCNU, vincristine, radiation and temozolomide. They also indicate that the tumor has a good prognosis and that there is a need to re-classify a tumour if thought to be an oligodendroglioma but not showing the co-deletions. In such a case, the tumour may be any one of the following : DNET, central neurocytoma, extraventricular neurocytoma, or clear cell ependymoma. There is no unifying genetic signature for ependymomas although loss of chr 22 is associated with spinal location. In embryonal tumours, abnormalities of 17p are associated with an aggressive course in medulloblastomas. Interestingly 75% of AT/RT have monosomy 22 or 22q deletion as detected by FISH. It separates the highly anaplastic and morphologically diverse AT/RT from PNET and medulloblastomas.

Expression of growth factors and growth factor receptors: The EGFR coded by the EGFR cellular oncogene is located on chromosome locus 7p12-p14. EGFR is amplified in 40%-60% of glioblastomas. The primary glioblastomas rarely contain EGFR over expression; patients with anaplastic astrocytomas or glioblastomas have a poor prognosis when EGFR gene amplification is present; amplification could be a significant prognostic factor in these tumors. Over expression of PDGFR-ά (platelet derived growth factor) is associated with LOH of chromosome 17p and p53 mutations in secondary glioblastomas. Other growth factors expressed in gliomas include fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), and vascular endothelial growth factor (VEGF).

Neuroblastoma

Two characteristic genetic events in neuroblastoma are the loss of a critical region on the distal short arm of chromosome 1 and amplification of the MYCN oncogene which is associated with an aggressive clinical course. These events are related, with LOH for 1p preceding the development of amplification. Other genetic abnormalities found in this tumor include gain of chromosome 17, hyperploidy or near triploidy and defects in expression or function of nerve growth factor receptor. Mutations of the p53 and RAS genes are found only rarely. Multiple pathways are likely to be involved in the oncogenesis of neuroblastoma. Surveys of gene expression profiles may ultimately identify patterns of transcription that can be used to optimize patient classification and correlate clinical outcome.

GASTROINTESTINAL TRACT

Colorectal carcinoma

Like breast and prostate cancer, colon cancer is much less prevalent in the developing world, as also in the richer Asian countries and Southern Europe. There are two distinct pathways for development of colon cancer, both of which result in step wise accumulation of mutations in series of oncogenes and tumor suppressor genes. Several hereditary cancer syndromes increase the risk for colon carcinoma more or less specifically, among them familial adenomatous polyposis coli (FAP), hereditary non-polyposis colon carcinoma (HNPCC), Peutz-Jeghers syndrome (PJS), and Cowden’s disease. The study of these moderately prevalent to very rare syndromes has greatly contributed to the understanding of sporadic colon cancer. The fundamental ‘gatekeeper’ step in colon cancer development leading primarily to formation of adenomas is the constitutive activation of the WNT signaling pathway. It is usually caused by loss of function of the classical tumor suppressor APC gene, less frequently by mutations in CTNNB1 oncogenically activating beta-Catenin gene, and rarely by other changes in the pathway. The activation appears to confer a kind of ‘stem-cell’ phenotype to the carcinoma cells. Further steps in the progression of the cancer are associated with mutations activating k-RAS, loss of function of TP53, and inactivation of the cellular response to TGF-beta.

Since deletions and LOH of chromosome 5q are among the most frequent alterations in colorectal cancer overall, following the identification of APC as the gene mutated in FAP, sporadic carcinomas were screened extensively for mutations in the gene. Today, it is assumed that both alleles of the gene are inactivated by point mutation and deletion/recombination, or occasionally promoter hypermethylation in 70%-80% of colon and rectal cancers, irrespective of whether they are familial or sporadic. Moreover, the frequency of APC mutations is almost the same in early and late stage tumors. Thus, APC is a prototypic tumor suppressor. Since its inactivation appears to be almost mandatory for the development of colorectal tumors and most probably takes place at an early stage, the designation of ‘gatekeeper’ is appropriate.

