Clinician Login

/Clinician Login
Clinician Login 2017-10-05T21:32:00+00:00

Welcome to the GMI Intranet for UCSF Clinicians

Our intention in this section is to provide you with all of the support you need to use genomic data in your clinical practice.

The Clinical Cancer Genomics Laboratory (CCGL) performs Clinical Oncology testing, including the development of Next-Generation Sequencing tests, for the UCSF community. The lab is located in the Mt Zion Cancer Research Building, 2340 Sutter Street in S151.

Research Samples — Request for Applications (RFA) – UCSF 500 Cancer Gene Panel

Thank you to all who submitted RFA’s from the Cancer Center and Department of Pathology.

PURPOSE

Department of Pathology members are invited to submit applications for research projects to the Clinical Cancer Genomics Laboratory (CCGL). The laboratory will utilize targeted capture followed by next generation sequencing to screen for mutations and copy number changes in 538 cancer-related genes (see http://gmi.ucsf.edu/clinicians/ for current list) in human tumor samples. Both frozen and FFPE tumor samples are acceptable (see http://gmi.ucsf.edu/clinicians/ for sample requirements/instructions). Matched normal tissues from the same patients will allow the identification of somatic variants but is not a requirement.

AVAILABLE FUNDING

Proposals with a projected direct cost of up to $20,000 will be considered.  Proposals will be reviewed by Boris Bastian and Scott Oakes.  Applicants will be informed of the committee’s decision within 1 week.  If approved, funding will commence on December 15 and samples should be submitted until December 22.  Any project already supported by another source will not be considered.  An award from this program may be adjusted to avoid overlapping funding.

DEADLINE

Applications were due on December 12, 2014.

APPLICATION INSTRUCTIONS

Each application should include the following:

  1. Cover page: Title of the project, principal investigator name and signature, amount requested.
  2. Estimated budget. The reagent cost for the sequencing is estimated at $400 per sample and $800 per tumor/normal pair and should be included in the budget. Technician costs for the sequencing portion will be covered by the Genomic Medicine Initiative and do not need to be included in the budget. The CCGL will provide FASTQ or BAM files for tumor and normal tissue. Additional bioinformatics analyses such as variant calling on tumor and normal samples with Unified Genotyper, somatic variant analysis with Mutect (for tumor/normal pairs only), and genome-wide copy number analysis with CNVkit is available for $100 per patient. Salary support is not eligible.
  3. Statement of Rationale, Background and Significance (2 pages maximum, excluding references):
    • How was (or will) this sample set (be) assembled? The samples need to be covered by an existing CHR (protocol or waiver).
    • Why are these samples of special interest for this program?
    • What is the unmet need to expand genetic annotation of this sample category not already covered by other large scale discovery efforts (TCGA, ICGC, etc.) d. What are the biological, pathologic, or clinical questions that may be clarified by this project? e. How will the data be analyzed?
  4. Other sources of current funding.

Applications should be submitted electronically to Boris Bastian and Scott Oakes.

PURPOSE

Cancer Center Members are invited to submit applications for research projects to the Clinical Cancer Genomics Laboratory (CCGL). The laboratory will utilize targeted capture followed by next generation sequencing to screen for mutations and copy number changes in 538 cancer-related genes (see http://cancer.ucsf.edu/CCGL for current list) in human tumor samples. Both frozen and FFPE tumor samples are acceptable. Matched normal tissues from the same patients will allow the identification of somatic variants but is not a requirement.

AVAILABLE FUNDING

Proposals with a projected direct cost of up to $25,000 will be considered. Applicants will be informed of the committee’s decision in 1 week. If approved, funding will commence on November 7 and samples should be submitted until December 1. Any project already supported by another source will not be considered. An award from this program may be adjusted to avoid overlapping funding.

DEADLINE

Applications were due on November 1 and will be reviewed by a panel composed of members of the Cancer Center leadership.

APPLICATION INSTRUCTIONS

Each application should include

  1. Cover Page — Title of the project, principal investigator name and signature, amount requested
  2. Estimated Budget — The reagent cost for the sequencing is estimated at $400 per sample and $800 per tumor normal pair and should be included in the budget. Technician costs for the sequencing portion will be covered by the Genomic Medicine Initiative and do not need to be included in the budget. The CCGL will provide FASTQ or BAM files for tumor and normal tissue. Additional bioinformatics analyses such as variant calling on tumor and normal samples with Unified Genotyper, somatic variant analysis with Mutect (for tumor normal pairs only), and genome-wide copy number analysis with CNVkit is available for $100 per patient. Salary support is not eligible.
  3. Statement of Rationale, Background & Significance (2 pages maximum, excluding references)
    • How was (or will) this sample set (be) assembled? The samples need to be covered by an existing CHR (protocol or waiver).
    • Why are these samples of special interest for this program?
    • What is the unmet need to expand genetic annotation of this sample category not already covered by other large-scale discovery efforts (TCGA, ICGC, etc.)?
    • What are the biological, pathologic, or clinical questions that may be clarified by this project?
    • How is the data being analyzed?
  4. Other sources of current funding

Applications should be submitted electronically to Kate Shumate

Initiated by Alan Ashworth and Boris Bastian

CCGL Test Menu

UCSF 500

The CCGL is currently accepting paper requisitions for a select number of cases for UCSF 500 cancer gene panel testing. This is limited to the acceptance of requests to patients that have exhausted treatment options and/or for which a therapeutic target may be identified.

