Changes

__TOC__

__TOC__

6.7.1.2.1 Initial diagnostic studies

6.7.1.2.1 Initial diagnostic studies

    a. Analysis

::a. Analysis

        i. Analyze 20 metaphase cells and/or a sufficient number of cells to characterize all abnormal clones and subclones.

:::i. Analyze 20 metaphase cells and/or a sufficient number of cells to characterize all abnormal clones and subclones.

        ii. If all cells show a complex karyotype where each cell is different, then analyze at least 10 cells with karyotyping.

:::ii. If all cells show a complex karyotype where each cell is different, then analyze at least 10 cells with karyotyping.

    b. Documentation

::b. Documentation

        i. For abnormal cells:

:::i. For abnormal cells:

            1. If only one abnormal clone is present: two karyotypes.

::::1. If only one abnormal clone is present: two karyotypes.

            2. If more than one related abnormal clone is present: at least one karyotype of the stemline and at least one of each sideline.

::::2. If more than one related abnormal clone is present: at least one karyotype of the stemline and at least one of each sideline.

            3. If unrelated clones are present: at least one karyotype for each stemline and one for each associated pertinent sideline.

::::3. If unrelated clones are present: at least one karyotype for each stemline and one for each associated pertinent sideline.

        ii. For normal cells:

:::ii. For normal cells:

            1. If only normal cells are present: two karyotypes.

::::1. If only normal cells are present: two karyotypes.

            2. If normal and abnormal cells are present: one karyotype of a normal cell plus karyotypes for abnormal clone(s) as described.

::::2. If normal and abnormal cells are present: one karyotype of a normal cell plus karyotypes for abnormal clone(s) as described.

6.7.1.2.2 Follow-up studies may be performed to assess stage of disease at the time of diagnosis or at the time of tumor recurrence.

6.7.1.2.2 Follow-up studies may be performed to assess stage of disease at the time of diagnosis or at the time of tumor recurrence.

    a. Analysis

::a. Analysis

        i. Analysis should include a minimum of 20 metaphase cells.

:::i. Analysis should include a minimum of 20 metaphase cells.

        ii. Additional cells may be scored for a specific abnormality identified in the diagnostic sample.

:::ii. Additional cells may be scored for a specific abnormality identified in the diagnostic sample.

        iii. In addition to looking for the known clonal aberration(s) from the diagnostic study, analysis of a sample after therapy should be performed with awareness of the possibility of new aberrations signifying clonal evolution and/or a new clonal process (i.e., therapy-related malignancy).

:::iii. In addition to looking for the known clonal aberration(s) from the diagnostic study, analysis of a sample after therapy should be performed with awareness of the possibility of new aberrations signifying clonal evolution and/or a new clonal process (i.e., therapy-related malignancy).

        iv. FISH analysis may be considered in lieu of conventional chromosomal analysis for diagnoses characterized by an abnormality for which FISH testing is available.

:::iv. FISH analysis may be considered in lieu of conventional chromosomal analysis for diagnoses characterized by an abnormality for which FISH testing is available.

    b. Documentation

::b. Documentation

        i. If both normal and abnormal cells or if only abnormal cells are present:

:::i. If both normal and abnormal cells or if only abnormal cells are present:

            1. One or two karyotypes from each abnormal clone with a minimum of two karyotypes.

::::1. One or two karyotypes from each abnormal clone with a minimum of two karyotypes.

            2. One karyotype of a normal cell, if a normal karyotype was not documented in a previous study.

::::2. One karyotype of a normal cell, if a normal karyotype was not documented in a previous study.

            3. If only normal cells are present: two karyotypes.

::::3. If only normal cells are present: two karyotypes.

6.7.2 FISH analysis

6.7.2 FISH analysis

6.7.2.1 FISH analysis may be used for primary, supplementary, or follow-up evaluation

6.7.2.1 FISH analysis may be used for primary, supplementary, or follow-up evaluation

    a. As a primary method for tumor evaluation, FISH is useful when (i) fresh tumor tissue is not available; (ii) rapid diagnostic information is needed to narrow the differential diagnosis; (iii) gene amplification or rearrangement for diagnostic or prognostic and/or therapeutic purposes is to be determined; (iv) no metaphase cells are obtained by culture of tumor material; or (v) conventional cytogenetic analysis yields a normal result.

::a. As a primary method for tumor evaluation, FISH is useful when (i) fresh tumor tissue is not available; (ii) rapid diagnostic information is needed to narrow the differential diagnosis; (iii) gene amplification or rearrangement for diagnostic or prognostic and/or therapeutic purposes is to be determined; (iv) no metaphase cells are obtained by culture of tumor material; or (v) conventional cytogenetic analysis yields a normal result.

    b. Supplemental FISH may be used as an adjunct to the initial conventional chromosomal analysis or CMA analysis to: (i) document a specific molecular event (e.g., gene rearrangement or fusion); (ii) provide a rapid result to aid in the differential diagnosis or planning of therapy; (iii) to assess gene copy number,; (iv) clarify level of clonality; or (v) confirm a microarray variant.

