***About molecular biology [HERE ]...it's an introductory page and with links to much other information within that web site.
About DNA [HERE]. A new 2013 ASCP practical pathology reference (#25, below) text for practicing pathologists by Dr. Heriot, HERE.
A warning about medical-testing reliability HERE.
Molecular TARGETS: The College of American pathologists has collaborated with a company to produce easy info as to available oncotherapy targeted therapy and clinical trials, HERE. Another info source, HERE25. There is also important molecular targeting of infectious organisms...often with clinically significant shortening of diagnostic turn around time (TAT)...as in ruling in or ruling out a mycobacterial etiology of a granulomatous tissue reaction23.
OOPS!! The idea of "personalized medicine" and "targeted therapy" became big in our community in August 2011 with a big upsurge of molecular testing, especially in NSCLC of adenocarcinoma type. As we grappled with cases, we were sometimes confronted with tumor IHC product expression heterogeneity issues in breast cancer. The March 8, 2012 issue of NEJMed19 came out with the blockbuster publication of a study showing some 40% of cancers have significant heterogenity in the molecular mutations on which the targeted therapy approach had assumed tumor mutational homogeneity. What follows still applies, but real thinking will need to be done as to what should be sampled in a stage IV situation in search of a "target" for treatment. In its first issue of 2013, JAMA has indicated the remarkable heterogeniety, need for more invasive (larger) sampling, and reservation of frozen tumor24. [extensive discussion on heterogeneity27] In some instances, there is a need to compare a patient's native (germline or "wild") DNA or RNA to his/her tumor DNA or RNA. The discussion below also attempts to make sense out of CPT4 billing issues. A source for genomic context as to humans.
Specimen DNA/RNA 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 which must be executed or handled correctly that lead to success. These links are to pertinent text, below:
- Patient BIOPSY TARGET, sampling and/or specimen "grossing".
- Prompt/expert sample handling to get the maximum quantity & quality.
- Evidence line #1: Histology sections needed for DX by H&E, special stains, & IHC.
- Evidence line #2: Histology for ISH morphological testing uses part of sample
- Evidence line #3: PCR or other "grind & bind" assays overview for molecular testing:
- Storing DNA for potential analysis
- RNA recovery for rtDNA testing (Oncotype)
- Quality DNA test sample component needed for PCR or MUTATION testing:
- since the DNA sample will include ALL DNA (both tumor and heterotopic tissues) from each and every cell type (desmoplastic post-piopsy proliferative repair tissue, benign normal cells, host immune response cells, etc.), it is important that a knowledgeable pathologist mark the specimen to be processed to help assure that only the areas of highest concentration of tumor nuclei is retrieved for the next steps.
- lyse the dissected cells & nuclei to release genomic DNA (gDNA)
- concentrate or purify the released DNA by magnetic microbeads or solutions that precipitate DNA (or other techniques) into a centrifuged pelette & discard all other confounding debris and proteins.
- use the purified DNA for molecular analysis
- CPT 4 CODING for BILLING for your WORK in molecular testing: [NOTE: 2012 was predicted to bring on new coding that would later replace the "stacking" approach to molecular coding] Many codes are "global', possessing a technical charge component (TC) and a professional charge coomponent (PC). Fewer are only PC or TC.
- searching archived ("signed out") specimens for best/correct material for use in molecular tests, 88363 or use 88363-26 (PC only when send to outside lab). Do not also charge this archival (88363) charge when charging for microdissection (88381).
- new tissue for DX and/or screening for triage or diagnosis, the routine histological stain for microscopy being the H&E stain & for cytology, the Pap stain (and/or some rapid stains):
- cytological specimens (FNAs, EUS, EBUS, fluids):
- 88172, initial or only intraoperative assessment for adequacy of FNA sample (was proper target diagnostic tissue obtained?).
- 88177: each intra-op. additional FNA pass, adequacy assessment.
- 88173: FNA interpretation (a professional component only code).
