1. Discuss renal tumors of infancy 2. PCR 3. NGS 4. Microarray 5. Liquid biopsy

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1. Renal Tumors of Infancy

Overview

Renal tumors of infancy and childhood encompass a distinct spectrum compared to adult renal tumors. The most common and best-studied is Wilms tumor (nephroblastoma), but several other entities are recognized, each with unique biology and clinical behavior.

A. Wilms Tumor (Nephroblastoma)

Definition & Epidemiology

Wilms tumor is the most common primary malignant renal tumor of childhood and the second most common solid tumor outside the brain in infants (behind neuroblastoma). It is an embryonal tumor arising from remnants of immature kidney (metanephric blastema).

Accounts for ~6-7% of all childhood cancers and 95% of all kidney cancers in children under 15
Average annual incidence: 8.0 per million
More than 80% are diagnosed before 5 years; median age 3.5 years
Slightly more frequent in females; higher incidence in Black children; lower in East Asian populations
Bilateral in ~5-7% of cases; median age at diagnosis is lower in bilateral disease
Overall 5-year survival: ~90%

(Campbell Walsh Wein Urology, 3-Volume Set)

Genetics

Multiple genetic mutations have been identified:

Gene (Chromosome)	Frequency	Somatic/Germline
IGF2 (11p15)	70%	Both
WTX (Xq11)	20%	Somatic only
WT1 (11p13)	20%	Both
CTNNB1 (3p21)	15%	Somatic only
TP53 (17p13)	4% overall; 70% anaplastic	Both

Most Wilms tumors arise from somatic mutations restricted to tumor tissue; a smaller fraction originate from germline mutations
Knudson's two-hit model does NOT explain most cases
10-15% have a germline pathogenic variant or early epigenetic alteration
~1-2% are familial

Key Predisposition Syndromes:

WAGR syndrome (Wilms, Aniridia, GU anomalies, intellectual disability) - due to 11p13 deletion including WT1
Denys-Drash syndrome - WT1 point mutations; mesangial sclerosis, nephropathy
Beckwith-Wiedemann syndrome (BWS) - 11p15.5 abnormalities (IGF2 overexpression/H19 loss); hemihypertrophy
Simpson-Golabi-Behmel syndrome
DICER1 mutations - associated with cystic variant

(Campbell Walsh Wein Urology)

Pathology - Nephrogenic Rests (NRs)

Nephrogenic rests are precursor lesions of Wilms tumor - remnants of embryonic renal tissue persisting after 36 weeks gestation.

Two types:

Perilobar nephrogenic rests (PLNRs) - found at the lobar periphery (elaborated late in embryogenesis); subcortical, sharply demarcated, blastema-tubule rich; associated with BWS and 11p15 mutations
Intralobar nephrogenic rests (ILNRs) - found anywhere within the lobe; stroma-rich, intermingle with parenchyma; associated with WT1 mutations, WAGR, and Denys-Drash; arise from earlier gestational aberrations

Nephroblastomatosis = multiple nephrogenic rests; diffuse overgrowth of PLNRs produces a thick rind enlarging the kidney while preserving its shape. Wilms tumor developed in 52% of such patients receiving adjuvant therapy.

Children <12 months with Wilms tumor AND nephrogenic rests (especially PLNRs) have markedly increased risk of contralateral disease.

Histology

Three classic components:

Blastemal (poorly differentiated small round blue cells)
Epithelial (tubular/glomerular structures)
Stromal (myxoid to fibroblastic)

Favorable histology = mixed triphasic or predominantly epithelial/stromal; any of the three elements may predominate.