APC/β-catenin pathway:

Loss of Adenomatous Polyposis Coli (APC) gene: The APC gene has been mapped to 5q21. The APC gene encodes a 2,843-amino acid protein that is important in cell adhesion and signal transduction; beta-catenin is its major downstream target. It is considered a gatekeeper gene, and the loss of APC is among the earliest events in the chromosomal instability (CIN) colorectal tumor pathway. The important role of APC in predisposition to colorectal tumors is supported by the association of APC germline mutations with familial adenomatous polyposis (FAP) and attenuated familial adenomatous polyposis (AFAP). Both conditions can be diagnosed genetically by testing for germline mutations in the APC gene in DNA from peripheral blood leukocytes. Most FAP pedigrees have APC alterations that produce truncating mutations, primarily in the first half of the gene. More than 300 different disease-associated mutations of the APC gene have been reported. The vast majority of these changes are insertions, deletions, and nonsense mutations that lead to frame shifts and/or premature stop codons in the resulting transcript of the gene. The most common APC mutation (10% of FAP patients) is a deletion of AAAAG in codon 1309; no other mutations appear to predominate.

Mutation of K-RAS: K-RAS plays an important role in intracellular signal transduction and is mutated in fewer than 10% of adenomas of less than 1 cm in size, in about 50% of adenomas larger than 1 cm, and in approximately 50% of carcinomas.

Loss of SMADs: A common allelic loss in colon cancer is on 18q21. SMAD2 and SMAD4, involved in TGF-alpha signalling are present in this locus.

Loss of p53: Losses at chromosome 17p have been found in 70% to 80% of colon cancers, but the loss is infrequent in adenoma. This suggests that mutations in p53 occur late in colon carcinogenesis.

Microsatellite Instability Pathway:

This pathway is characterized by genetic lesions in DNA mismatch repair genes. It is involved in 10% to 15% of sporadic cases and in the HNPCC syndrome. These genes have been classified as caretaker genes because of their role in genetic proofreading during DNA replication. The genes that have been implicated in HNPCC include hMSH2 on chromosome 2p22; hMLH1 on chromosome 3p21; hPMS1 and hPMS2 on chromosomes 2q31 and 7q22, respectively; hMSH6 on chromosome 2p21. 90% of mutations involve MSH2 and MLH1. Mutations in mismatch repair genes cause alteration of micro-satellites which are fragments of repeat sequences in the human genome leading to micro-satellite instability.

Molecular techniques have been extensively studied for screening, staging, testing for submicroscopic disease and assessment of sentinel lymph nodes in colorectal carcinomas. However technical difficulties and lack of specificity and correlation have limited the progress. However some direction in management is suggested by the molecular tests. Overall survival among patients with MSS or MSI-L tumours is improved by adjuvant chemotherapy although there is no benefit among patients whose tumours are MSI-H. Similarly resistance to adjuvant chemotherapy with fluorouracil has been shown to correlate with high thymidylate synthase mRNA levels. In tumours harbouring mismatch repair deficiencies, other drugs such as topoisomerase-1 inhibitors may prove better therapeutic options. Finally, a small molecule termed RITA has been shown to restore the apoptosis inducing function of p53 gene in tumour cells that retain the wild type gene. There may eventually be a role for mutational analysis of TP53 to identify patients most likely to benefit from therapy with this class of compounds.

Stomach

The two categories of gastric carcinomas differ in their epidemiology. They also have different clinico-pathological and underlying mechanisms of carcinogenesis. Intestinal type carcinomas have MSI present in 15%-50% tumours often associated with TGFβR11, IGFIIR, BAX, MSH6, MSH3 and E2F4. They also have a higher prevalence of TP53 mutations and hTERT expression. Diffuse type carcinomas show less frequent microsatellite instability. They also harbour functionally relevant mutations in the CDH1 gene encoding E-cdherin in upto 50% of cases. These are often associated with down regulation of expression resulting from hypermethylation of the gene promoter.

The clinical utility of molecular methods in staging, screen and SN biopsy are unproven. However, molecular demonstration of free tumour cells in the peritoneal cavity has been used to identify high risk patients most likely to benefit from adjuvant chemotherapy.

Molecular techniques are also being employed to differentiate between collision tumours and composite tumours of the gastroesophageal junction.