- and, from these might come nonroutine studies involving histology slides as well as cytology slides for special histochemical/cytochemical stains, IHC, ISH, cytogenic, flow cytometric, and the below "grind & bind" molecular assays.
- surgical specimens, biopsies or excisions: gross & micro diagnosis, 88304-88309. From these might come nonroutine studies involving histology slides as well as cytology slides for special histochemical/cytochemical stains, IHC, ISH, cytogenic, flow cytometric, and the below "grind & bind" molecular assays.
- hematology specimens (at receipt...by pathologist at an originating lab and not in a reference lab):
- 85060, peripheral blood smear, dispositional (professional specimen-receipt consult)...checking the requisition and data (or screening an abnormal CBC sample from the ER or an office or urgent care facility), the pathologist quickly advises as to advantageous (confirming that the requisition will generate the most appropriate testing to save on time and costs) changes needed as opposed to what may have been generically ordered. And, this is quite likely to be a different pathologist than the one who does the full interpretation of the hematology study at a different date.
- 85097, bone marrow smear, dispositional (professional specimen-receipt consult)...checking the requisition and data, the pathologist quickly advises as to advantageous (confirming that the requisition will generate the most appropriate testing to save on time and costs) changes needed as opposed to what may have been generically ordered. And, this is quite likely to be a different pathologist than the one who does the full interpretation of the hematology study at a different date.
- morphological cytogenetic (ISH by FISH, SISH, or CISH) search for gene amplification, gene deletion, gene rearrangement (as in ALK in NSCLC):
- 88367, computer-assisted quantification (gene average per nucleus), each specific gene probe (88367-26 if local pathologist interprets [PC] and another lab does & bills the technical [TC] work).
- 88368, manual quantification (gene average per nucleus), each specific gene probe (88368-26 if local pathologist interprets and another lab does and bills the technical work).
- Efforts, separate techniques, and interpretation for "grind and bind" or chromosomal assays for gene mutations ("stacking codes", 83890-83914, were expected to be phased out after 2012, initially by reporting both "stacked" and new codes or other payor demands):
- macrodissection ( gross fresh specimen), direct for chromosomal/molecular target (pick out likely fetal-component product of conception (POC) tissue or selecting the node component away from fat and other tissue & reported once for “each tissue preparation.” If two separately identified GYN [for POC] or sentinel nodes are submitted and a separate prep performed on each for molecular testing, then report 88387 or 88388 however many times there are different preparations; and, this should be amply described, including the lab to which preps are being sent, within the path. report):
- 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.
- microdissection of key, tumor-rich area for molecular tests, cytopreps or surgicals:
- 88313, slide: group 2 special stain on a deparafinized slide, if needed to grossly highlight a DNA-rich target area for selective scraping off of rich areas & suspension in liquid and pipetting into carrier tube for lysis, etc. Or,
- 88381-26, slide: Do not also charge the archival (88363) charge when charging this code. local professional-component (1) slide = manual dotting of key target area on routinely stained slide (slide microdissection, manual, for manual removal by other/reference lab's technicians for ascertainment) & noting on the slide the percentage of the circled tissue that is tumor; or (2) 88381, block: trimming target area out of parafin block and forwarding straight to other/reference lab for ascertainment. In (1), if (for whatever reason) only the block was sent, an alternative is to do marking, as above, on local slide & e-mail to ref. lab as an attached case slide photo or an attached scan from a color copier. A FAX is much less likely to work clearly enough.
- 88380, slide: laser capture highly selective target cell removal directly from within the histology section frame on slide for ascertainment.
- techniques21 of process:
- 83907, lysis of target cells (e. g., (1) scraped from specific area dotted or special-stained on slide or (2) from trimmed parafin embedded tissue) to release DNA from nuclei.
- 83890, extraction...ascertainment (via magnetic beads, etc.) of the optimal DNA/RNA versus,
- 83891, isolation into highly purified DNA/RNA.