::b. Supplemental FISH may be used as an adjunct to the initial conventional chromosomal analysis or CMA analysis to: (i) document a specific molecular event (e.g., gene rearrangement or fusion); (ii) provide a rapid result to aid in the differential diagnosis or planning of therapy; (iii) to assess gene copy number,; (iv) clarify level of clonality; or (v) confirm a microarray variant.

    c. Follow-up FISH studies may be indicated to assess recurrent disease or disease progression and/or to differentiate recurrence of a tumor from a new disease process.

::c. Follow-up FISH studies may be indicated to assess recurrent disease or disease progression and/or to differentiate recurrence of a tumor from a new disease process.

    d. If initial studies failed to identify the clonal process unique to the tumor, then follow-up studies may provide another opportunity.

::d. If initial studies failed to identify the clonal process unique to the tumor, then follow-up studies may provide another opportunity.

6.7.2.2 Characterization of interphase FISH aberrations and FISH signal patterns. Characterization of interphase FISH aberrations and the FISH signal patterns in diagnostic samples is useful for future monitoring of disease. Gene fusions may confirm a specific tumor diagnosis. If a particular patient’s tumor has a unique FISH signal pattern, documentation of the pattern at diagnosis can prevent misinterpretation of FISH analysis at follow-up.

6.7.2.2 Characterization of interphase FISH aberrations and FISH signal patterns. Characterization of interphase FISH aberrations and the FISH signal patterns in diagnostic samples is useful for future monitoring of disease. Gene fusions may confirm a specific tumor diagnosis. If a particular patient’s tumor has a unique FISH signal pattern, documentation of the pattern at diagnosis can prevent misinterpretation of FISH analysis at follow-up.

6.7.2.3 Sample types. Sample types that may be used for FISH include (i) paraffin-embedded tissue sections; (ii) touch preparations (TP); (iii) cytospin preparations; (iv) cultured or direct harvest tumor cells; (v) fixed cytogenetically prepared cells; or (vi) fresh-frozen tumor tissues.

6.7.2.3 Sample types. Sample types that may be used for FISH include (i) paraffin-embedded tissue sections; (ii) touch preparations (TP); (iii) cytospin preparations; (iv) cultured or direct harvest tumor cells; (v) fixed cytogenetically prepared cells; or (vi) fresh-frozen tumor tissues.

    a. Paraffin-embedded tissue3

::a. Paraffin-embedded tissue3

        i. Before scoring a paraffin-embedded FISH slide, it is crucial for a pathologist to review a hematoxylin and eosin–stained slide and delineate the region of tumor cells that should be scored because it can be difficult to differentiate normal cells from malignant cells using only DAPI counterstain. The technologist should be clear, before scoring the slide, where the malignant cells of interest are located on the slide.

:::i. Before scoring a paraffin-embedded FISH slide, it is crucial for a pathologist to review a hematoxylin and eosin–stained slide and delineate the region of tumor cells that should be scored because it can be difficult to differentiate normal cells from malignant cells using only DAPI counterstain. The technologist should be clear, before scoring the slide, where the malignant cells of interest are located on the slide.

        ii. Formalin-fixed, paraffin-embedded tissue is acceptable for FISH analysis. Tissues preserved in B5 fixative or decalcified are not suitable for FISH.

:::ii. Formalin-fixed, paraffin-embedded tissue is acceptable for FISH analysis. Tissues preserved in B5 fixative or decalcified are not suitable for FISH.

        iii. Tumor sections cut 3 to 4 µm thick and mounted on positively charged organosilane-coated (silanized) slides work well. The cytogenetics laboratory should request several unstained sections and one hematoxylin and eosin–stained sequentially cut section from the submitting laboratory.

:::iii. Tumor sections cut 3 to 4 µm thick and mounted on positively charged organosilane-coated (silanized) slides work well. The cytogenetics laboratory should request several unstained sections and one hematoxylin and eosin–stained sequentially cut section from the submitting laboratory.

    b. Touch preparations

::b. Touch preparations

        i. A pathologist should make the TP or should be involved in selecting the tissue for TPs.

:::i. A pathologist should make the TP or should be involved in selecting the tissue for TPs.

        ii. TPs are helpful when tissue architecture is not crucial.

:::ii. TPs are helpful when tissue architecture is not crucial.

        iii. TPs should be made by lightly touching the piece of tumor to a glass slide without smearing, followed by air drying.

:::iii. TPs should be made by lightly touching the piece of tumor to a glass slide without smearing, followed by air drying.

    c. Cytospin preparations

::c. Cytospin preparations

        i. Cytospin preparations are useful for a concentration of samples with very low cellularity (e.g., cerebrospinal fluid).

:::i. Cytospin preparations are useful for a concentration of samples with very low cellularity (e.g., cerebrospinal fluid).

    d. Fixed cytogenetically prepared cells

::d. Fixed cytogenetically prepared cells

        i. Such preparations have multiple uses for both interphase and metaphase FISH evaluation including confirmation and clarification of suspected chromosome aberrations or characterization of an apparently abnormal clone. Metaphase cell evaluation may help clarify specific chromosome rearrangements.