Gastrointestinal Stromal tumor:

The identification of c-KIT mutations and platelet derived growth factor- ά (PDGFR) mutations in these tumors constitutes significant progress in understanding their pathogenesis. c-KIT is the receptor for stem cell factor and belongs to type III receptor tyrosine kinase (RTK) subfamily, whose members include platelet-derived growth factor receptors and ß (PDGFR and PDGFRß). As in other RTK, stem cell factor induces dimerisation of KIT followed by transautophosphorylation of the cytoplasmic tyrosine kinase domain, leading to activation of multiple signaling pathways, such as the PI3K/AKT and c-Jun N-terminal kinase/STAT pathways, promoting proliferation and inhibiting apoptosis. Activating mutations of kit gene in GIST occur in exons 11, 9, 13 and 17. The meaning of KIT activation is highlighted by the recent introduction of an inhibitor of RTKs, STI-571 which can induce regression of GISTs. Even advanced disease stabilises with this therapy and the quality of life improves.

Another member of the RTK family, PDGFRά, is involved in the pathogenesis of GIST and mutations in c-KIT are mutually exclusive with those in PDGFRA. Interestingly, these two genes are located in the same chromosomal region (4q12). Both in vitro and in vivo studies have shown that the type of mutation in c-KIT or PDGFRά genes may predict the response to treatment with imatinib.

Esophageal carcinoma:

Most of these cases overexpress p53 gene and epidermal growth factor receptor (EGFR).

Hepatocellular carcinoma:

Pre-neoplastic changes such as hepatocyte dysplasia result from point mutations in selected cellular genes, loss of heterozygosity in tumor suppressor genes, DNA methylation changes and constitutive expression of hepatocyte growth factor (HGF) and TGF-ά. Alteration of p53 gene is particularly seen in areas with high prevalence of hepatitis B infection.

Currently the role of molecular pathology in oesophageal, small intestinal and pancreatic tumours is not clinically relevant.

URINARY SYSTEM

About 1%-4% of renal cell carcinomas (RCC) occur as a component of an inherited predisposition syndrome which should be suspected whenever an individual has multi-focal RCC, history of previous renal tumour or has close relatives diagnosed with RCC.

Biallelic inactivation of the VHL gene is specific for clear cell RCC whether occurring in the context of von Hippel-Lindau syndrome or sporadically. Molecular analysis is routinely not required in the diagnosis though it can be used to identify clear cell RCC with unusual morphology. However, mutational analysis of the VHL gene and global gene expression profiling has been shown to provide prognostic information that more accurately predicts patient survival than the TNM staging. Microarray analysis has shown gene profile of non-aggressive (100% survival at 5 years) and aggressive clear cell RCC (0% at 5 years) in a set of approximately 40 genes. VHL mutations are associated with a different spectrum of clinical disease and may correlate with the response to anti-VEGF therapy.

Sporadic papillary RCC can have a number of differential diagnosis – clear cell RCC, collecting duct carcinoma, carcinomas associated with TEF3 fusion genes and mesonephric adenoma. +7, +17, -Y characteristic of type I papillary RCC, differentiates it from mesonephric adenoma. When combined with LOH at 3p (characteristic of clear cell RCC), it can distinguish sporadic type I papillary RCC with prominent clear cell component from clear cell RCC. TFE3 fusion transcripts separates tumours harbouring this fusion gene from type I papillary RCC as they are difficult to differentiate morphologically.

Chromophobe RCC shows a pattern of chromosome loss unique to itself, that is, loss of whole chromosomes 1, 2, 6, 10, 13, 16, 21 and Y. This helps to differentiate it from renal oncocytoma.

RCC associated with Xp11.2 rearrangements primarily affect children and young adults. They are composed of clear cells with papillary growth pattern. They account for 24%-41% of RCC in children. The translocation which commonly involves ASPL-TFE3 gene in a reciprocal translocation is characteristic of this tumour and diagnostic too. The diagnosis is by FISH and IHC. In this context is also another renal neoplasm defined by molecular technique I.e.: by the TFEB fusion gene. However, it has strong nuclear immunoreactivity which may supplant molecular identification.

