Molecular Diagnostics and Cytogenetics

Slide 1 · Molecular Diagnostics

and Cytogenetics

Diagnostic classification · prognostic stratification · molecular monitoring · precision therapy

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

Select an assay that detects the relevant class of genetic alteration.

Explain the diagnostic role of clonality, translocations, FISH, PCR, arrays, and sequencing.

Distinguish prognostic, predictive, and residual-disease applications.

Integrate germline testing and liquid biopsy with morphologic assessment.

Slide 3 · Molecular testing is an adjunct to pathology

Histology and cytology establish most cancer diagnoses.

Molecular methods add decisive information in selected diagnostic problems.

A result is interpreted in relation to specimen, morphology, and clinical question.

The same molecular lesion may carry different implications in different tumor contexts.

Slide 4 · Six clinical questions for a molecular laboratory

What is the tumor?

How may it behave?

Is disease still present?

Is risk inherited?

Is a target present?

Has resistance emerged?

Diagnosis · prognosis · monitoring · predisposition · treatment · resistance

Slide 5 · Start with the clinical question, not the technology

Patient + lesion

Morphology and phenotype

Defined molecular question

Appropriate assay

Integrated report

A test is useful only when its analytical target answers the diagnostic or therapeutic question.

Slide 6 · Biologic material determines feasible testing

Material

Advantages

Important considerations

Tumor tissue

Morphology and tumor-specific DNA/RNA

Tumor fraction and fixation affect analysis

Blood / marrow cells

Accessible hematologic tumor material

Cell population must reflect the lesion

Cell-free plasma DNA

Serial, minimally invasive sampling

Detected material may be sparse

Slide 7 · Molecular lesion classes require different methods

Alteration

Illustrative detection methods

Point mutation

PCR / sequence-based assays

Gene fusion / translocation

Cytogenetics, FISH, RT-PCR or sequencing

Copy-number gain or loss

FISH, arrays or sequence-based assays

Clonal receptor rearrangement

PCR for IG or TCR rearrangements

Slide 8 · Assay selection: What can each technique answer?

Technique

Most direct readout

Typical clinical use

PCR / RT-PCR

Defined DNA or RNA sequence

Clonality, fusions, disease burden

Karyotyping

Large-scale chromosomal change

Characteristic acquired rearrangement

FISH

Targeted chromosomal interval

Fusion/rearrangement or amplification

SNP / tiling array

Genome-wide copy number

Deletions, gains and allelic imbalance

Targeted NGS

Sequences in many selected genes

Molecular disease subclassification

Slide 9 · Clonality is a diagnostic question in lymphoid lesions

Neoplastic B- and T-cell proliferations arise from a single founding clone.

That clone carries a characteristic antigen-receptor gene rearrangement.

Reactive lymphoid tissue contains many clones with distinct rearrangements.

PCR analysis can therefore help distinguish clonal and polyclonal proliferations.

Slide 10 · Why antigen-receptor rearrangements act as clonal markers

Antigen-receptor rearrangement

Unique sequence in one lymphocyte

Expansion of descendant cells

Repeated identical rearrangement

Schematic: Shared rearrangement acts as a molecular fingerprint of a lymphoid clone.

Slide 11 · Immunoglobulin gene rearrangement testing

B-cell antigen-receptor loci undergo somatic rearrangement.

PCR targets rearranged immunoglobulin gene sequences.

A restricted clonal pattern supports a B-cell clonal expansion.

The assay contributes to, but does not replace, full lymphoma assessment.

Slide 12 · T-cell receptor gene rearrangement testing

Each T-cell clone carries characteristic rearranged TCR gene sequences.

PCR provides evidence of a dominant T-cell population.

A reactive infiltrate is expected to include numerous different T-cell clones.

Interpretation requires knowledge of the cellular context of the sample.