Cases identified that meet this acceptance criteria should be emailed to Jessica Van Ziffle and Boris Bastian for approval.
Please click here to submit input on gene variants specific to your area of expertise

All of the exons of the genes listed are targeted. Precise numbers for coverage are being determined: some exons that are GC rich are hard to capture, so they may not be covered well.

Click here to view the following gene list in Excel

Cancer Gene Panel v1

Please click here to submit input on gene variants specific to your area of expertise

Click Here to view the Specimen Requirements in a Word file.

Specimen Requirements for the UCSF 500 Cancer Gene Panel

  1. For the UCSF 500 Cancer Gene Panel, the Clinical Cancer Genomics Laboratory will accept FFPE tissue blocks or cut unstained sections. These specimens must be accompanied by an H&E stained section for evaluation by a pathologist. FNA cytology smears are acceptable if adequate cells are present. Peripheral blood, buffy coat cells, or buccal swabs are also acceptable. Ideally, samples should yield at least 250 ng of DNA for library preparation. For research samples only, extracted DNA is also acceptable.
  2. FFPE and FNA tissue quality and tumor quantity must be adequate as determined by a pathologist. The pathologist identifies areas (tumor and normal) of the specimen for analysis. Samples may be enriched for tumor content by macro-dissection.
    • In areas of tumor, the percentage of tumor cells among all nucleated cells in the area should be at least 40% for optimal sensitivity.  Specimens with less than 40% tumor cells may be tested with reduced sensitivity.
  3. FFPE tissue and cytology smear slides require no special transport or storage. These samples are stable indefinitely at room temperature. Buffy coat will be stored frozen. Peripheral blood and buccal swabs are stable to ship at room temperature, but should be stored at 4oC if stored for longer than 24 hours.
  4. Inadequately labeled specimens are unacceptable. Minimum information required on the requisition form includes two unique patient identifiers. Specimens that are not labeled correctly or received without a requisition form will not be processed until specimen identification is verified by the submitting physician.
  5. Specimens are accepted on business days between the hours of 8AM – 5PM.
  6. DNA extraction has a minimum turnaround time of 2 days to provide material for downstream assays (Turn around time does not include time needed for analysis of DNA quantity and quality).

Protocol for Small Biopsies with Intent for Molecular Testing/Research or Other Sendout with Immediate Assessment by Pathologist 

  1. Clinician or Clinical Research Coordinator contact the FNA Clinic front desk one day ahead to alert of an upcoming biopsy with special testing requirements.  This includes any requests for tissue obtained beyond that necessary for diagnosis.   FNA CLINIC:  885-7301, hours 8:30 a.m.- 5 p.m.
  2. FNA Clinic front desk directs the Clinician or Clinical Research Coordinator to the appropriate Cytopathologist on-call at the appropriate hospital the day biopsy is scheduled.
  3. Clinician or designate contacts the Cytopathologist and sends specific specimen requirement documents (written requirements from study, industry sponsor or commercial laboratory) and expresses written priority for tissues via e-mail or fax to the Cytopathologist.
  4. Cytopathologist performs on-site evaluation and divides specimen based on priorities and requirements specified by Clinician.
  5. Cytopathologist hand-carries specimens to Cytology laboratory and/or Surgical Pathology gross room for accessioning and will communicate and specific processing requirements (i.e. no decalcification, snap frozen & store at -80C, conserve tissue–do not perform IHC).
  6. If specimens are to be stored at -80C, a waiver of liability must be signed by the patient prior to the procedure.  Frozen storage is available only as a courtesy and only for 3 months.
  7. Gross room executes as per instructions by Cytopathologist and inserts notes in CoPath to instruct histology lab as to special processing.

Download to print this protocol

Pathogenic Mutation
Alterations with sufficient evidence to classify as pathogenic (capable of causing disease)

Variant, Likely Pathogenic (VLP)
Alterations with strong evidence in favor of pathogenicity

Variant, Unknown Significance (VUS)
Alterations with limited and/or conflicting evidence regarding pathogenicity

Variant, Likely Benign (VLB)
Alterations with strong evidence against pathogenicity.

Benign
Alterations with very strong evidence against pathogenicity

We need input from the Oncology Community on which variants need to be specified as “No Pathogenic Mutations Found”.
Please go to our Contact page and use the form to make a comment.

Methods used in the research analysis pipeline

SEQUENCING READ ALIGNMENT AND FILTERING

Paired-end sequencing reads are aligned to the human reference genome, UCSC version hg19, using BWA-MEM. The reads are sorted by position and converted to compressed BAM format using samtools. Likely PCR and optical duplicate read pairs are marked using the Picard command MarkDuplicates and removed using SAMBAMBA. Finally, reads near potential indels are realigned using the Genome Analysis Toolkit (GATK) command IndelRealigner.

Software Versions

  • BWA: 0.7.10-r789
  • Samtools: 1.1 (using htslib 1.1)
  • Picard tools: 1.97(1504)
  • SAMBAMBA: 0.4.7
  • GATK: 2014.4-3.3.0-0-ga3711aa

Output Files

  • <SampleID>.deduplicated.realign.bam — alignment of sequencing reads, in BAM format
  • <SampleID>.deduplicated.realign.bam.bai — index of reads in the BAM file

References
BWA-MEM
Picard (no paper)
Samtools
SAMBAMBA (software only)
GATK

VARIANT CALLING

Single-nucleotide variants (SNVs) and small sequence insertions and deletions (indels) are called in each sample using the GATK command HaplotypeCaller in normal or germline samples and UnifiedGenotyper in tumor samples. Where a matched tumor-normal pair is provided, somatic SNVs and indels are called using MuTect and SomaticIndelDetector, respectively, both part of the GATK-based Somatic Analysis Toolkit (SATK). The variant calls are stored in VCF files and annotated using Annovar.