Recent cDNA microarrays have shown that RCC subtypes have discrete gene expression profiles. A molecular diagnostic algorithm (MDA) can accurately distinguish between main RCC subtypes and oncocytoma especially when used in combination with histopathology. Also currently MDA can not consistently distinguish chromophobe RCC from oncocytoma. The MDA is based upon ratios of CA9 (carbonic anhydrase 9), AMACR (ά methyl coenzyme A racemase), CLCNKB (kidney specific basolateral chloride channel) with each being quantited by QT-PCR. Hence, in future, renal masses can be accurately classified preoperatively on needle biopsies by a combination of morphology, IHC, gene expression profile and QT-PCR; thereby helping in decision for ablative therapy (or observation only) depending upon the molecular classification obtained.

Wilm’s tumour

It is a nephroblastoma that arises from nephrogenic rests which are parts of the developing kidney and have failed to differentiate in young children. Therefore, the tumor consists of several components resembling tissue structures in the fetal kidney, e.g. blastema, mesenchymal stroma, and tubular structures. Wilms tumors can be unilateral or bilateral, and the latter situation occurs more often with germ-line mutations. Nephroblastomas were one of the original paradigms of the two hit model of cancer formation but it is now thought that a number of genetic events contribute to its formation. Mutations at several loci predispose to Wilm’s tumors.The genes are WT1 11p13, WT2 11p15, FWT1 17q13 and FWT2 19q13. WT1 encodes a transcription factor which is involved in the development and

differentiation of the genitourinary tract. WT1 is expressed specially. Its function is necessary for the formation of renal tubules from the metanephric mesenchyme under the influence of the branching ureter bud. The understanding of the function of WT1 is complicated by the presence of several different isoforms resulting from differential splicing and translational initiation. Accordingly, consequences of mutations in the WT1 gene vary depending on their location. Wilm’s tumour with anaplastic morphology usually shows TP53 mutations. WT2 is related to a locus that causes Beckwith-Wiedemann syndrome; a condition of fetal overgrowth, metabolic disregulation, and predisposition to childhood tumors; although the precise relationship is unclear.

It has been claimed that tumors with WT1 mutations may be more aggressive than others even though they tend to have fewer chromosomal alterations. It is, however, not clear, whether this is a property of tumors with WT1 mutations or not – more likely – a property of the subtype to which they belong. Mutations in WT1 are almost exclusively found in tumors with a

high stromal content that are associated with intralobular nephrogenic rests. These tumors appear to fail epithelial differentiation completely and then, in a more or less stochastical fashion, differentiate into some mesenchymal lineage. They arise earlier than the blastema-rich tumors associated with perilobular nephrogenic rests. These are very often characterized by loss of imprinting and overexpression of IGF2 and appear to respond better to chemotherapy. In contrast to the former group, WT1 is normally expressed indicating that the block in differentiation is caused by a different defect.

Renal/Extrarenal/Malignant rhabdoid tumour (MRT) : Rhabdoid tumour of the kidney (RTK) is characterized by biallelic alteration of the hSNF5 locus at 22q11. hSNF5 encodes a protein tht is member of the ATP-dependant chromatin remodeling complex. This genetic alteration is also characteristic of MRT of CNS and soft tissues.

Urothelial tumors of bladder:

30% to 60% of tumors show monosomy or deletion of 9p and 9q as well as deletions of 17p, 13q, 11p and 14q. Deletion 9p involves tumor suppressor gene p16INK4a, which encodes an inhibitor of cyclin dependant kinase. The 9q deletion involves numerous other tumor suppressor genes, but their identity is not known. Many invasive urothelial cell carcinomas show deletion of 17p, including the region of the p53 gene, which contribute to progression of these tumors. Increased expression of RAS and epidermal growth factor receptor (EGFR) is also seen. On the basis of this, a model for bladder carcinogenesis has been proposed. The first pathway is initiated by deletion of tumor suppressor genes on 9p and 9q, leading to superficial papillary tumors, few of which then acquire p53 mutation and progress to invasion. The second pathway is possibly initiated by p53 mutation, leads to CIS and with loss of chromosome 9, progresses to invasion. Molecular genetics has been used to identify occult metastasis and increase sensitivity in urine cytology for early malignancies of the bladder.

BREAST

Genetic analysis of individual genes has little role in the diagnosis of individual tumours. The molecular evaluation of individual loci does play an important role in stratifying patients into appropriate treatment groups. Analysis of HER2/neu is the best example. Analysis of TP53 may have a similar role because specific mutations of the gene are associated with clinical outcome response to specific chemotherapy regimens, and response to radiation therapy.