Slide 13 · Clonality and malignancy are not synonymous questions

Molecular result

Supported interpretation

What remains necessary

Clonal pattern

Dominant related lymphocyte population

Morphology, phenotype and clinical context

Polyclonal pattern

Multiple lymphocyte populations

Assessment of sampling and diagnostic suspicion

Inconclusive analysis

No definitive clonal assignment

Alternative or complementary evaluation

Slide 14 · Recurrent translocations are tumor-associated markers

Many leukemias and lymphomas harbor characteristic chromosomal translocations.

A rearrangement may create a fusion oncogene or dysregulate a gene.

Routine cytogenetics and FISH can demonstrate selected abnormalities.

Finding a characteristic lesion can substantially refine tumor classification.

Slide 15 · What routine cytogenetics contributes

Obtain suitable dividing tumor cells

Examine metaphase chromosomes

Identify large structural changes

Interpret tumor-specific pattern

Conventional analysis surveys the karyotype; Obtaining metaphases can be difficult in some solid tumors.

Slide 16 · FISH: Target-specific hybridization

Select fluorescent DNA probe

Hybridize to complementary locus

Visualize labeled signal

Interpret chromosome-region pattern

Schematic method; FISH is applicable to metaphase preparations and interphase nuclei.

Slide 17 · Metaphase and interphase FISH

Preparation

Useful feature

Constraint

Metaphase chromosomes

Chromosomal context for a target locus

Depends on available metaphases

Interphase nuclei

Examines nondividing nuclei, including fixed tissue

Probe signal must be interpreted without full karyotype

Slide 18 · FISH probe configurations are question-specific

Locus copy number

Example: Increased locus signals

More locus signals can indicate amplification; Interpretation requires appropriate controls.

Break-apart strategy

Example: Spatially separated probe components

Separated signal components can indicate rearrangement involving the interrogated locus.

Schematic only — not a patient FISH micrograph

Slide 19 · Chromosomal rearrangements: Method trade-offs

Method

Strength

Question to ask

Karyotype

Broader structural chromosome survey

Can tumor metaphases be obtained?

FISH

Targeted locus analysis in cells or tissue

Which rearrangement is suspected?

RT-PCR

Detection of a particular fusion transcript

Is its expressed transcript testable?

Slide 20 · Round blue cell tumors: Morphology may be insufficient

Several childhood sarcomas show overlapping small round cell morphology.

A specific chromosomal rearrangement may distinguish a particular entity.

Molecular findings are combined with the morphologic differential diagnosis.

This is especially valuable when preparation of tumor chromosomes is difficult.

Slide 21 · Ewing sarcoma: A characteristic translocation

Small round cell tumor

Morphologic differential

Test for t(11;22)(q24;q12)

Support Ewing sarcoma diagnosis

The characteristic rearrangement is a confirmatory molecular clue—not a substitute for tumor classification.

Slide 22 · A translocation can generate a fusion transcript

Two chromosomal loci rearrange

Fusion gene is formed

Fusion RNA is transcribed

RNA-based assay detects transcript

Schematic principle; The molecular target can be DNA rearrangement or expressed fusion RNA.

Slide 23 · PCR and reverse-transcription PCR are distinct

PCR

RT-PCR

Starts with DNA template

Starts with RNA; Makes complementary DNA

Amplifies a selected DNA locus

Amplifies a selected expressed RNA target

Examples: Receptor gene rearrangements

Examples: BCR-ABL fusion transcripts

Slide 24 · Anchored RT-PCR expands fusion transcript detection

RNA from fixed tumor tissue

Generate complementary DNA

Anchored amplification

Identify fusion transcript

Anchored RT-PCR as particularly useful with formalin-fixed paraffin-embedded tissue.

Slide 25 · Fixed tissue expands access—and complicates RNA assays

Formalin-fixed, paraffin-embedded tumor tissue is widely available.

Anchored RT-PCR can identify a broad range of tumor-associated fusion transcripts.

RNA is comparatively prone to degradation during tissue processing.

Method selection must account for the available analyte and preservation.

