19.3.09

Importance of Tissue fixation in Cancer management

“Do you want a quick result, or the right result?”
There is always a pressure on the laboratory (and the pathologist) to turn specimens around quickly, so that decisions can be relayed to anxious and expectant patients.

Proper specimen handling and preparationhas been under-appreciated by clinicians and pathologists alike.How preanalytical variables such as tissue handing and tissue fixation have the potential to significantly and adversely affect the accurate assessment of therapeutic targets such as ER/Her2neu in tumor specimens.,must be understood properly.

Following are the Consensus Recommendations on Estrogen Receptor Testing in Breast Cancer By Immunohistochemistry by Members of the Standardization Ad-Hoc Consensus Committee.


  1. As soon as they are available from the operating room, breast specimens with a known cancer, or a suspicion of cancer, should be oriented, inked, carefully sectioned at 0.5 to 1 cm intervals and placed in the appropriate volume of fixative with gauze pads or paper towels in between slices to assist with the penetration of fixative into all areas of the tissue.

  2. In addition,if gross tumor is easily identifiable, the pathologist (or pathology assistant) should remove a 2-mm thick sample of tumor, together with a 2-mm thick sample of benign tissue and place both into the same cassette at the time of the initial evaluation, thus ensuring that normal breast elements are available as appropriate internal tissue controls for subsequent breast marker testing.

  3. Breast core biopsies should be fixed and processed in an identical manner to excision specimens.

  4. Only 10% aqueous phosphate-buffered 4% formaldehyde pH 7.0-7.4 (10% phosphate-buffered formalin) should be used as the fixative for breast tissue samples.

  5. Minimum fixation times of at least 8 hours optimally 24 to 48 hours, not to extend to more than 72 hours are recommended for all laboratories accepting breast tissue samples.

There is a misconception that smaller biopsy samples will fix more quickly than larger resection specimens and therefore require less time in formalin. It is true that formalin will penetrate smaller samples more quickly than larger samples, but actual fixation is a chemical reaction that takes time.Penetration time of formalin is of little importance than the chemical reaction time .For example, a single layer of cells is penetrated by formalin extremely rapidly, however the chemical reaction requires 24 hours to complete. A 4-mm thick slice of tissue is fully penetrated within an hour and requires 25 hours for the chemical reaction to complete.


Having a record of the fixation time for each breast tissue sample is expected to prove valuable for interpreting and troubleshooting aberrant and/or unexpected ER results.One such example would be when an ER IHC assay is found to be negative in a well-differentiated cancer such as a tubular carcinoma or a classic lobular carcinoma. This result would be unexpected, and having access to the details on tissue fixation, such as a long delay before the initiation of fixation or short fixation time in formalin would be valuable in interpreting the validity of the unexpected results for such a patient.


Reference:


Consensus Recommendations on Estrogen Receptor Testing in Breast Cancer By Immunohistochemistry;Hadi Yaziji et al.Apllied immunohistochem Mol.Vol16,Dec.2008

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Interesting Case

Clinical History:

53 years male,History of hypertension and tachycardia,MRI abdomen:-Left adrenal mass:- size 5.8 cm Right renal mass:- size-3.0cm Microscopic examination of the renal mass showed vascular tumor with diffuse sheets of clear cells having Fuhrman grade III nuclei. There was no evidence of necrosis within the tumor. There was no evidence of extraparenchymal invasion.
Gross examination of the left adrenal gland revealed cortically centered, solid and multinodular mass measuring 6.5 x 6.0 x 5.0 cm and weighing 122 grams. The tumor was encapsulated but showed evidence of extraparenchymal penetration. The tumor had golden brown cut surface with areas of hemorrhage and necrosis. The partial nephrectomy showed 3.0cm x 3.0cm x 3.0 cm yellow solid mass which did not invade into the perinephric adipose tissue.
Microscopically, the adrenal mass had predominant diffuse sheets and focal trabecular arrangements. The former pattern was present in about third of tumor. The cells had clear cytoplasm and round to ovoid nuclei with conspicuous nucleoli. Mitotic rate was 9/50 HPF and included atypical forms. Gross necrosis and capsular invasion were documented microscopically. There was no evidence of lymphovascular invasion. Considering the above mentioned features, a Weiss histopathologic score2 of 7/9 was applied.



Discussion:

The differential diagnosis included Renal Cell Carcinoma (RCC) with contralateral adrenal metastasis, Adrenocortical carcinoma (ACC) with contralateral renal metastasis, synchronous RCC and ACC or synchronous RCC and adrenocortical adenoma. A panel of immunohistochemical stains was performed to sort out the diagnosis. Adrenal tumor demonstrated strong Vimentin positivity and is negative for CK7, CK20, E1/AE3, EMA, Synaptophysin and S100.Renal cell carcinoma was positive for CK7, AE1/AE3, EMA (weak) and Vimentin. It was negative for CK20, Synaptophysin and S100. The difference of immunoprofile between the two tumors documented that they originated from two different primaries.

Final Diagnosis:

The diagnosis of synchronous RCC and ACC rather than metastasis influences the prognosis.

Prognosis:

The longest disease free interval after removal of contralateral adrenal metastasis was 12.1 years8 and the longest crude survival was 14.3 years. In contrast non metastazing RCC has an excellent prognosis if no metastasis developed.