- 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 morphologic (FISH/SISH/CISH) identification (above): (1) each probe, 88365 qualitative (as with cytology [HPV] or tissue); & (2) quantitative, each probe, computer-assisted = 88367 or, (3) quantitative, each probe, manual = 88368...see above.
- by finding segment or point mutations of a nucleotide(s):
- by sequencing identification, each segment, 83904.
- by alle-specific or codon-specific IHC identification (88342), each separate marker.
- Interpretation of "stacking code items" composite results for a specific test such as KRAS gene mutations (use code again for another item such as ALK if on a different date of service)21:
- if M. D. interprets, use 83912-26 for payment from the physician fee schedule21.
- if technologist or PhD clinical scientist interprets, use 83912 for payment from the clinical laboratory fee schedule21.
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 & histone modification is another example24).
FROM THE PATIENT, BIOPSY TARGET: First of all, tissue actually containing adequate amounts of the lesion/abnormality of interest for accurate diagnosis 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 of whch target to sample is hugely important, a sizeable, nonnecrotic target being preferable. For the pathologist, a huge dilemma 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. Though the modern medical psyche strains toward "least-invasive", a new molecular sample which can be frozen may become needed24. When dealing with a larger, fresh specimen in which it is uncertain where the cellular malignancy is, the specimen can be sliced and placed in 10% NBF containing toluidine blue (1 cc. of 15 toluidine blue in 200 cc of 10% NBF). The areas with richest amounts of blue staining contain the highest amounts of DNA. Under investigation in late 2012 is the tumor DNA source of plasma circulating tumor DNA [free or within circulating tumor cells?]24.
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) fresh tissue transport from the OR (keep "cold ischemia" time as short as possible); (2) refrigeration (as in PCA3 rt-PCR testing), (3) 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), (5) air-drying of cytology preps for rapid Wright's stain, (6) alcohol fixation of smears, (7) promptness of 10% NBF fixation of thin-sliced tissue blocks, and (8) avoidance of acidic or heavy metal fixatives (any mercuric or zinc [GI FIX] fixative & probably avoid acidic Hartmann's fixative). Nontumor 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 old time, pre-IHC, cytosol testing for ER & PR), they lack the critical quality control aspect of morphospecific concordance. So, 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% or 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. Microarray testing may also be confounded by the presence of extraneous, non-tumor (not-the-target) tissue.
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 or next generation nucleotide (next gen. = 454, illumina, or solid) sequencing.
Yet, at least when searching for evidence of loss of functional protein, "grind and bind" molecular testing may be less helpful than IHC!
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, the test may be falsely analyzed as "negative"). 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 .
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:
Specimen Plans & acquisition & reserving for testing:
- FNAs/fluid concentrates for cytopreps for diagnosis & reserve any excess sample until morphological idea of what was obtained (a prime example is the alert processing of high volume effusion samples most likely to contain malignant cells).
- aliquot a piece for frozen section for slides for reserving DNA, as above.
- 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:
Then there is the purifying the DNA  for (1) their own in-house testing or (2) forwarding of the purified lysate extract to a reputable reference molecular testing lab4 (insiders sometimes refer to such commercial or research labs as CORE labs) for either ISH testing or for testing-target amplification
for DNA sequencing in search of point or SNP mutations.
The sequencing results must be graphically displayed to ascertain that the sequencing worked; then the graphics must be searched by human or computer and interpreted as to mutations.
- 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).
- 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). So, as with the Oncotype test for breast cancer, there is now a morphospecific online calculator, the Magee equation.
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 acid 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.