Slide 26 · Arrays survey copy-number changes across the genome

DNA microarrays can map deletions and amplifications at multiple loci.

Tiling designs sample regions distributed throughout the genome.

SNP arrays also provide information about polymorphic alleles.

Genome-wide arrays differ from a FISH assay directed at one or a few loci.

Slide 27 · Array comparative genomic hybridization

Label tumor + reference DNA

Hybridize both to genomic probes

Compare relative hybridization

Call regions of gain or loss

Schematic; Relative signal imbalance indicates copy-number change, not a patient measurement.

Slide 28 · SNP arrays add allele-specific information

Genotype SNP loci

Assess overall copy number

Assess allele proportions

Infer gain, loss or allelic imbalance

SNP markers can reveal loss of heterozygosity in addition to copy-number abnormalities.

Slide 29 · Copy-number loss versus loss of heterozygosity

Finding

What is observed

Interpretive point

Copy-number loss

Reduced quantity of DNA at a locus

Deletion is one potential mechanism

Loss of heterozygosity

Loss of mixed allelic state

May occur even with retained diploid copy number

Both together

Reduced DNA amount and allelic diversity

Concordant pattern supports an allelic deletion

Slide 30 · FISH and arrays answer different scales of question

FISH

Genomic array

Interrogates selected chromosome regions

Surveys many intervals genome-wide

Displays signals within nuclei or chromosomes

Reports locus-level hybridization/genotype patterns

Supports targeted rearrangement or amplification evaluation

Maps regional gains, losses and allelic changes

Slide 31 · NGS permits broad molecular characterization

Next-generation sequencing reads many DNA molecules in parallel.

Tumor genomes can contain numerous simultaneous alterations.

Targeted sequencing enables evaluation of selected panels of cancer-relevant genes.

NGS increasingly contributes to molecular classification, especially in hematologic disease.

Slide 32 · NGS: Biological sample to sequence reads

Extract DNA

Prepare DNA library

Separate + amplify templates

Parallel sequencing

Produce short sequence reads

Simplified schematic of a common massively parallel short-read sequencing approach.

Slide 33 · NGS bioinformatics produces clinically interpretable variants

Sequence reads

Align to reference

Call variants

Annotate significance

Issue contextual report

Depth, allele fraction, annotation and the clinical setting determine interpretability.

Slide 34 · Sequencing depth affects variant detection

Depth denotes the number of reads covering a genomic location.

Greater depth can support identification of lower-frequency variants.

Variant allele frequency describes the proportion of reads carrying a variant.

Low-frequency variation requires careful analytical interpretation.

Slide 35 · Targeted tumor panels prioritize interpretable genes

Targeted NGS commonly examines exons from selected cancer-associated genes.

Focused panels concentrate sequencing resources on clinically relevant loci.

Alterations may support diagnosis, prognosis, or selection of therapy.

Each molecular finding still requires disease-specific interpretation.

Slide 36 · Hematologic neoplasms increasingly have molecular categories

Acute leukemias and some myeloid neoplasms require detailed genetic assessment.

Targeted sequencing can contribute to placement in molecularly defined groups.

Cytogenetic lesions and sequence variants can complement the morphologic diagnosis.

A molecular category must be assigned using the applicable disease framework.

Slide 37 · Molecular evidence must be integrated—not isolated

Morphology

Immunophenotype

Cytogenetic + molecular findings

Integrated pathologic classification

Clinical context, sample quality and the applicable entity definition govern the final interpretation.

Slide 38 · Diagnostic, prognostic and predictive biomarkers

Use

Question

Illustration

Diagnostic

What neoplasm is this?

Ewing-associated translocation

Prognostic

What clinical behavior is anticipated?

MYCN amplification in neuroblastoma

Predictive

Which treatment may work?

EGFR mutation or ALK fusion in lung cancer

Slide 39 · Neuroblastoma: MYCN amplification

Amplification of the MYCN oncogene is associated with an adverse prognosis.

