Director General Armed Forces Medical Services & Senior Colonel Commandant, Army Medical Corps, Professor of Pathology, ArmyHospital (R&R), Delhi Cantt-110010.

Editorial

“Cancer biomarkers” constitutes one of the most rapidlyadvancing fields in clinical diagnostics. They can be usedto screen asymptomatic individuals in the generalpopulation, to assist in early and specific diagnosis insuspect cases, to select patients who may benefit fromspecific treatments, to predict prognosis and responseto therapy and finally to monitor patients after primarytherapy.

The introduction of advanced sophisticatedtechnologies like microarray, mass spectrometry andautomated DNA sequencing have opened new avenuesin the field of “Cancer biomarkers”. But by this worldof mechanization, is light microscopy being lost in thethick dense forests? Let us critically analyze the assetsand limitations of all these newer methodologies.

Conventional histopathology based on assessingmorphology has remained the standard diagnosticmethod for many years. The use of enzyme histo-chemistry and electron microscopy expanded the primarymicro-anatomic evaluation to include biochemical andsub-cellular ultra-structural features. More recently, wehave progressed and immuno-histochemistry,cytogenetics, analysis of DNA ploidy and moleculargenetic assays have been added as valuable adjuncts tolight microscopy in cancer diagnosis.

Oncologic imaging has also undergone remarkableadvances. The imaging paradigm is shifting fromanatomic and spatial 2D and 3D images to a focus onmolecular, functional, biologic and genetic imaging.Various modalities in diagnostic and prognostic oncologywill be reviewed. These bear interest to all laboratorymedicine experts, medical and surgical oncologists andallied specialties.

Immuno-histochemistry is a well-established techniquebased on the detection of specific protein sequences(antigenic determinants) of tumors by the use of anti-sera and monoclonal antibodies directed against them.It is of paramount importance in unclassified tumors such

as undifferentiated tumors, small round blue cell tumorsand lymphoid malignancies in particular. The commonimmunohistochemical panels used are cytokeratin forepithelial malignancies, leucocyte common antigen forlymphomas, S-100 protein for neural and neuro-ectodermal differentiation, HMB-45 for melanomas,desmin and vimentin for tumors exhibiting muscle andmesenchymal differentiation respectively. This also helpsin metastatic tumors of unknown primary to direct furthertherapeutic decisions by delineating the origin of thetumor.

Immuno-histochemistry has been utilized extensivelyto determine estrogen, progesterone and Her-2 neureceptor status in breast cancer in predicting responseto therapy [1].

Yet other antibodies directed against proteins involvedin the regulation of cell cycle like cyclin D1 and E havebeen reported to be of prognostic significance in breastcancer and squamous cell carcinoma of head and neck.

Molecular Oncology is another field where distinctiveabnormalities of genetic structure and gene expressionof the cancer cell are studied. The resultant abnormalitiesof the cell cycle lead to dysregulated proliferation ofcancer cells. Solid tumors are characterized by multiplespecific and non specific changes, while lymphomas andleukemias are distinguished by highly specificcytogenetic and molecular genetic rearrangements.These changes are being analyzed on chromosomes,DNA or RNA and are finger printed by moleculartechniques.

Chromosomal aberrations are frequently encounteredin malignant cells and are often distinctive of a specifictumor type. Chromosomal alterations can be of variedtypes. These include, duplications (addition ofchromosome), deletions (loss of whole or parts ofchromosomes), segmental amplifications (randomreiteration of segments or extra fragments),translocations (exchange between chromosomes) and

Recent Advances in Diagnosis of CancerLt Gen JR Bhardwaj, PVSM, AVSM, VSM, PHS

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Key Words: Oncology; Molecular; Markers

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inversions (reversal of orientation). Bone marrowaspirate samples can be used in hematologicalmalignancies to recognize individual abnormalities.Banding analysis of metaphase chromosome has beenthe traditionally performed method for detection ofchromosome abnormalities [2]. However, an analysisof chromosomal abnormalities in solid tumors hashistorically been taxing and laborious using tissuesections.