CELLULAR ELEMENTS DETECTION:
histochemical stains = H&E, et. al. (@)
the classical stains in pathology labs
IHC (immunohistochemical) stains for protein expression= (@)
there are large #s of reagent antibody markers with variable performance characteristics & they are widely available in many community histology labs & allow labeling of all sorts of cell components
cytochemical stains (leukemia Dx) for cell expression of various products= (@)
classical stains in hematology & cytopathology labs; supravital stains for smear adequacy appear NOT to injure DNA
flow cytometry for cell membrane expressions of proteins/products
biological fluid or liquid supensions of solid-tissue cellularity; cell membrane & cytoplasmic labeling and counting of vast numbers of cells
protein electrophoresis (an expression)
mostly on serum, urine, & CSF
|branched chain DNA assay
(bDNA)||does not require amplification of the DNA or RNA test target [HERE]
|RFLP (restriction fragment length polymorphisms)
|Dot blot (DNA Dot-blot hybridization)
|SB (immunoblot...Southern Blot)
||a labled DNA method in 1970s by Dr. E. M. Southern to
detect DNA segments [HERE ]
|NB (immunoblot...northern Blot)
||a labled DNA method to detect mRNA segments [HERE ]
|WB (immunoblot...western Blot)
||a labled antibody method to detect specific cell
proteins [HERE ]
|SWB (immunoblot...southwestern Blot)
||a labled DNA probe method to detect specific cell proteins
immunotransfer blot assay)
|gene arrays (Oncotype, Mammaprint, Mammastrat)...slide or film based microarrays.
||[HERE ] and [HERE]
DNA & RNA AMPLIFICATION:
||qualitative PCR: present or absent
|real time PCR (QPCR)
||PCR in quantitative mode [HERE]
|DNA enrichment by allele specific hybridization (DEASH)
||said to be able to enrich to detect mutations when as little as only 1% of the specimen DNA is from undamaged malignant nuclei.
|TMA (transcription-mediated DNA amplification)
|LCR (ligase chain reaction DNA amplification)
|SDA (strand displacement amplification) and MDA (multiple displacement amplification) appear to be the same.
||said to be more applicable to whole-genome studies [HERE].
|rt-PCR (reverse transcriptase PCR)
||detects RNA and reverses it to genomic DNA & then amplifies the DNA [HERE ]
DNA IN-SITU HYBRIDIZATION:
|Methods, visual (morphospecific):
|FISH (fluorescent in-situ hybridization) = (@)
||need fluorescent scope
|CISH (chromogenic in-situ hybridization) = (@)
||permanent slide preps
|SISH (silver in-situ hybridization) = (@)
||permanent slide preps
DNA Nucleotide SEQUENCING:
|PCR (polymerase chain reaction DNA amplification) with first generation Sanger sequencing (developed 1975).
||detects actual genomic DNA and amplifies it (a shallow sequencing technique) with lots of analytical test "noise"; needs at least 40% of nuclei in DNA sample to be invasive tumor nuclei
|PCR (polymerase chain reaction DNA amplification) with second generation or next generation (Roche 454, then Illumina, Life Technologies Corp, Pacific Biosciences) sequencing (NGS5) (came out 2006). Wikipedia listing.
||sequences the genomic DNA and allows quantification (a variably deeper sequencing technique ["amplicon sequencing"]) with far less analytical test "noise" and capabilty to even estimate percentage of cells carrying mutation; needs at least 40% of nuclei in DNA sample to be invasive tumor nuclei; gene panels are coming out for "targeted sequencing" of a limited number of gene coding elements, such as Ion Torrent's AmpliSeq (46 genes on panel with 739 mutations) for cancer or others more restricted, such as for JAK2, KRAS, EGFR, and BRAF; this may require research labs providing testing in translational mode; Mayo is working on a panel for 22 genes associated with hereditary CRC; NCGR may come out with a 450 gene panel of rare diseases which couples can use to see if either or both have positivity; FOX Chase Ca. Ctr. is validating the Ion Torrent on parafin embedded tissue & looking at about 50 caancer related genes; FDA & CMS regs loom as a likely severe hobbler of what could be rapid translation to the clinic 12. Capillary "sequencing" is an old method20.
|third generation technologies:
||Cell Signalling Technology, Inc. has just begun developing IHC antibodies for specific mutations.