Amplification creates additional copies of the oncogene in neoplastic cells.

MYCN status contributes to neuroblastoma risk assessment.

Amplification is a prognostic finding, not a standalone histologic diagnosis.

Slide 40 · Neuroblastoma: Loss of chromosome 1p

Chromosome 1p deletion is another unfavorable genomic feature.

Loss of a chromosomal region represents a copy-number alteration.

Its prognostic implication differs from the molecular detection method.

Risk assessment draws on multiple tumor and patient characteristics.

Slide 41 · Oligodendroglioma: Combined chromosome-arm losses

Loss of chromosome arms 1p and 19q is an important tumor-associated alteration.

The source associates 1p/19q loss with improved therapeutic response and survival.

The clinical implication is distinct from those of EGFR amplification in the comparison.

Interpret this finding within the integrated glioma diagnosis.

Slide 42 · TP53 alterations often carry adverse implications

Mutation or deletion involving TP53 is linked to worse outcomes in many cancers.

TP53 defects can be associated with resistance to treatment.

The prognostic meaning varies with neoplasm type and the broader molecular context.

Molecular results refine risk rather than fully determining an individual outcome.

Slide 43 · Selected prognostic alterations: Different mechanisms

Tumor setting

Genetic feature

Use emphasized in source

Neuroblastoma

MYCN amplification

Adverse prognosis

Neuroblastoma

Chromosome 1p deletion

Adverse prognosis

Oligodendroglial tumors

1p/19q loss

Favorable response / longer survival in source comparison

Various cancers

TP53 mutation or deletion

Often unfavorable prognosis

Slide 44 · Molecular monitoring of minimal residual disease

Identify tumor-specific molecular marker

Treat patient

Test follow-up specimen

Evaluate molecular persistence or recurrence

The marker must be detectable with an appropriate, sufficiently sensitive method.

Slide 45 · CML: BCR-ABL transcript monitoring

CML with BCR-ABL fusion

Therapy

Quantitative RT-PCR for fusion RNA

Longitudinal disease assessment

RT-PCR can measure residual leukemia-associated transcript burden over time.

Slide 46 · Baseline diagnosis and post-treatment monitoring

At diagnosis

During follow-up

Establish the biologic identity of the tumor

Track a known tumor-specific genetic signature

Integrate molecular finding with initial pathology

Use serial molecular measurements to assess disease burden

Question: Which entity or target?

Question: Residual disease or emerging relapse?

Slide 47 · Somatic tumor testing and hereditary testing

Tumor (somatic) testing

Constitutional (germline) testing

Asks which alterations exist in the neoplasm

Asks whether a predisposition allele is inherited

May select targeted cancer therapy

May affect screening, prevention and family counseling

Interprets tumor-specific molecular biology

Has implications beyond the tested individual

Slide 48 · BRCA1 and BRCA2: Hereditary susceptibility

Pathogenic germline variants in BRCA1 and BRCA2 predispose to particular cancers.

Testing may identify individuals requiring specialized risk assessment.

Findings can inform surveillance or discussion of preventive interventions.

At-risk relatives may also need appropriately structured genetic counseling.

Slide 49 · RET: A proto-oncogene in inherited cancer risk

Some inherited mutations in the RET proto-oncogene increase risk of specific neoplasms.

Detection may enable evaluation before symptomatic cancer develops.

A family-oriented management strategy can include targeted screening.

Clinical decisions require variant-specific interpretation and counseling.

Slide 50 · Hereditary predisposition testing has consequences beyond diagnosis

Recognize inherited-risk context

Identify germline variant

Counsel and evaluate patient

Consider risk reduction

Discuss testing of relatives

Presymptomatic testing has important ethical and familial implications.

Slide 51 · Predictive testing connects a lesion to a drug target

Identify tumor genetic lesion

Establish altered oncoprotein

Determine therapeutic relevance

Select context-appropriate treatment

A predictive molecular marker directs a treatment decision only in the relevant cancer setting.