Copy number variants and alterations are called in each sample using CNVkit. Copy ratios are inferred from read depth in across the genome, using both on- and off-target reads, and normalized to a reference profile derived from a pool of 18 normal samples sequenced on the CCGL platform.

Software Versions

  • SATK (MuTect, SomaticIndelDetector): 2013.1-10-gd6fa6c3
  • GATK (HaplotypeCaller, Unified Genotyper): 2014.4-3.3.0-0-ga3711aa
  • Annovar: v2014Nov12
  • CNVkit: 0.3.3

GATK and SATK Output Files

  • <SampleID>.deduplicated.realign.recal.bam, .bam.bai — mapped reads with base quality score recalibration (BQSR), and their index
  • <SampleID>.deduplicated.realign.recal.UGT.vcf — SNVs and indels called by GATK UnifiedGenotyper (if tumor sample)
  • <SampleID>.deduplicated.realign.recal.HC.vcf — SNVs and indels called by GATK HaplotypeCaller (if germline sample)

SATK Output Files (if tumor-normal pair)

  • <SampleID>.deduplicated.realign.bam.mutect.vcf — SNVs called by MuTect
  • <SampleID>.deduplicated.realign.bam.mutect.vcf — indels called by SomaticIndelDetector

Annovar Output Files

  • <SampleID>.deduplicated.realign.recal.UGT.snps.hg19_multianno.txt
  • <SampleID>.deduplicated.realign.recal.HC.snps.hg19_multianno.txt
  • <SampleID>.deduplicated.realign.bam.mutect.snps.hg19_multianno.txt
  • <SampleID>.deduplicated.realign.bam.sid.snps.hg19_multianno.txt

CNVkit Output Files

  • <SampleID>.cnr — bin-level log2 ratios
  • <SampleID>.cns — segmentation
  • <SampleID>-diagram.pdf — ideogram of each chromosome’s copy number and gene-level CNAs
  • <SampleID>-scatter.pdf — whole-genome copy number profile visualized as a scatter plot
  • <SampleID>.nexus  — BioDiscovery Nexus Copy Number input file (in “basic” format)
  • <SampleID>-gainloss.tsv — tab-separated table of genes with estimated log2 copy ratio value above +0.3 or below -0.3
  • <SampleID>-breaks.tsv — tab-separated table of genes that contain a copy number breakpoint

References
GATK
MuTect
Annovar
CNVkit

SEQUENCING QUALITY CONTROL

For each sample, the sequencing read alignment is analyzed for a number of sequencing quality metrics using the Picard tools MarkDuplicates, CollectAlignmentSummaryMetrics, CalculateHsMetrics, and CollectInsertSizeMetrics.

Software Versions

  • Picard tools: 1.97(1504)

Output Files

  • <SampleID>.duplication_metrics.txt — report from Picard MarkDuplicates
  • <SampleID>.deduplicated.alignment_metrics.txt — report from Picard CollectAlignmentSummaryMetrics
  • <SampleID>.deduplicated.hs_metrics.txt — report from Picard CalculateHsMetrics
  • <SampleID>.deduplicated.target_coverages.csv — per-target coverage statistics from Picard CalculateHsMetrics
  • <SampleID>.deduplicated.insert_metrics.txt — report from Picard CollectInsertSizeMetrics
  • <SampleID>.deduplicated.insert_histogram.pdf — plotted histogram of insert size distribution, from Picard CollectInsertSizeMetrics

References
Picard (no paper)

SUPPORTING DATA FILES

UCSF500 targeted genomic regions (BED format)
UCSF500 baited genomic regions (BED format)

Please return the completed Consent Form and Requisition Form
Cases identified that meet this acceptance criteria should be emailed to Jessica Van Ziffle and Boris Bastian for approval. Outside pathology specimens must receive a Pathology confirmatory slide review. This can be ordered concurrently with processing for UCSF 500 testing.

PCR-Based Tests

TEST SUMMARY

CPT code:  81210
Specimen Type
Formalin-fixed, paraffin-embedded tissue. Tissue block or unstained slides. If sending unstained slides, we require five (5) 10-micron tissue sections on uncharged glass slides. One adjacent H&E stained slide should be included. Cytology smears may also be used for testing if there is sufficient tumor present. Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if testing on a cytology smear is desired.
Methodology: Sanger sequencing
Turnaround Time: 7-10 business days

TEST INDICATIONS

This test detects the V600E mutation in the BRAF gene from formalin-fixed paraffin-embedded tumor tissue. BRAF V600E mutation has been shown to be present in a number of tumors, including thyroid and colorectal carcinomas, melanomas, histiocytic tumors, and certain brain tumors.

In the thyroid, presence of the mutation is associated with more aggressive clinical behavior. BRAF mutation in Lynch syndrome-associated colorectal cancers is extremely rare, and the presence of BRAF mutation is a strong indication of a sporadic (i.e., non-Lynch) colorectal cancer. However, note that not all MSI-H colorectal cancers that lack BRAF mutation are due to Lynch syndrome. BRAF mutation in colon cancer is associated with a lack of response to EGFR therapy. In melanoma, BRAF V600E mutation predicts response to vemurafenib. BRAF mutation testing may also be useful in evaluation for EGFR therapy in lung cancer.