Unfavorable histology = Anaplasia:

Defined by markedly enlarged, hyperchromatic nuclei and abnormal multipolar mitotic figures
Focal anaplasia - only in a discrete area; better prognosis
Diffuse anaplasia - TP53 mutations in 70%; highest risk; significant chemoresistance
Present in ~5% of all Wilms tumors, but disproportionately affects survival

Clinical Presentation

Abdominal mass (most common presenting sign; >85% are symptomatic)
Abdominal pain
Hematuria (~20%)
Hypertension (~25%; from elevated plasma renin)
Fever, anorexia, weight loss (~10%)
Acute abdominal pain from tumor rupture
Varicocele or hepatomegaly - may indicate IVC obstruction from tumor thrombus

Physical exam should assess for signs of associated syndromes: aniridia, hemihypertrophy, genitourinary anomalies.

Coagulopathy: 8% have acquired von Willebrand disease - check coagulation studies preoperatively.

Staging (COG/NWTSG)

Stage	Description
I	Limited to kidney; completely excised; capsule intact
II	Extended beyond kidney; completely excised
III	Residual non-hematogenous tumor; regional node involvement; spillage
IV	Hematogenous metastases (lung, liver, bone, brain)
V	Bilateral renal involvement

SIOP (European) uses a pre-chemotherapy staging system, while COG (North American) stages at primary surgery.

Imaging

All solid renal tumors of childhood share common radiographic features and Wilms tumor cannot be reliably distinguished from other renal tumors (RCC, CCSK) by imaging alone. In the SIOP-9 study, 5.4% of patients started on preoperative chemo for "Wilms tumor" had a different malignancy at nephrectomy.

Ultrasound - first-line; identifies intrarenal origin, venous extension
CT/MRI - staging, contralateral kidney evaluation, IVC involvement
Chest CT - pulmonary metastases
MRI has potential to distinguish hyperplastic nephrogenic rests from Wilms tumor (elliptical/lenticular vs spherical shape)

Treatment

COG approach (North American): Primary surgery (upfront nephrectomy) followed by adjuvant chemotherapy +/- radiotherapy

SIOP approach (European): Preoperative chemotherapy followed by nephrectomy

Chemotherapy backbone:

Stages I-II favorable histology: Vincristine + Actinomycin-D (VA) x 18 weeks
Stage III-IV favorable histology: VA + Doxorubicin + Radiotherapy
Anaplastic: Cyclophosphamide + Etoposide + Carboplatin added
Blastemal-predominant (post-chemo SIOP): Intensified regimen

Surgery:

Radical nephrectomy is standard; lymph node sampling mandatory
Nephron-sparing surgery in bilateral disease (Stage V) or solitary kidney
Persistent blastemal-predominant or anaplastic tumors require complete nephrectomy (chemoresistant)

Radiotherapy:

Flank/whole-abdomen RT for stage III, peritoneal spill
Whole-lung RT for pulmonary metastases

Late Effects of Treatment

Cumulative incidence of all chronic health conditions: 65% at 25 years post-therapy
Severe chronic conditions: 24%
Cardiac: doxorubicin cardiomyopathy, radiation heart disease
Renal failure risk (especially bilateral disease, solitary kidney)
Second malignancies in radiation field
Fertility: gonadal radiation causes hypogonadism, temporary azoospermia; alkylating agents + pelvic RT cause ovarian failure; offspring of irradiated females at risk for low birth weight

(Campbell Walsh Wein Urology)

B. Other Renal Tumors of Infancy

1. Cystic Nephroma

Rare benign cystic neoplasm; bimodal distribution
In children <4 years: M:F ratio 2:1
Encapsulated multilocular mass; locules lined by flattened/cuboidal/"hobnail" cells
Septa: connective tissue with atrophic renal tubules
Benign but rare malignant transformation
Treatment: complete excision

2. Cystic Partially Differentiated Nephroblastoma

Rare benign cystic neoplasm histologically identical to cystic nephroma
Key distinction: Contains Wilms tumor elements within septa
Mostly in children <2 years of age
Cured by complete excision (Brenner and Rector's The Kidney)