The technique of Fluorescence in situ hybridization(FISH) is applicable to interphase cells. Hence, it is moresensitive compared to conventional cytogenetics [3].Comparative genomic hybridization (CGH) is a newlydescribed method that globally assays for chromosomalgains and losses in genomic complement. These neweradvanced techniques appear more promising as of today.

Identification of the cancer gene along with itsstructural aberrations has become essential to identify,as many mutations are not discernible at the cytogeneticlevel. Documented gene alterations are based onanalyses of oncogenes, tumor suppressor genes, DNArepair genes and regulators of apoptosis.

Molecular methods detect signature nucleotidesequences within the repertoire of the nucleic acidcontent of a cell and hence enable us to distinguishbetween benign and malignant cells. Cellular DNA isanalyzed using Southern Blot (SB) procedure orPolymerase Chain Reaction (PCR)[4]. Messenger RNA(mRNA) detection of genes and their products is doneby the techniques like northern blot, reversetranscription-PCR (RT-PCR) and in situ hybridization.

Relatively newer techniques like microarray, allowmeasurement of differential expression of a distinct genecomplement in different histo-morphological types andgrades of a particular tumor[5]. This method can alsoprofile the degree and type of genes which are activatedor suppressed at a given point of time in a spectrum ofmalignancies. All these methods are highly sensitive butcritical analysis needs to be performed beforeestablishing a cause effect relationship. Diagnosticmolecular markers for some malignancies are depictedin Table 1.

Measurement of tumor markers levels, when usedalong with other diagnostic tests, can be useful in thedetection and diagnosis and follow up of some type ofcancers. They are biologic or biochemical substancesproduced by tumors and secreted into blood, urine, otherbody fluids or body tissues of some patients with certaintypes of cancer in higher than normal amounts. A tumormarker may be produced by tumor itself, or by the bodyin response to the presence of cancer or certain non-cancerous conditions [6]. Some examples of the most

commonly measured tumor markers are presented inTable 2.

However, in most instances tumor marker levels aloneare not sufficient to diagnose cancer as it may showfalse elevation in non-neoplastic conditions as manytumor markers are proteins, over expressed not only bycancer cells, but also by normal tissues e.g. CA-125 isalso elevated in conditions like endometriosis and non-malignant ascites besides epithelial ovarian cancer.Some markers may be elevated in more than one typeof cancer, thereby decreasing the diagnostic accuracye.g. elevated CEA levels are found in multiplemalignancies of gastrointestinal origin. Also, manymarkers share cross-reacting epitopes with products ofnormal tissues, which leads to errors in their quantitativeestimation.

In conclusion, the minds of young pathologists areconstantly receiving various inputs. The subdivisions andsuper-specialities are increasing rapidly. However, theclinical context with applied basic histo-morphologyshould form the foundation stone to diagnosis of cancer.The application of ancillary techniques should be carefully

Table 1Selected molecular genetic markers in cancer diagnosis

Disease Marker Method

CML t(9;22) (q34; q11) SB, RT-PCR, FISH[BCR/ABL]

CLL Trisomy 12 FISHALL t(9;22) [BCR/ABL] RT-PCR, FISH

t(1;19) [E2A/PBX]t(8;14), t(2;8), t(8;22) SB, FISHt(4;11) RT-PCR, FISH

AMLM2 t(8;21) [AML1/ETO] SB, RT-PCR, FISHM3 t(15;17) [PML/RARA]M4 Eo inv 16 [MYH 11/CBFb]

NHL all cases Antigen receptor gene SB, PCRrearrangement

Follicular NHL t(14;18) [BCL2/IGH] SB, PCRBurkitt’s lymphoma t(8;14), t(2;8), t(8;22) SB, FISH

[MYC; IGH/IGK/IGL]Ewings family of t(11;22) [FL11/EWS] SB, FISHtumoursNeuroblastoma MYCN amplification SB, FISHBreast Cancer HER2/NEU/ERBB2 SB, FISH

AmplificationBladder Cancer p53 mutation PCRHead and neck p53 mutation PCRcancersColon K ras mutation PCR

CML - Chronic myeloid leukemia; CLL - Chronic lymphocyticleukemia; ALL - Acute lymphoblastic leukemia; AML - Acutemyeloid leukemia; NHL - Non-Hodgkin’s lymphoma; SB - Southernblot hybridization; FISH - Fluorescent in-situ hybridization; PCR -Polymerase chain reaction; RT-PCR - Real time Polymerase chainreaction; Genes are represented in capital letters withinparentheses.

