***About molecular biology [HERE ]...it's an introductory page and with links to much other information within that web site. About DNA [HERE].

Specimen quality is supremely important! Forensic TV shows have created somewhat of a myth that molecular techniques can flawlessly find the needle in the haystack from the crudest specimen imaginable. NOT SO!! There are many factors handled correctly that lead to success. These links are to pertinent text, below:

1. Patient BIOPSY TARGET, sampling.
2. genomic context as to humans.
3. histological sections for ISH morphological testing
4. PCR or "grind & bind" assays
   • Storing DNA for potential analysis
   • RNA recovery for rtDNA testing (Oncotype)
   • Quality DNA needed for PCR or MUTATION testing
     1. lyse the dissected cells to release genomic DNA (gDNA)
     2. concentrate or purify the selected DNA by magnetic microbeads or solutions that precipitate DNA into a centrifuged pelette
5. use the purified DNA for molecular analysis
6. CPT 4 CODING for BILLING for your WORK:
• searching archived ("signed out") specimens for molecular tests, 88363.
• new tissue for DX and/or testing:
  • cytological specimens:
    • 88172, intraop assessment for adequacy of FNA sample (was proper tissue obtained?).
    • 88177: each additional FNA pass, adequacy assessment.
    • 88173: FNA interpretation
  • surgical specimens, biopsies or excisions: gross & micro diagnosis, 88304-88309.
• morphological cytogenetic (ISH by FISH, SISH, or CISH) search for gene amplification, deletion, rearrangement:
  • 88367, computer-assisted quantification (gene average per nucleus), each specific gene probe.
  • 88368, manual quantification (gene average per nucleus), each specific gene probe.
• "grind and bind" assays for gene mutations ("stacking codes", 83890-83914, to be phased out in 2012, initially by reporting both "stacked" and new codes):
  • microdissection of key, tumor-rich area for molecular tests, cytopreps or surgicals:
    • gross fresh specimen, direct for molecular target:
      • 88387 (molecular sentinel node or genetic [POC]¹⁵.
      • 88388 (molecular sentinel node or genetic [POC] in conjunction with intraoperative diagnosis by FS or cytology TP¹⁵.
    • 88313: group 2 special stain on a dparafinized slide, if needed to grossly highlight a DNA-rich target area for selective scraping off
    • 88381 manual dotting of key target area on routinely stained slide or trimming target area out of block (slide microdissection, manual) for manual removal by others for ascertainment.
    • 88380 laser capture target cell removal from on slide for ascertainment.
  • 83907, lysis of target cells (e. g., from parafin embedded tissue) to release DNA.
  • 83890, extraction (ascertainment) of the optimal DNA/RNA versus,
  • 83891, isolation DNA/RNA, highly purified.
• separation of DNA or RNA:
  • by gel separation, 83894.
  • by single segment sequencing, each segment, 83904.
• amplification or enrichment of DNA or RNA:
  • DNA by PCR, 83898.
  • RNA by rt-PCR, 83902.
  • 88112, slide prep for cell concentration and target enrichment for such as UroVision FISH probe testing.
• mutation identification:
  • by ISH (FISH/SISH/CISH) identification: each, computer-assisted, 88365 or 88367; ISH, each, manual, 88368.
  • by sequencing identification, each segment, 83904. Then interpretation, 83912.
  • by alle-specific or codon-specific IHC identification (88342).

GENOMIC CONTEXT ¹²: The human genome has 3.3 gigabases; entire genome sequencing is thought likely to find an abnormal base in every thousand bases (3.3 miliion abnormals)! If one only searched for the protein-encoding genes (exome), it is thought that 90% of disease associated abnormals would be found. But, do we yet even know all of the exome? One expert thinks that some 500 genes have been identified as cancer associated¹⁷. The gene for familial retinoblastoma covers about 250,000 bases. Influences external to cells and their DNA...epigenetic factors...can cover & block or unmask genetic interaction sites as well as turn genetic "switches" on and off (DNA "methylation" being an example).