HOW THE TEST WORKS

A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas of tumor. DNA is extracted from these areas and the region of BRAF exon 15 that includes codon 600 is PCR amplified. Sequencing of PCR products is performed using the Sanger method.

LIMITATIONS OF THE TEST

Results of this test should be correlated with the patient’s other clinical and laboratory information. This assay is not intended to test for BRAF mutations other than the valine to glutamate substitution at amino acid position 600 (V600E). However, non-V600E mutations at or near codon 600, including V600K, V600R, c.1794_1796dupTAC (insT), and K601E may also be detected with this assay.

The test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST

In general, the tumor should be a minimum of 0.4 cm in size. Blocks selected for BRAF Mutation Testing must contain tumor tissue. Areas of tumor must contain at least 50% tumor cells. The lab can remove adjacent non-tumor tissue, so the entire slide does not need 50% tumor cells, just the area with tumor. Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if  the specimen suitability is uncertain. Label slides with pathology case number and block identification.

Specimen rejection criteria include: All required slides not included. Insufficient tumor tissue present on slide as determined by pathologist. Outside slides not labeled or not accompanied by printed copy of test order.

HOW TO ORDER THE TEST

UCSF Clinicians — this test can be ordered through APeX
Outside Physicians — this test can be ordered through the GMI website

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

TEST SUMMARY

CPT code: 81235
Specimen Type: Formalin-fixed, paraffin-embedded tissue. Tissue block or unstained slides. If sending unstained slides, we require five (5) 10-micron tissue sections on uncharged glass slides. One adjacent H&E stained slide should be included. Cytology smears may also be used for testing if there is sufficient tumor present. Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if  testing on a cytology smear is desired.
Methodology: Sanger sequencing
Turnaround Time: 7-10 business days

TEST INDICATIONS

EGFR mutations are commonly seen in non-small cell carcinomas of the lung, usually in adenocarcinomas, but much less frequently in adenosquamous carcinoma as well.  They are extremely rare in squamous cell carcinoma.  Some mutations in EGFR, such as the L858R mutation and exon 19 deletion, have been shown to be predictive of response to EGFR-directed tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer.  Other EGFR mutations, such as T790M and exon 20 insertion, are associated with resistance to EGFR TKIs.

EGFR mutation testing is not indicated for EGFR monoclonal antibody therapy in patients with metastatic colorectal carcinoma.  For these patients, KRAS mutation testing (with consideration of BRAF mutation testing) is recommended.

HOW THE TEST WORKS

A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas of tumor. DNA is extracted from these areas and regions of EGFR exons 19, 20 and 21 that contain the most common EGFR mutations are PCR amplified. Sequencing of PCR products is performed using the Sanger method.  This assay is clinically validated for the detection of wild-type EGFR, point mutations in exon 18 (G719A/S), point mutations in exon 20 (S768I and T790M), point mutations in exon 21 (L858R and L861Q), insertion mutations in exon 20, and deletion mutations in exon 19.

LIMITATIONS OF THE TEST

Results of this test should be correlated with the patient’s other clinical and laboratory information.  This test has only been validated for wild type detection and the detection of the following mutations: exon 19 deletions, exon 20 insertions, L858R, L861Q, S768I, T790M and G719A/S.

This test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation.

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST

In general, the tumor should be a minimum of 0.4 cm in size. Blocks selected for EGFR Mutation Testing must contain tumor tissue. Areas of tumor must contain at least 50% tumor cells. The lab can remove adjacent non-tumor tissue, so the entire slide does not need 50% tumor cells, just the area with tumor.Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if  the specimen suitability is uncertain. Label slides with pathology case number and block identification.

Specimen rejection criteria include: All required slides not included. Insufficient tumor tissue present on slide as determined by pathologist. Outside slides not labeled or not accompanied by printed copy of test order.

HOW TO ORDER THE TEST

UCSF Clinicians — this test can be ordered through APeX
Outside Physicians — this test can be ordered through the GMI website

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

TEST SUMMARY

CPT code:  81275
Specimen Type
Formalin-fixed, paraffin-embedded tissue. Tissue block or unstained slides. If sending unstained slides, we require five (5) 10-micron tissue sections on uncharged glass slides. One adjacent H&E stained slide should be included. Cytology smears may also be used for testing if there is sufficient tumor present. Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if testing on a cytology smear is desired.
Methodology: Sanger sequencing
Turnaround Time: 7-10 business days

TEST INDICATIONS

KRAS mutations at codons 12 and 13 have been shown to be predictive of resistance to EGFR-directed therapy in patients with metastatic colorectal and non-small cell lung cancer. Mutations in codons 12 and 13 are the most commonly reported mutations in KRAS in colorectal cancer. Limited data exists for codon 61, but studies of patients with metastatic colorectal cancer with codon 61 mutation have shown decreased response to EGFR therapy.

HOW THE TEST WORKS

A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas of tumor. DNA is extracted from these areas and areas of KRAS exons 2 and 3 that include codons 12, 13 and 61 are PCR amplified. Sequencing of PCR products is performed using the Sanger method.