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weighed and performed only when of therapeuticrelevance. A complete and comprehensive knowledgeor expert consultation should always be called for asand when required. Economic restraints and availabilityof these methodologies should not compromise on patienttherapy but judicious use of these, is the need of thehour.

References1. Diaz, Leslie K MD, Sneige, Nour MD. Estrogen Receptor

Analysis for Breast Cancer: Current Issues and Keys toIncreasing Testing Accuracy. Advances in Anatomic Pathology2005; 12(1):10-19

2. Marcucci, Guido A, Mrozek, Krzysztof A, Bloomfield, ClaraD. Molecular heterogeneity and prognostic biomarkers in adultswith acute myeloid leukemia and normal cytogenetics. CurrentOpinion in Hematology 2005; 12(1):68-75.

3. Lestou VS, Ludkovski O, Connors JM, Gascoyne RD, Lam

Table 2Selected important tumor markers

Tumor Marker Half life Malignancies Non-malignant conditions Conditions

AFP 5-7 days Hepatoblastoma,nonseminomatous Cirrhosis, hepatitis Levels>1000ng/ml in large HCC(Alpha feto protein) germ cell tumour (NSGCT) testis, while 40% with small respectable

non-dysgerminomatous germ cell tumors have normal levels.tumour of ovary, hepatocellular ~40% of patients with NSGCTcarcinoma (HCC), others like have elevated AFP. Levels ofgastric, pancreatic and lung AFP along with α-hCG and LDH

help in risk stratification of germcell tumours of testis

β-HCG (Human chronic 18-48 hrs Choriocarcinoma, hydatidiform Hypogonadism High levelsgonadotropin) mole,testicular germ cell tumors, (>103mIU/ml) in chorio-

others like bladder, prostate carcinoma and H-mole. Used forand kidney risk stratification of gestational

trophoblastic neoplasmsCEA (Carcinoembrionic 2 weeks Colorectal cancers, others like breast, Smokers, fatty liver, Used to follow up the colorectalantigen) cholangiocarcinoma , and stomach. hepatitis cancers rather than diagnoses

Also in liver metastases, malignantascites and pleural effusion

WL, Horsman DE. Characterization of the recurrenttranslocation t(1;1)(p36.3;q21.1-2) in non-Hodgkin lymphomaby multicolor banding and fluorescence in situ hybridizationanalysis. Genes Chromosomes Cancer 2003; 36(4):375-81.

4. Dabritz J, Hanfler J, Preston R, Stieler J and Oettle H. Detectionof Ki-ras mutations in tissue and plasma samples of patientswith pancreatic cancer using PNA-mediated PCR clampingand hybridisation probes. British Journal of Cancer 2005; 92:405-12.

5. Xie W, Rimm DL, Lin Y, Shih WJ and Reiss M. Loss of SmadSignaling Is Associated With Poor Outcome In HumanColorectal Cancer A Tissue Microarray Analysis. Cancer J2003; 9(4): 302-12.

6. Hazel B. Nichols, Amy Trentham-Dietz, Richard R. Love,John M. Hampton, Pham Thi Hoang Anh, D. Craig Allred,Syed K. Mohsin and Polly A. Newcomb. Differences in BreastCancer Risk Factors by Tumor Marker Subtypes amongPremenopausal Vietnamese and Chinese Women. CancerEpidemiology Biomarkers and Prevention 2005; Vol. 14: 41-7.