FROM THE PATIENT, BIOPSY TARGET: First of all, tissue actually containing adequate amounts of the lesion/abnormality of interest is mandatory! In deep lesions, this sampling is the job of surgeons, radiologists, and physicians trained in endoscopic ultra-sound-guided biopsies. Surgeon or Radiologist determination ofwhch target to sample is hugely important, a sizeable, nonnecrotic target being preferable. For the pathologist, a huge delimma appears when BOTH (1) a specific diagnosis must be made AND (2) molecular testing MIGHT be needed. The pathologist plays a huge role in on-site (1) intra-procedural decisions as to sample adequacy and (2) ANTICIPATING the possible need for molecular testing (a malignant diagnosis may not have yet been made & a medical oncologist may not have yet been selected). This is the game-breaker pre-analytical factor from which any inadquacy cannot be recovered except by scheduling and performing another procedure.

THINKING AHEAD, TUMOR SAMPLING: Forward-thinking stewardship of the genomic nucleic acid and protein expression targets within ever-smaller specimen samples will be increasingly important! Therefore, for both diagnosis and molecular testing, intra-operative assessment is important (FNA = 88172 & each additional needed assessment, 88177; intra-op. consult, 88329; intra-op. frozen 88331 & each additional 88332; intraoperative touch prep diagnosis & STAT report, 88333 or touch preps as part of case workup but not STAT report, 88161).To the extent that it is judged adequately safe for the patient, the assisting pathologist in deep biopsy procedures may need to be fairly aggressive in intra-procedurally requesting additional material.

STORING DNA/RNA BY THE PATHOLOGY TEAM: Prompt (1) refrigeration (as in PCA3 rt-PCR testing), (2) deep freezing (Tissue Bank), (3) cryostat freezing at about minus 26 degrees F for frozen sectioning with sections promptly fixed in 70% alcohol (can be kept long periods in a dessicator jar), (4) air-drying of cytology preps for rapid Wright's stain, (5) alcohol fixation of smears, (6) promptness of 10% NBF fixation of thin-sliced tissue blocks, and (7) avoidance of acidic or heavy metal fixatives (any meruric or zinc [GI FIX] fixative & probably avoid acidic Hartmann's fixative). Products contributed from the sample preparation can result in decreased signal or increased background fluorescent noise from the sequencing reactions. Attention to each/all such details tends to optimize DNA preservation for assay accuracy...an initial & critical factor for success.This is another critical pre-analytical factor.

Since many test methods are "grind and bind" (as with pre-IHC cytosol testing for ER & PR), the pathologist's pre-analytic impact on case sensitivity & accuracy for detecting molecular alterations that are actually present becomes critical. He/she must select tumor foci with (1) the least amount of admixed tumor-cell apoptosis/necrosis, or with the least amount of less-well-fixed tumor (the least amount of damaged DNA). The rt-PCR test is confounded by lots of admixed DNA from (2) non-invasive tumor components with hormone or other marker-expression quantities differing from the invasive (e.g., HER-2 neg. IDC overshadowed by HER-2 pos. DCIS) or (3) neotissue (desmoplasia or endothelial) cells or (4) inflamatory cells. Factors (3) & (4), above, add plenty of proliferative expression component to the test-sample "soup" and need to be avoided. If the local pathologist is NOT going to play any of the critical role of enrichment through microdissection, he/she should forward the block with the greatest amount of well-fixed invasive tumor.

RNA RECOVERY: In such as the Oncotype DX invasive breast cancer multi-gene assays to predict risk of recurrence (through analysis of gene EXPRESSION), the whole slide section (microtomb section frame) can be used when the tissue section is free of troublesome components and has 50% of greater of the tissue surface area composed of the invasive target lesion. Blood, necrosis, and fat have scant RNA and do not interfere¹⁴. Proliferative biopsy track repair tissue, macrophages, other nucleated cells, skin, muscle, or noninvasive component epithelium greater than 50% of sample DO/MAY interfere¹⁴ and must be manually left behind on the slide during target retrieval by microdissection. What is carefully removed from the tissue slide frame for analysis is done so in order to (1) enrich the specimen with the correct cellularity and (2) get rid of (leave behind) the incorrect, undesirable cellularity. The invasive target retrieved/removed must be no smaller than 2 MM (0.2CM). This process is very similar to retrieval of target DNA. Slide sections are about 6 microns thick, and about 30 microns in aggregated thickness is needed for sample adequacy (about the thickness of a human hair). That aggregate of enriched or removed tissue will then have all RNA extracted and DNA and other stuff washed away, making the purified RNA sample.