LIMITATIONS OF THE TEST

Results of this test should be correlated with the patient’s other clinical and laboratory information. This assay is not intended to test for KRAS mutations outside of codons 12, 13 and 61.

This test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST

In general, the tumor should be a minimum of 0.4 cm in size. Blocks selected for KRAS Mutation Testing must contain tumor tissue. Areas of tumor must contain at least 50% tumor cells. The lab can remove adjacent non-tumor tissue, so the entire slide does not need 50% tumor cells, just the area with tumor. Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if  the specimen suitability is uncertain. Label slides with pathology case number and block identification.

Specimen rejection criteria include: All required slides not included. Insufficient tumor tissue present on slide as determined by pathologist. Outside slides not labeled or not accompanied by printed copy of test order.

HOW TO ORDER THE TEST

UCSF Clinicians — this test can be ordered through APeX
Outside Physicians — this test can be ordered through the GMI website

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

TEST SUMMARY

CPT code:  81479
Specimen Type: Formalin-fixed, paraffin-embedded tissue.  Tissue block or unstained slides.  If sending unstained slides, we require five (5) 10-micron tissue sections on uncharged glass slides. One adjacent H&E stained slide should be included.
Methodology: Bisulfite treatment followed by high resolution melting curve analysis.
Turnaround Time: This assay is run once per month

TEST INDICATIONS

MLH1 promoter methylation is common in sporadic microsatellite unstable tumors, including colorectal cancer and endometrial cancer, and is associated with loss of MLH1 protein expression.  It is rarely detected in Lynch syndrome (hereditary nonpolyposis colorectal cancer or HNPCC).  Therefore, in tumors with high-level microsatellite instability (MSI-H) and/or loss of MLH1 staining by immunohistochemistry, the absence of MLH1 promoter methylation is associated with an increased likelihood of germline mutation in a DNA mismatch repair protein.

HOW THE TEST WORKS

A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas with sufficient tumor for detection.  DNA is extracted from tumor areas on adjacent unstained slides and treated with bisulfite, which converts unmethylated cytosine residues to uracil.  Methylated cytosines are protected from conversion.  Treated DNA is then amplified by real-time PCR.  A 100 bp segment of the MLH1 promoter known to contain eight CpG dinucleotides and be subject to methylation is targeted.  The amplified product is detected with the intercalating dye EvaGreen®.  The melt characteristics of the generated amplicons are determined by loss of EvaGreen® fluorescence as the double-stranded molecule is heated and becomes single-stranded.  The temperature at which the amplicon dissociates is dependent upon the sequence, and indicates whether the sequence was methylated or unmethylated.

LIMITATIONS OF THE TEST

Lack of MLH1 methylation identification by this assay is not sufficient for a diagnosis of Lynch syndrome.  Additionally, up to 1% Lynch syndrome cases appear to be caused by constitutional MLH1 promoter methylation.  Therefore, a diagnosis of Lynch syndrome cannot be definitively confirmed or ruled out on the basis of the MLH1 promoter methylation testing alone.  Correlation with patient history and other testing (including mismatch repair immunohistochemistry, microsatellite instability, MLH1 gene sequencing, and in cases of colorectal carcinoma, BRAF mutation) is recommended.  This assay cannot rule out the presence of an epigenetic alteration outside of the promoter region analyzed.  This test only detects methylation, and will not detect point mutations, insertions, deletions, or inversions.

This test is intended for use in tumors that show high-level microsatellite instability (MSI-H) and/or loss of MLH1 staining by immunohistochemistry.

This test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST

In general, the tumor should be a minimum of 0.4 cm in size.  Blocks selected for MLH1 Promoter Methylation testing must contain tumor tissue.  Areas of tumor must contain at least 50% tumor cells. The lab can remove adjacent non-tumor tissue, so the entire slide does not need 50% tumor cells, just the area with tumor.  Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if the specimen suitability is uncertain.  Label slides with pathology case number and block identification.

Specimen rejection criteria include: All required slides not included. Insufficient tumor tissue present on slide as determined by pathologist. Outside slides not labeled or not accompanied by printed copy of test order.

HOW TO ORDER THE TEST

UCSF Clinicians — this test can be ordered through APeX
Outside Physicians — this test can be ordered through the GMI website

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

TEST SUMMARY

CPT code: 81301
Specimen Type
Formalin-fixed, paraffin-embedded tissue. Tissue block or unstained slides. If sending unstained slides, we require five (5) 10-micron tissue sections on uncharged glass slides. One adjacent H&E stained slide should be included.
Methodology
Multiplex PCR with fragment analysis by capillary electrophoresis. The Microsatellite Instability by PCR assay utilizes a multiplex PCR reaction which amplifies five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27).
Turnaround Time: 7-10 business days

TEST INDICATIONS

Microsatellite Instability by PCR is designed to aid in the identification of patients who harbor germline mutations in mismatch repair (MMR) proteins that are associated with HNPCC. It is important to note that defective MMR resulting in MSI can also arise spontaneously in a tumor (i.e., without inheritance of a mutant MMR gene), and therefore the presence of MSI is not diagnostic for Lynch syndrome/Hereditary Non-Polyposis Colorectal Cancer (HNPCC).