3. Congenital Mesoblastic Nephroma (CMN)

Most common renal tumor in the first 3 months of life
Two subtypes: classic (fibrous, similar to infantile fibromatosis) and cellular (similar to congenital fibrosarcoma; ETV6-NTRK3 fusion)
Cellular type has malignant potential
Treatment: nephrectomy; cellular type may recur

4. Clear Cell Sarcoma of the Kidney (CCSK)

"Bone-metastasizing renal tumor of childhood"
Peak age: 1-4 years; M>F
BCOR internal tandem duplication in most cases
Metastasizes to bone (distinctive), brain, lung, liver
More aggressive; requires doxorubicin in treatment
No histologic resemblance to clear cell RCC

5. Rhabdoid Tumor of the Kidney (RTK)

Highly malignant; predominantly in infants (<2 years)
Loss of SMARCB1/INI1 (22q11) - immunohistochemistry shows loss of nuclear INI1 staining
Associated with CNS malignancies (synchronous posterior fossa tumors)
Very poor prognosis

(Campbell Walsh Wein Urology; Brenner and Rector's The Kidney)

2. PCR (Polymerase Chain Reaction)

Principle

PCR is a technique for exponential in vitro amplification of a specific DNA sequence using repeated cycles of:

Denaturation (94-96°C) - double-stranded DNA is separated into single strands
Annealing (50-65°C) - short oligonucleotide primers bind to complementary sequences flanking the target region
Extension (72°C) - DNA polymerase (Taq polymerase, thermostable) synthesizes new DNA from each primer

Each cycle doubles the number of target DNA copies. After 30-40 cycles, an approximately billion-fold amplification is achieved.

Key Components

Template DNA (target sequence)
Two oligonucleotide primers (forward and reverse; ~20 nucleotides each)
Thermostable DNA polymerase (Taq from Thermus aquaticus)
Deoxynucleotide triphosphates (dNTPs) - building blocks
Buffer with MgCl₂ (cofactor)

Variants

Type	Description	Application
RT-PCR (Reverse Transcription PCR)	RNA is first converted to cDNA by reverse transcriptase, then PCR amplified	mRNA expression, viral RNA detection (influenza, SARS-CoV-2)
Real-time PCR (qPCR)	Fluorescent detection during amplification; quantifies DNA/RNA levels	Viral load quantification, gene expression, MRD
Multiplex PCR	Multiple primer sets in one reaction	Simultaneous detection of multiple pathogens
Nested PCR	Two sequential rounds of PCR with inner/outer primers	Increased sensitivity
Digital PCR (dPCR)	Partitions sample into thousands of reactions; absolute quantification	ctDNA, rare variant detection
Allele-specific PCR	Primers designed to amplify only mutant or wild-type allele	Single-nucleotide variant detection

Applications in Pathology/Medicine

Infectious disease diagnosis - RT-PCR for influenza, SARS-CoV-2; PCR for TB, HPV, MRSA
Oncology - Detection of fusion transcripts (BCR-ABL in CML), mutations, MRD monitoring
Prenatal diagnosis - Single-gene disorder screening (Creasy & Resnik)
Clonality assessment - PCR-based IgH/TCR rearrangement for lymphoid neoplasms
Forensic/identity testing - STR (short tandem repeat) profiling
Chimerism testing after bone marrow transplantation
Pharmacogenomics - CYP2D6, CYP2C19 genotyping

Limitations

Requires prior knowledge of target sequence (primers must be designed)
Susceptible to contamination (false positives)
Cannot detect unknown mutations or genome-wide changes

(Quick Compendium of Clinical Pathology 5th edition; Jawetz Melnick & Adelberg's Medical Microbiology)

3. Next-Generation Sequencing (NGS)

Definition

NGS (also called massively parallel sequencing or second-generation sequencing) refers to high-throughput DNA sequencing technologies that can simultaneously sequence millions to billions of DNA fragments, representing a quantum leap from Sanger ("first-generation") sequencing.