Slide 52 · PML-RARA in acute promyelocytic leukemia

The PML-RARA fusion identifies a molecular lesion relevant to treatment selection.

Its demonstration can be urgent when acute promyelocytic leukemia is suspected.

A specific retinoic acid receptor-associated rearrangement underlies drug sensitivity.

Cytogenetic/FISH or molecular assays contribute to targeted management.

Slide 53 · BCR-ABL: Both therapeutic target and monitoring marker

BCR-ABL is a characteristic fusion in chronic myeloid leukemia.

It also occurs in a subset of acute lymphoblastic leukemia.

Detection identifies an oncogenic lesion relevant to targeted treatment.

Fusion-transcript measurement provides a separate longitudinal monitoring function.

Slide 54 · EGFR mutations in lung carcinoma

Selected lung cancers contain activating mutations involving EGFR (ERBB1).

The mutation can identify a therapeutically targetable abnormality.

The marker is predictive in a defined clinical and tumor context.

Subsequent molecular testing may identify resistance-associated variants.

Slide 55 · ALK rearrangement in lung carcinoma

A subset of lung cancers expresses abnormal ALK fusion oncoproteins.

Molecular testing can detect ALK gene rearrangements.

ALK protein immunohistochemistry can provide complementary evidence.

The result can identify tumors potentially responsive to ALK inhibitors.

Slide 56 · BRAF alterations in melanoma

Some melanomas harbor activating BRAF mutations.

The mutation can identify a candidate for an oncoprotein-directed therapy.

A molecularly similar lesion does not guarantee equal therapeutic effect in every histologic type.

The pathology diagnosis remains important to treatment interpretation.

Slide 57 · ERBB2 / HER2: Molecular and protein-level assays

Immunohistochemistry

FISH

Detects HER2 protein overexpression in tumor cells

Assesses ERBB2 gene amplification

Measures a phenotypic oncoprotein product

Measures the corresponding genomic copy-number lesion

Can guide therapy in relevant breast tumors

May complement or clarify tissue protein testing

Slide 58 · Same pathway lesion, different histologic setting

Tumor

BRAF-associated treatment pattern

Hairy cell leukemia

Often sustained responses

Melanoma

Responses may be transient

Colon carcinoma

Limited responses in the described comparison

Slide 59 · Liquid biopsy: Tumor material enters circulation

Tumor cell turnover

Cells or DNA enter blood

Plasma / cell collection

Sensitive molecular analysis

Cell-free tumor DNA derives in part from dying cancer cells; Detection depends on tumor shedding.

Slide 60 · Circulating tumor cells versus circulating tumor DNA

Circulating tumor cells

Circulating tumor DNA

Intact tumor-derived cells in blood

Tumor-derived fragments within cell-free DNA

May provide cellular material for study

Can be tested for selected driver or acquired mutations

Requires cell-based recovery

Requires highly sensitive nucleic acid detection

Slide 61 · Serial ctDNA can reveal emergent resistance

Find targetable lesion

Start matched therapy

Repeat plasma testing

Find resistance-associated variant

Reassess therapy

In some settings, a resistance variant can be detected before clinically apparent relapse.

Slide 62 · Tissue biopsy and liquid biopsy are complementary

Tumor tissue

Plasma-based molecular test

Shows architecture and morphologic phenotype

Permits repeat, minimally invasive sampling

Documents invasion and tumor–stroma context

May reflect tumor-derived variants released into blood

Requires tissue collection

Does not by itself reproduce tissue histopathology

Slide 63 · Important interpretive constraints

A negative targeted assay does not examine every possible lesion.

Selected methods require an appropriate target or prior knowledge of a locus.

Tissue processing and analyte preservation influence what can be measured.

Technical findings must be reconciled with morphologic and clinical evidence.

Slide 64 · A clinically useful integrated testing pathway

Define tissue diagnosis + problem

Choose genomic target class

Select validated assay

Interpret result with context

Communicate clinical implication

The molecular report should distinguish diagnostic, prognostic, predictive and monitoring uses.