For first generation Sanger sequencing of the test DNA in search of mutations, the diligent selection of optimal target areas in the block by the pathologist is critical. This is so that he/she (or reference molecular lab technicians) can properly complete the macro- or micro-dissection of the focus containing the cell-pure, cell-rich lesion area to be tested. This is another critical pre-analytical factor.

It is said to be less important to have pure target lesion tissue in second generation (next gen. = 454, illumina, or solid) sequencing.

QUALITY DNA NEEDED: It is best to have at least 2 square mm. of malignant-cell-rich tissue or target-cell-containing smear with 50% or more (no less than 20%) of nuclei being invasive malignancy nuclei that (1) about 500 micrograms of tumor DNA (2) might deliver, among all the DNA retrieved, at least 25% mutated DNA (if a significant target mutation is present). If less than 100 malignant nuclei, there may not be enough target DNA to detect mutations [remember that any abnormal DNA is already diluted because 50% of all sample DNA is from one parent and 50% from the other]. If pathologist must return to archived (filed) materials to select the sample with appropriate DNA target tumor, a charge can be made [88363].

Some ideas for in-lab efforts to (1) volumetrically expand small samples to allow more histological sectioning or (2) even create samples for future testing include:

1. FNAs/fluid concentrates for cytopreps for diagnosis & reserve any excess sample until morphological idea of what was obtained.
2. aliquot a piece for frozen section for slides for reserving DNA, as above.
3. with agar, stack or suspend sufficient cores/particles sample so that many histology slides can be prepared for target DNA harvesting (selecting and purifying).
   
   Test sample selection:

The local general pathology lab is in a prime position to perform all of the important front-end work of selecting target-DNA-rich block/slide areas:

1. by macrodissection¹⁵: this is SELDOM used, [1.via choosing the correct block, 88387 ][2.via intra-op FS or cytoprep, 88388]. The code was originally for molecular sentinel node testing (which failed) but can be used when special exam or dissection is required as all or part of a non-microscopic analytical studies on tissue (example: selecting villi from POC sample for genetic testing¹⁵).
2. by microdissection [1. by instrument-assisted, such as laser capture, 88380][2. by manual means, 88381].

Morphospecific confirmation is the process by which a pathologist skilled in tumor morphology (1) actually documents that molecular abnormalities as with FISH are negative in TUMOR cells and/or (2) that invasive-tumor-cell-rich areas are accurately designated for retrieval from slides are other media as an integral part of any microdissection process acquiring the test sample for the "grind and bind" assays.

[HERE] is a quick review of evolution of sequencing, including some animations.

Tests which allow morphospecific confirmation (@) hold a distinct advantage when carefully judged in overall case concordance estimations. Rapid toluidine blue staining of reagent frozen section or other slides is a classical way to look for morphospecific assurance that tumor-rich DNA is being selected. Comparative viewing of unstained & deparafinized FFPE slides with a neighboring H&E slide is a quicker, approximate way to select out target DNA. Romanowsky-stained smears of tumor allow morphospecific viewing AND are able to provide high-quality DNA for the gind & bind PCR-type tests³. The target DNA selection process (microdissection) results in the sample for analysis. From that sample, all DNA is retrieved and purified (other tissue debris, proteins, and agents removed using the magnetic micro-balls or other separation strategy). From that purified DNA, the loci of interest are selected out by various technical methods and amplified (e. g. by PCR).

The patient is always well served when the pathologist mentally "tests" the case's specialty-test results for concordance with what is known about the tumor H&E findings (example for lung NSCLC [HERE] and lung cancer decision tree [HERE]).

Molecular tests depending on preserved RNA are depending on nucleic aid preservation that is much more subject to degradation than DNA. Molecular method source: [HERE]. Here are some tests for genomic (the molecular blue-print) proteins, RNA & DNA, and the various bio-molecular tissue expressions of those blue-prints.