Historical Context

Sanger sequencing was automated in the late 1980s; coupled with capillary electrophoresis, it formed the basis of the Human Genome Project
NGS enabled by the concept of "short read, massively parallel" sequencing
Superior sensitivity for detecting low-frequency variants in clinical samples
Cost-effective when multiple targets are of interest (vs. multiple separate PCR assays)

(Quick Compendium of Clinical Pathology 5th edition)

Workflow: Three Steps

Step 1 - Library Construction

Patient DNA is fragmented into small pieces (75-400 bp)
Fragments are size-selected
Regions of interest are enriched (for targeted sequencing) using capture probes or PCR with specified primers
Ligated to adapter sequences at both 5' and 3' ends
Immobilized on solid support or bead
Flanked fragments become known as "inserts" - this collection is the "library"

Step 2 - Sequencing

Sequencing reactions occur in a flow cell containing the immobilized DNA library
Data generated one nucleotide at a time
Millions of fragments sequenced simultaneously in parallel
Uses sequencing-by-synthesis (Illumina), ion semiconductor, or other platforms

Step 3 - Analysis (Pipeline)

Data analysis occurs in several computational phases:

Primary analysis - base calling (converting signals to nucleotide sequences)
Secondary analysis - alignment to reference genome; variant calling
Tertiary analysis - clinical interpretation

Types of Abnormalities Detected

Abnormality	Abbreviation
Single nucleotide variants	SNVs
Small insertions/deletions	Indels
Copy number alterations	CNAs
Structural rearrangements/fusions	SVs

Types of NGS Assays

Assay	Description
Whole genome sequencing (WGS)	All ~3 billion base pairs; highest resolution; highest cost
Whole exome sequencing (WES)	All protein-coding regions (~2% of genome); captures most disease-causing variants
Targeted gene panels	Specific cancer-relevant genes (e.g., 50-500 genes); most cost-effective for oncology
RNA sequencing (RNA-seq)	Transcriptome; detects fusions, splice variants, expression

Clinical Applications

Oncology - somatic tumor profiling for actionable mutations (EGFR, KRAS, BRAF, ALK, etc.), tumor mutational burden (TMB), microsatellite instability (MSI)
Inherited disease - germline exome/genome for rare diseases, congenital abnormalities
Hematopathology - lymphoma/leukemia classification, clonality, MRD
Infectious disease - metagenomic sequencing for unknown pathogens
Prenatal - cell-free DNA sequencing for chromosomal aneuploidies (NIPT)
Pharmacogenomics - comprehensive drug metabolism profiling
Liquid biopsy - ctDNA detection and quantification

Advantages over PCR

No prior sequence knowledge needed (WGS/WES)
Detects multiple variant types simultaneously
Higher sensitivity for low-frequency variants
Comprehensive genomic profiling in one assay

(Quick Compendium of Clinical Pathology 5th edition; Harrison's Principles of Internal Medicine 22E)

4. Microarray (Chromosomal Microarray Analysis / Array CGH)

Principle

Microarray analysis allows genome-wide surveys for chromosomal imbalances (copy number variations) without prior knowledge of which chromosomal regions are affected - unlike FISH, which requires a specific target.

Types of Microarray Platforms

Three general platforms in clinical use (Creasy & Resnik's Maternal-Fetal Medicine):

1. Array CGH (Comparative Genomic Hybridization)

Principle:

Equal amounts of test DNA (patient) and reference DNA (normal control) are labeled with two different fluorescent dyes (e.g., Cy3 green and Cy5 red)
Both are mixed and co-hybridized to an array spotted with thousands of DNA probes spanning the genome at regularly spaced intervals
At each probe location, binding of the two labeled DNA samples is compared
Equal (diploid): All spots fluoresce yellow (equal mix of green + red)
Deletion: Spots show skewing toward red (less test DNA)
Duplication: Spots show skewing toward green (more test DNA)
Resolution depends on probe density (probes every 200 bp >> probes every 1 kb)
Usually requires several contiguous probes to be involved for detection