Criteria for HNPCC testing (revised Bethesda guidelines)

  1. Colorectal cancer diagnosed in a patient who is less than 50 years of age.
  2. Presence of synchronous, metachronous colorectal, or other HNPCC-associated tumors, regardless of age. HNPCC-associated tumors include colorectal, endometrial, stomach, ovarian, pancreas, ureter and renal pelvis, biliary tract, and brain (usually glioblastoma as seen in Turcot syndrome) tumors; sebaceous gland adenomas and keratoacanthomas in Muir-Torre syndrome; and small bowel carcinoma.
  3. Colorectal cancer with the MSI-H histology diagnosed in a patient who is less than 60 years of age. Note that inclusion of the age criteria is controversial.
  4. Colorectal cancer in a patient with one or more first-degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed under age 50 years.
  5. Colorectal cancer in a patient with two or more first- or second-degree relatives with HNPCC-related tumors, regardless of age.

UCSF Clinical criteria for HNPCC testing in gynecologic tumors
Nearly half of women with HNPCC will present with a gynecologic tumor, rather than a colon cancer.

  1. Endometrioid histology at any GYN location in a patient under 50 in a definitive surgical procedure (i.e., not just biopsy, wait for primary surgery).
  2. Any adenocarcinoma at any GYN location in a patient with colon cancer.
  3. Clinician suspicion for Lynch syndrome.

HOW THE TEST WORKS

A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas of tumor and normal tissue. DNA is extracted separately from tumor and normal areas on adjacent unstained slides and amplified in the multiplex PCR reaction. Fluorescently labeled PCR products are identified and sized by capillary electrophoresis. Markers are scored for stability or instability by comparison of marker allele sizes from tumor and normal samples. The presence of a marker allele size in the tumor sample that is not present in the normal sample is indicative of instability for that marker. The number of unstable markers is used to determine the presence or absence of instability.

Marker instability is interpreted as follows

  • Microsatellite stable: No markers show instability
  • MSI-Low: Instability in one marker
  • MSI-High: Instability in two or more markers

LIMITATIONS OF THE TEST

This test is typically run in conjunction with immunohistochemical staining for mismatch repair proteins, performed by the UCSF Pathology Laboratory. Microsatellite Instability by PCR may also be ordered as a separate test.

Results from this test should be correlated with the patient’s clinical presentation, family history and any findings from immunohistochemical staining for mismatch repair proteins in the tumor. Genetic counseling may be recommended for some cases.

BRAF Mutation Testing (for colorectal samples only) or MLH1 Methylation Testing may be performed on samples that are MSI-High and show loss of MLH1 by immunohistochemistry to distinguish the sporadic MSI-high cases (BRAF mutation positive/MLH1 methylation positive) from the MSI-high cases associated with Lynch syndrome (BRAF mutation negative/MLH1 methylation negative). Note that not all MSI-H colorectal cancers that lack BRAF mutation or detectable MLH1 methylation are due to Lynch syndrome.

This test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST

Testing should be ordered only on surgical excision samples. Biopsy material is not appropriate. Blocks selected for MSI PCR testing must contain both tumor and normal tissue. If only tumor is present on the block, select an additional block with normal tissue.

Areas of tumor must contain at least 50% tumor cells. The lab can remove adjacent non-tumor tissue, so the entire slide does not need 50% tumor cells, just the area with tumor. Label slides with pathology case number and block identification.

Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if  the specimen suitability is uncertain. Label slides with pathology case number and block identification.

Specimen rejection criteria include

  • All required slides not included
  • Insufficient tumor or normal tissue present on slide as determined by pathologist
  • Outside slides not labeled or not accompanied by printed copy of test order

HOW TO ORDER THE TEST

UCSF Clinicians — this test can be ordered through APeX
Outside Physicians — this test can be ordered through the GMI website

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

TEST SUMMARY

CPT code: 81265
Specimen Type:  FFPE, maternal and fetal tissue required
Methodology:  STR genotyping
Turnaround Time:  1-2 weeks

TESTING INDICATIONS

Hydatidiform mole genotyping is used to evaluate for molar pregnancy (hydatidiform mole) in products of conception with abnormal villous morphology.  Molar pregnancy is a form of gestational trophoblastic disease that results from a defective fertilization process where the zygote inherits two copies of paternal DNA instead of one.  Molar pregnancies are associated with an increased risk of persistent gestational trophoblastic disease (GTD) including persistent mole, invasive mole, and choriocarcinoma.

Challenging histologic cases are often sent for DNA ploidy analysis.  However, ploidy analysis does not provide the parental origin of the chromosomal sets; thus, non-molar triploid pregnancies that contain two chromosomal sets from the mother (digynic gestations, which account for approximately 1/3 of all triploid pregnancies) can be erroneously diagnosed as partial hydatidiform moles.  Additionally, ploidy analysis cannot determine whether a complete mole is of monospermic or dispermic origin, the latter of which is believed to have a greater risk of persistent GTD (ref. 1).   Genotyping of short tandem repeats (STRs) has been validated as an accurate method of diagnosis for molar pregnancies (2, 4).