Additionally, as one searches some molecular labs on-line, here is an example of some "new" lingo noted: "X Lab performs genotyping of human DNA polymorphisms in support of biomedical research, typing Single Nucleotide Polymorphisms (SNPs), diallelic Insertion/Deletion Polymorphisms (indels) and Short Tandem Repeat Polymorphisms (STRPs) (also called Microsatellites); we have also developed a powerful panel of Ancestry Informative Markers (AIMs), based on SNPs, which is excellent for determining geoancestry. In addition, we offer both whole genome scans and fine mapping."
- See Wikipedia on-line
community-created encyclopedia, beginning with category of "science",
then "laboratory", then "laboratory techniques".
- molecular & morphology overview, Histopathology 53(1):1-19, July 2008.
- Betz BL, et. al., "The application of molecular diagnostic studies interrogating EGFR and KRAS mutations to [Romanowsky...Giemsa and Wright's stains] stained cytologic smears of lung carcinoma.", Am J Clin Pathol., 136(4):564-71, October 2011 [HERE].
- I'm indebted to our colleague, Dr. Phil J. Buckhaults, for an afternoon of orientation mentorship on 15 Nov. 2011 which begins the second phase of our group's ongoing relationship with this molecular expert.
- US Gov. CDC/MMWR specimen steps HERE.
- US Gov. genetics info home page HERE.
- GeneTests.org...hosted on the NCBI web site, sponsored by U. Washington, Seattle, listing genetics labs and all known gene tests, HERE.
- National Center for Biotechnology Information (NCBI) is HERE.
- NIH Genetic Test Registry, HERE.
- Cancer Cytogenomic Microarray Consortium, HERE.
- American College of Medical Genetics, HERE.
- Next generation sequencing lead article, CAP Today, November 2011, HERE.
- an example of a software web site HERE.
- GHI website, on left margin, click on "pathology review and RNA extraction"; brochure; andpersonal communications.
- Synovec MS, CAP expert in CPT-4 coding (co-chair, AMA-CPT Molecular Pathology Coding Workgroup), personal communication, late 2011.
- Vanderbuilt U. My Cancer Genome website.
- Lung cancer testing, lead article, CAP Today, November 2011, HERE.
- Genetics Home Reference is the National Library of Medicine's web site for consumer information about genetic conditions and the genes or chromosomes related to those conditions."
- Tumor Molecular Mutational Heterogeneity: Longo DL, Editorial, "Tumor Heterogeneity and Personalized Medicine", NEJMed 366(10):956-957; and Gerlinger M, et. al., "Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing", same NEJMed 366(10):883-892,
March 8, 2012.
- Mardis ER, Next-Generation Sequencing, 2012 video update lecture.
- Pathology/Lab Coding Alert, 13(4):25-32, April 2012.
- Djordjevic B, et. al., "Clinical assessment of PTEN loss in endometrial carcinoma: immunohistochemistry outperforms gene sequencing", Modern Pathology 25(5):699–708, May 2012, HERE.
- Mycobacterial (and other organisms) rapid molecular identification overview, HERE.
- Goetz L, et. al, "Viewpoint: Rebooting Cancer Tissue Handling in the Sequencing Era", JAMA 309(1):37-38, 2 January 2013, HERE.
- Cancer Genome Atlas Resaerch Network, HERE.
- Heriot K, Practical Surgical Pathology: Integrating Molecular Pathology into Your Morphologic Practice [chapter 43 "Clinical Genetics", p.435-451; chapter 44 "Molecular Path. I: Foundations", p.452-468; chapter 45 "Molecular Path. II: Techniques", p. 469-508; "Molecular Path. III: Applications and Diagnoses ", p.509-548; "Molecular Path. IV: Frontiers and Personalized Medicine ", p.549-559, ASCP Press, 592 pages, 2013.
- "Tumor Heterogeneity: Mechanisms and Bases for a Reliable Application of Molecular Marker Design", Int J Mol Sci. 2012; 13(2): 1951–2011, Published online 13 February 2012.
(posted 2004; latest addition or adjustment 27 June 2013)