(Robbins, Cotran & Kumar Pathologic Basis of Disease)

2. SNP Genotyping Arrays

Probes designed to identify SNP sites genome-wide
Provides both copy number AND genotype (haplotype) information
Can detect copy-neutral loss of heterozygosity (LOH) - which CGH cannot
SNPs occur approximately every 1000 nucleotides in the genome
Can determine transmission of alleles from parent to child

3. Combination Platforms (CGH + SNP)

Both oligonucleotide CGH probes AND SNP probes
Best of both - copy number and genotype information simultaneously
Currently preferred in prenatal diagnosis and constitutional studies

What Microarrays Detect

Copy number variants (CNVs) - deletions and duplications
Microdeletions/microduplications (too small for karyotype)
Chromosomal aneuploidy (trisomy, monosomy)
Uniparental disomy (UPD) - via SNP arrays
SNP arrays also provide genotyping data for consanguinity assessment

What Microarrays CANNOT Detect

Balanced translocations or inversions (no copy number change)
Polyploidy (triploidy) - unless SNP array
Point mutations / single nucleotide variants (NGS required)
Low-level mosaicism below detection threshold

Applications

Constitutional/postnatal diagnostics - developmental delay, intellectual disability, autism, dysmorphic features; first-line test (replaced karyotype in many guidelines)
Prenatal diagnosis - recommended when fetal structural anomaly detected on ultrasound (SMFM/ACOG)
Oncology - tumor profiling for CNVs, LOH; monitoring clonal evolution
Preimplantation genetic testing (PGT-A) - aneuploidy screening of embryos

Comparison: Karyotype vs. FISH vs. Array CGH vs. NGS

Feature	Karyotype	FISH	Array CGH	NGS
Resolution	~5-10 Mb	Single locus	50 kb - 1 Mb	1 bp
Prior knowledge needed	No	Yes	No	No (WGS/WES)
Detects balanced rearrangements	Yes	Partial	No	Yes (WGS)
Detects CNVs	Large only	Single	Genome-wide	Yes
Detects SNVs	No	No	No	Yes

(Robbins, Cotran & Kumar Pathologic Basis of Disease; The Developing Human - Clinically Oriented Embryology; Creasy & Resnik's MFM)

5. Liquid Biopsy

Definition

Liquid biopsy is the analysis of tumor-derived material from body fluids (predominantly blood) instead of conventional tissue biopsy, providing non-invasive or minimally invasive access to tumor molecular information.

"The liquid biopsy approach extracts molecular information from the tumor by detailed analysis of circulating tumor-derived genetic material in the bloodstream." - Tietz Textbook of Laboratory Medicine 7th Edition

Rationale

Conventional tissue biopsies are invasive, risky, and may not capture tumor heterogeneity (spatial or temporal)
Tumors release various analytes into body fluids
Liquid biopsy allows serial, repeated sampling for real-time disease monitoring

Components of Liquid Biopsy

1. Circulating Tumor Cells (CTCs)

First reported by Thomas Ashworth in 1869
Rare cells that originate from primary and metastatic tumors and enter the circulation
Can potentially invade distant organs (metastasis formation)
CTC counts at diagnosis and post-surgery correlate with relapse risk (breast cancer: >5 CTCs/7.5 mL blood = 6x increased recurrence risk)
FDA-cleared CellSearch system - uses immunomagnetic selection via EpCAM (epithelial marker)
Label-independent systems - exploit physical properties: size (CTCs generally 8-10 μm vs leukocytes), density, deformability
Challenge: Epithelial-to-mesenchymal transition (EMT) causes downregulation of EpCAM, making CTCs undetectable by standard methods

Applications of CTCs:

Prognosis (independent of primary tumor response to therapy)
Predictive marker - HER-2 status on CTCs may differ from primary tumor
Companion diagnostics (trastuzumab, lapatinib in HER2-negative primary/HER2-positive CTCs trials)
Detection of glioblastoma extracranial spread (organ donor exclusion)