 HOW THE TEST WORKS

The hydatidiform mole genotyping assay utilizes a DNA identification assay that looks at 15 STRs.  Comparison of the STR alleles present in the fetal and maternal DNA is used to determine the ploidy and parental composition of the fetal DNA.  A summary of potential results is below

 

CONDITION

PLOIDY

#MATERNAL ALLELES

#PATERNAL ALLELES

NOTES

Non-molar bi-parental hydropic abortus Diploid 1 1
Monospermic complete mole Diploid 0 2, homozygous ~90% of complete moles
Dispermic partial mole Diploid 0 2, heterozygous ~10% of complete moles; greater risk of persistent GTD than monospermic complete mole
Dispermic partial mole Triploid 1 2, heterozygous ~90% of partial moles
Monospermic partial mole Triploid 1 2, homozygous ~10% of partial moles
Digynic triploid non-molar pregnancy Triploid 2 1 ~1/3 of triploid pregnancies

LIMITATIONS OF THE TEST

  1. This assay does not detect cases of hydatidiform mole which demonstrate a biparental diploid genotype.  These cases are extremely rare (<2% of molar pregnancies) and may occur as a result of mutation in the NLRP7 (NALP7) gene on chromosome 19 (3).  p57 immunohistochemistry may be useful in detection of NLRP7-associated molar pregnancies, with p57 loss reported in such cases (2).
  2. This assay is only accurate when the pregnancy is being carried by the biological mother.  The test does not work in cases with fetal tissue derived from donor eggs/embryos, or with surrogate mothers.  The fetal DNA must be compared with biologically maternal DNA for an accurate diagnosis.

 SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST

  1. Both chorionic villi and endometrial/decidual tissue must be present on H&E slide(s).  Good physical separation of maternal and fetal tissue is necessary (see figures below), but separation may also be possible by testing separate blocks.  The slides need not consist entirely of fetal or maternal tissue, only that these tissues can clearly be separated from one another during manual dissection without a microscope.  Note that any non-tumor maternal sample (e.g., inflammatory skin biopsy, appendix) may substitute for decidua.  If there is too much mixing of fetal and maternal tissues, the test cannot be performed.  There must also be sufficient tissue for analysis (at least ~0.4 cm2 each of fetal and maternal tissue).  If you are uncertain which block(s) to use for testing, you may allow the laboratory to select for you.
  2. For each block being tested, we require formalin-fixed, paraffin-embedded tissues on glass slides (five unstained sections cut at 10 µm thickness on uncharged slides, along with an adjacent H&E stain).

HOW TO ORDER THE TEST

  1. For expert consultation along with genotyping and p57 immunohistochemistry, the case (all slides and blocks) should be sent to the UCSF Gynecologic Pathology Service.
  2. For genotyping with p57 immunohistochemistry, the case (all slides and blocks) should be sent to the UCSF Gynecologic Pathology Service.
  3. If genotyping only is desired, stained and unstained slides should be sent to the UCSF Applied Genomics Clinical Laboratory with the attached requisition form completed.  If you would prefer the laboratory select block(s) for testing, please send all H&E slides and corresponding blocks to the lab.

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

TEST SUMMARY

CPT code: 81265, 88381
Specimen Type: FFPE, blood, buccal swab, cytology smear, etc.

Genotyping of polymorphic short tandem repeats (STRs) is used for positive identification of samples.  Since the test determines whether two samples come from genetically identical individuals, it is necessary to have a reference sample to compare with the unknown sample.  This could be a known sample from the same patient, such as a prior specimen or a blood draw.  If the issue is a possible floater, the reference could be the remaining tissue on the slide.  Please contact the Clinical Cancer Genomics Laboratory (415.502.3252) to request testing and to explain the question you wish to have answered.  This will determine the specimen(s) that will need to be sent to the laboratory.  You will likely need to speak to a pathologist there to describe the tissues that are to be evaluated and to determine if testing is feasible.
Methodology: This test uses 15 short tandem repeat (STR) sequences to determine if two samples come from genetically identical or different individuals.
Turnaround Time: 14 calendar days

TEST INDICATIONS

The Clinical Cancer Genomics Laboratory can perform Specimen Identity PCR on FFPE samples of uncertain identity.  Examples where this could be helpful include

  • A possible “floater” of carcinoma on a slide that otherwise shows benign tissue.
  • A loose tissue fragment outside of a cassette.
  • Two tissue blocks with the same labeling.

HOW THE TEST WORKS

The specimen identity test utilizes a DNA identification kit from Life Technologies called the AmpFlSTR Identifiler kit that genotypes 15 different STR loci. Comparison of the alleles at each STR locus between two samples is used to determine whether identity matches or mis-matches.

LIMITATIONS OF THE TEST

Samples with very little tissue or intermixed tissues that cannot be macrodissected away may not be suitable for testing.

Tumor samples with microsatellite instability may give erroneous results due to altered microsatellites when compared with the same patient’s normal DNA (V. Bossuyt, N. Buza et al, 2013).  If suspected source(s) of tissue contamination or a possible swapped specimen are provided for testing, it will improve the ability to make positive matches when dealing with tumor samples that could be microsatellite unstable (colon and endometrial carcinoma, most commonly).  Immunohistochemistry for DNA mismatch repair proteins may also be helpful in such cases if sufficient tissue is present.

In all cases, providing suspected contamination sources/possible swapped specimen(s) and related known reference samples (e.g., patient blood draws) should aid in interpretation of this test.

This test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST

In general, the tissues in question should be a minimum of 0.3 cm in size if slide macrodissection is required.  If tissue can be removed directly from a tissue block, or if macrodissection is not required, it may be possible to test smaller areas.  Contact the laboratory at 415.502.3252 or CCGL@ucsf.edu if the specimen suitability is uncertain.  Label slides with pathology case number and block identification.