Slide 65 · Case 1 · Lymphoid proliferation

Hypothetical vignette

A lymph-node biopsy shows a lymphoid proliferation whose reactive or neoplastic nature remains uncertain.

Which molecular feature may help establish whether a dominant lymphocyte clone is present?

Consider antigen-receptor gene rearrangements.

Slide 66 · Case 1 · Interpretation

PCR can evaluate immunoglobulin or T-cell receptor rearrangements.

A dominant rearrangement supports clonality of the relevant lymphocyte population.

A heterogeneous rearrangement pattern supports polyclonality.

The result must be integrated with morphology and immunophenotype.

Slide 67 · Case 2 · Childhood small round cell tumor

Hypothetical vignette

Biopsy of a childhood soft-tissue mass shows a malignant small round cell tumor. Morphology is not sufficiently specific.

What characteristic rearrangement can support Ewing sarcoma?

Select a fusion-associated cytogenetic target.

Slide 68 · Case 2 · Interpretation

The t(11;22)(q24;q12) rearrangement is a characteristic diagnostic clue.

PCR-based molecular detection can help confirm Ewing sarcoma.

FISH or an appropriate transcript assay can provide complementary testing pathways.

The molecular finding supports—not replaces—the overall tumor diagnosis.

Slide 69 · Case 3 · Treated CML

Hypothetical vignette

A patient with known chronic myeloid leukemia is receiving treatment. The question is whether molecular disease persists.

Which analyte should be followed serially?

The tumor has a characteristic expressed fusion gene.

Slide 70 · Case 3 · Interpretation

BCR-ABL fusion RNA can be quantified using a reverse-transcription PCR approach.

Serial assays evaluate molecular disease burden rather than initial tumor morphology.

A changing transcript measurement may indicate residual or recurrent disease.

Interpretation depends on assay and clinical follow-up context.

Slide 71 · Case 4 · Drug resistance in lung cancer

Hypothetical vignette

A patient with molecularly targeted treatment for lung carcinoma develops concern for emerging resistance. Repeated tissue biopsy is undesirable.

How can the tumor genotype be reevaluated from blood?

The blood may contain DNA shed by neoplastic cells.

Slide 72 · Case 4 · Interpretation

A plasma-based liquid biopsy can interrogate circulating tumor DNA.

Sensitive molecular analysis can seek a resistance-associated sequence variant.

Repeated sampling can potentially detect genomic evolution before overt relapse.

Tissue remains important when morphology or broader evaluation is needed.

Slide 73 · One clinical question → one molecular strategy

Clinical question

Informative target

Representative strategy

Is the infiltrate clonal?

IG / TCR rearrangement

PCR

Does the tumor harbor a hallmark fusion?

Chromosomal / RNA fusion

FISH, cytogenetics, RT-PCR

What is the molecular category?

Selected sequence variants

Targeted NGS

Is disease still present?

Clone-specific transcript

Quantitative RT-PCR

Is treatment resistance evolving?

New tumor-derived variant

Sensitive liquid-biopsy assay

Slide 74 · Core synthesis

Match the molecular technique to the alteration and the clinical decision.

Clonality, translocation, copy-number and sequence assays answer different questions.

The same test result can serve diagnostic, prognostic or predictive roles depending on context.

Molecular data increase—not eliminate—the need for histopathologic interpretation.

Slide 75 · Primary Robbins source and supporting methods

Robbins, Cotran & Kumar. Pathologic Basis of Disease. 11th ed. (2026).

Primary section: Neoplasia — Molecular Diagnostics and Cytogenetics.

Technical methods: Genes and Human Diseases — Molecular Genetic Diagnosis.

Adjacent topics consulted: Immunohistochemistry, Molecular Profiling of Tumors and Neuroblastoma.

All schematics in this lecture are original editable teaching diagrams, not clinical images.