:::i. Such preparations have multiple uses for both interphase and metaphase FISH evaluation including confirmation and clarification of suspected chromosome aberrations or characterization of an apparently abnormal clone. Metaphase cell evaluation may help clarify specific chromosome rearrangements.

    e. Fresh-frozen tumor tissues

::e. Fresh-frozen tumor tissues

        i. Such tissues may be useful in sequential analysis of recurring tumors or in evaluation of archived samples.

:::i. Such tissues may be useful in sequential analysis of recurring tumors or in evaluation of archived samples.

6.7.2.4 Documentation. Analysis and documentation of FISH results should be in accordance with Section E9 of these Standards and Guidelines for Clinical Genetics Laboratories.4

6.7.2.4 Documentation. Analysis and documentation of FISH results should be in accordance with Section E9 of these Standards and Guidelines for Clinical Genetics Laboratories.4

6.7.3.2 Sample types that may be used for CMA analysis include (i) fresh tumor tissue; (ii) paraffin-embedded tumor tissue; (iii) frozen tumor; and (iv) cultured cells, chromosomally characterized when possible.

6.7.3.2 Sample types that may be used for CMA analysis include (i) fresh tumor tissue; (ii) paraffin-embedded tumor tissue; (iii) frozen tumor; and (iv) cultured cells, chromosomally characterized when possible.

    a. Fresh tumor tissue

::a. Fresh tumor tissue

        i. If the tumor is homogeneous, fresh tumor is the optimal sample for CMA and can be procured at the time of sample processing for chromosomal analysis. A small piece of identified tumor should be transferred to the microarray laboratory as soon as possible for DNA isolation. For heterogeneous tumors with areas of necrosis, normal tissue, or prominent stoma, DNA isolation from histologically characterized formalin-fixed paraffin-embedded material may be needed to ensure that isolated DNA is from the tumor.

:::i. If the tumor is homogeneous, fresh tumor is the optimal sample for CMA and can be procured at the time of sample processing for chromosomal analysis. A small piece of identified tumor should be transferred to the microarray laboratory as soon as possible for DNA isolation. For heterogeneous tumors with areas of necrosis, normal tissue, or prominent stoma, DNA isolation from histologically characterized formalin-fixed paraffin-embedded material may be needed to ensure that isolated DNA is from the tumor.

    b. Paraffin-embedded tumor

::b. Paraffin-embedded tumor

        i. A pathologist should review the hematoxylin and eosin-stained section of the tumor to identify an area of concentrated tumor for DNA isolation.

:::i. A pathologist should review the hematoxylin and eosin-stained section of the tumor to identify an area of concentrated tumor for DNA isolation.

    c. Fresh-frozen tumor

::c. Fresh-frozen tumor

        i. Frozen stored tumor should provide high-quality DNA for CMA. A pathologist’s review of the original H&E-stained slides can assure the frozen sample contains adequate tumor.

:::i. Frozen stored tumor should provide high-quality DNA for CMA. A pathologist’s review of the original H&E-stained slides can assure the frozen sample contains adequate tumor.

    d. Cultured tumor cells

::d. Cultured tumor cells

        i. Tumor cells that have been placed into culture may be used for DNA isolation and CMA as long as they remain viable. An early decision to use cells for CMA is best to minimize growth of normal tissue components.

:::i. Tumor cells that have been placed into culture may be used for DNA isolation and CMA as long as they remain viable. An early decision to use cells for CMA is best to minimize growth of normal tissue components.

        ii. DNA from cultured and harvested tumor cells that have been chromosomally characterized as abnormal may be used for CMA.

:::ii. DNA from cultured and harvested tumor cells that have been chromosomally characterized as abnormal may be used for CMA.

6.7.3.3 Documentation: analysis and documentation of CMA studies should be in accordance with Section E11 of these Standards and Guidelines for Clinical Genetics Laboratories.5

6.7.3.3 Documentation: analysis and documentation of CMA studies should be in accordance with Section E11 of these Standards and Guidelines for Clinical Genetics Laboratories.5

6.8.1.2 TAT guidance:

6.8.1.2 TAT guidance:

    a. Because of the multiplicity of tumor types and the different tumor growth characteristics in culture, TATs will vary. However, the final report for each tumor should be available as soon as possible given such factors. Final results should be available within 28 calendar days.

::a. Because of the multiplicity of tumor types and the different tumor growth characteristics in culture, TATs will vary. However, the final report for each tumor should be available as soon as possible given such factors. Final results should be available within 28 calendar days.

    b. Tumor FISH analysis results should be available within 1 to 4 days for most tumors and within 7 days for paraffin-embedded tumors.

::b. Tumor FISH analysis results should be available within 1 to 4 days for most tumors and within 7 days for paraffin-embedded tumors.

    c. Preliminary verbal reports may be appropriate for some case studies. If preliminary results are communicated, then the date of preliminary report should be documented in the final report. The content of the preliminary report should be documented if it differs significantly from that of the final report.

::c. Preliminary verbal reports may be appropriate for some case studies. If preliminary results are communicated, then the date of preliminary report should be documented in the final report. The content of the preliminary report should be documented if it differs significantly from that of the final report.

6.8.2 Reporting

6.8.2 Reporting