Specimen rejection criteria include

  • All required slides not included
  • Insufficient tissue present on slide as determined by pathologist
  • Outside slides not labeled or not accompanied by printed copy of test order

HOW TO ORDER THE TEST

UCSF pathologists and outside physicians: contact the laboratory at 415.502.3252 or CCGL@ucsf.edu to initiate ordering of Specimen Identity testing

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

Molecular Tumor Board

Molecular Tumor Board Meetings — Chair: Michael Korn

TO ATTEND IN PERSON (Lunch is Provided)
When: 
12-1:30pm on Fridays
Location:
Mission Hall, Room 6500
550 16th Street, Mission Bay Campus

TO PARTICIPATE REMOTELY
WebEx invites are sent the Wednesday before each meeting

Box Folder, Distribution List, Comments or Questions
Presentations and de-identified case summaries from previous meetings are in the “Molecular Tumor Board” Box folder. To get access to the folder, to be added to the MTB distribution list, or if you have comments or questions, contact Kristen McCaleb

CASE REVIEW
Request a case review:

  • Type in the above text so we know you are human!

Clinician Input

Requesting important tumor-specific hotspots of clinical relevance. The UCSF 500 Pipeline calls variants in 510 genes using multiple bioinformatic algorithms. In addition to reporting the called variants, we can report wild-type status for critical variants submitted below.

Please submit your list of genetic variants that you consider critical for patient management. This will allow us to manually confirm wild-type status.

See the current Hotspot Database by Tumor Board (updated January 27th, 2015)

Please submit your disease-specific hotspots or request for an addition to the gene panel for the next version

Ordering Guidelines

This UCSF 500 Consent Form is required for the UCSF 500 Panel. No other CCGL tests require a consent form.

UCSF 500
Ordering is not yet live in APeX. Please review Ordering Information
Search for CCGL UCSF 500 (will have screenshot when test code is ready)

Sample Requirements
Listed under “UCSF 500” above
UCSF 500 Consent Form

Data
Will be returned in APeX in 3-4 weeks

Discussion of Results with the Molecular Tumor Board
Request review

Single Gene tests — order in CoPath or APeX
UCSF 500 — order in APeX

Orders made by Clinicians outside of UCSF — use the CCGL Requisition Form 
COMPONENTS
Order to block receipt by laboratory
 — Clinician dependent
Block receipt to DNA (including slide recuts) — 2 business days
DNA to Post-hyb (Capture Library) — 9 business days
HiSeq  1.1 business days
Data — 0.2 business days
Report (Subset Panel) — 1 business day
Report (UCSF 500) —  2-3 business days
TOTAL TURNAROUND — after receipt of pathology block
Subset panel = 13.3 business days
UCSF 500 = 15.3 business days
Ordering

CCGL Leadership

CLINICAL CANCER GENOMICS LABORATORY (CCGL)
CONTACT EMAIL PHONE
Executive Director Boris Bastian Boris.Bastian@ucsf.edu 415.502.0267
Medical Director J.P. Grenert James.Grenert@ucsf.edu 415.502.0747
Molecular Pathology Director Nancy Joseph Nancy.Joseph@ucsf.edu 415.502.3119
Technical Director Jessica Van Ziffle Jessica.VanZiffle@ucsf.edu 415.502.0747
Bioinformatics Director
Iwei Yeh YehI@derm.ucsf.edu 415.353.7989

CCGL Laboratory Staff

Email the Lab
Lab Phone: 415.502.3252

Arthur Delance
Alexander Gagnon
Allan Gopez
Emmy Luu
Lisa Martell
Sonia Mirza
Janyaporn Phuchareon
Caryll Pineda
Jennifer Rasmussen
Swapna Vemula
Jingly Weier

CCGL Laboratory Staff

[/fusion_tab]

UCSF Exome Testing

  1. Refer patients to the UCSF Personalized Genomics Clinic (PGC) for an evaluation of genetic testing approach based on clinical and family history and consent process.
  2. Biological parents (if available) and relatives may be asked to attend the PGC visit for potential testing and consent.
  3. Clinical sequencing results will be discussed with the patient and/or family and clinical recommendations provided to the referring provider.

To Refer

IN APEX

Enter: “Ambulatory Referral to Ped and Adult Genetics
In comments specify: “Referral to the Personalized Genomics Clinic for UCSF Clinical Exome Sequencing

[/fusion_builder_row_inner]
Ordering

CONTACT INFORMATION

Scheduling — 415.476.2757

Clinical Geneticist: Anne Slavotinek — anne.slavotinek@ucsf.edu
Clinical Geneticist: Joseph Shieh — joseph.shieh2@ucsf.edu
Genetic Counselor: Marta Sabbadini — marta.sabbadini@ucsf.edu

Ordering Process Guidelines

  1. The Fast Exome test is currently being developed – please check back for updates.
[/fusion_builder_row_inner]
Ordering

BILLING

Inpatient (billed to UCSF) Outpatient (billed to insurance / MediCal / Medicare)
TBD TBD TBD
TBD TBD TBD

Newborn Genetics Leadership

 GENOMIC MEDICINE LAB CONTACT EMAIL PHONE
Genetic Counselor  Marta Sabbadini Marta.Sabbadini@ucsf.edu 415.476.8342
Medical Director Heather Pua Heather.Pua@ucsfmedctr.org 415.476.5373
Technical Director Jessica Van Ziffle Jessica.VanZiffle@ucsf.edu 415.502.0747
Bioinformatician Diya Vaka Dedeepya.Vaka@ucsf.edu
[/fusion_tabs]