(Tietz Textbook of Laboratory Medicine 7th Edition)

2. Circulating Tumor DNA (ctDNA)

A subset of cell-free DNA (cfDNA) - first described in blood in 1948 by Mandel and Metais
Tumor-derived DNA first observed in plasma of cancer patients in the late 1980s
Released mainly by apoptotic or necrotic tumor cells
cfDNA is naturally fragmented with lengths correlating with DNA wrapped around individual nucleosomes
Challenge: ctDNA must be detected against a large background of normal (germline/hematopoietic) cfDNA

Detection methods:

NGS-based methods - most sensitive; detect multiple variants simultaneously; digital sequencing, error-correction algorithms
Digital PCR (ddPCR) - absolute quantification of specific mutations
Targeted sequencing panels

Clinical Applications of ctDNA (Harrison's Principles of Internal Medicine 22E; Tietz):

Application	Details
Early cancer detection	Multi-cancer early detection (MCED) tests
Diagnosis assistance	Lung cancer - "liquid biopsy" to detect EGFR, ALK, ROS1 mutations non-invasively
Treatment selection	Identify actionable mutations when tissue biopsy insufficient
Resistance monitoring	Serial sampling to detect emergence of resistance mutations (EGFR T790M, ESR1)
Minimal residual disease (MRD)	Post-surgery/therapy ctDNA to detect occult residual disease
Disease monitoring	ctDNA levels track tumor burden in real time
NIPT	Fetal cell-free DNA for chromosomal aneuploidies in maternal plasma

3. Circulating miRNAs

Small non-coding RNAs; highly stable in circulation
Tumor-specific miRNA profiles can serve as biomarkers
Examples: miR-21 (overexpressed in many cancers)

4. Extracellular Vesicles (Exosomes)

Membrane-bound vesicles (30-150 nm) released by viable tumor AND normal cells
Contain proteins, lipids, mRNA, miRNA, DNA
CTCs can contribute to the exosome pool
Role in pre-metastatic niche formation
Currently less clinically validated than ctDNA

Sources of Liquid Biopsy (Beyond Blood)

Fluid	Applications
Urine	Bladder carcinoma, genitourinary tumors (urinary cfDNA)
CSF	CNS tumors, leptomeningeal carcinomatosis
Pleural/peritoneal fluid	Thoracic/abdominal cancers
Stool	Colorectal cancer (fecal DNA multi-analyte assays)
Saliva	Head and neck cancers
BAL	Lung cancer

CTC vs. ctDNA Comparison

Feature	CTCs	ctDNA
Detection difficulty	More difficult (rare cells)	Easier (plasma extraction)
Information type	Cellular/protein/transcriptional	DNA sequence only
Tumor heterogeneity	Individual cell analysis possible	Reflects bulk tumor average
EMT challenge	Yes (EpCAM loss)	No
Serial monitoring	Difficult	Easy
Resistance detection	mRNA/protein level changes	DNA mutations

"The present over-competition between researchers working in different biomarker fields (ctDNA versus CTCs) appears to be counterproductive" - capturing the complementary nature of these modalities.

(Tietz Textbook of Laboratory Medicine 7th Edition; Harrison's Principles of Internal Medicine 22E; Schwartz's Principles of Surgery 11th Edition)

Summary: Relationship Between These Technologies in Oncology

Technology	Primary Role
PCR	Targeted detection of known mutations/sequences; fast, sensitive
NGS	Comprehensive genomic profiling; unknown variants; multiple targets
Microarray	Genome-wide copy number variants; no sequence data
Liquid biopsy	Non-invasive tumor monitoring via blood; uses PCR, ddPCR, NGS as detection platforms

These technologies are increasingly used together in precision oncology: microarray for constitutional CNV analysis, tumor NGS panels for somatic profiling, liquid biopsy (ctDNA + NGS) for dynamic monitoring, and PCR for rapid targeted confirmation.

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