Routine pathologic parameters can predict Oncotype DX recurrence scores in subsets of ER positive patients: who does not always need testing?

Oncotype DX(TM) is an RT-PCR-based assay used to predict chemotherapy benefit in patients with estrogen receptor (ER) positive breast cancers. We were interested if routinely available pathologic parameters could predict Oncotype DX Recurrence Scores (RS) in subsets of patients. We identified 173 breast cancers with available RSs and used 104 of these as a test set and 69 cases as a validation set. Pathologic characteristics including size, histologic type, Nottingham grade, and lymphatic invasion were recorded. Test set cases were stained for ER, progesterone receptor (PR), HER2, Ki67, CyclinD1, BCL2, D2-40, and P53. Statistical correlations with RS and regression tree analysis were performed. The validation set was subjected to analysis on the basis of grade, PR, and Ki67. In the test set, grade, PR levels and Ki67 had the strongest correlation with RS (P = 0.0002-0.0007). Regression tree analysis showed grade and PR as factors that could segregate cases into RS categories, with Ki67 adding value in certain subsets. A subset of cancers with a high likelihood of having a low RS (0-18) was identified with the following characteristics: grade 1, strong PR expression (Allred score ≥ 5) and Ki67 ≤ 10%. No cases with these characteristics had a high RS (≥ 31) and 73% had a low RS. Cancers highly likely to have a high RS were grade 3, low to absent PR expression (Allred score <5) and Ki67 > 10%. 80% of cases with these characteristics had a high RS and no cases had a low RS. Our validation set had similar findings in these two subsets. In conclusion, When cost and time are a consideration and the added value of Oncotype DX(TM) testing is in question, it may be reasonable to assume the results of this test in two specific subsets of breast cancers: (1) grade 1, high PR, low Ki67 cancers (low RS), and (2) grade 3, low PR, high Ki67 cancers (high RS).

Genotypic and phenotypic progression in endometrial tumorigenesis: determining when defects in DNA mismatch repair and KRAS2 occur.

We set out to determine the relative timing of loss of DNA mismatch repair and KRAS2 mutation in endometrial tumorigenesis. We studied endometrial carcinoma (CA) and synchronous atypical endometrial hyperplasia (AEH), the premalignant precursor of endometrial cancer. Carcinoma and hyperplasia were investigated for loss of mismatch repair as evidenced by microsatellite instability (MSI) and for KRAS2 mutations. Endometrial cancers previously shown to be MSI-positive were evaluated for KRAS2 codon 12 and 13 mutations. DNA was isolated from foci of AEH concomitant with, but physically remote from, the cancers by use of tissues prepared by laser capture microdissection (LCM). The AEH DNAs were then assessed for MSI and KRAS2 mutations. Of 210 endometrial CAs investigated, 51 (26%) were MSI-positive, and among those, 21 (41%) arose concomitantly with AEH. Of 41 foci of AEH (mean, two foci per patient) investigated, 34 (83%) were MSI-positive. KRAS2 mutations were seen in 5/51 (10%) MSI-positive carcinomas. From the five patients informative for both KRAS2 mutation and MSI, 10 foci of AEH were available for investigation. All 10 AEH specimens (100%) were MSI-positive, and six (60%) had the KRAS2 mutation present in the coexisting CA. The observation that some MSI-positive AEH specimens lack the KRAS2 mutation seen in the coexisting CA supports a model in which loss of DNA mismatch repair precedes KRAS2 mutation. However, in addition to the absence of KRAS2 mutations in AEH, we discovered mutations in LCM hyperplasia and carcinoma specimens that were not present in the portion of the cancers originally investigated. These discordant genotypes suggest genetic heterogeneity in endometrial hyperplasia and concomitant cancer.

Quantitative amplification of genomic DNA from histological tissue sections after staining with nuclear dyes and laser capture microdissection.

Laser capture microdissection (LCM) allows the selective sampling of tissue from histological sections. A prerequisite for this technique is the availability of histological dyes that do not interfere with downstream analysis of the sampled genetic material. We have examined the effect of four histological nuclear dyes (methyl green, hematoxylin, toluidine blue O, azure B) on TaqMan polymerase chain reaction amplification of beta-actin genomic DNA prepared from fixed and frozen tissue. Tissue sampled from the histological sections by manual dissection was compared with tissue sampled by LCM. As previously reported, when manually dissected tissue sections were analyzed, polymerase chain reaction amplification of DNA after hematoxylin staining was inferior to that after staining with the other dyes. In contrast, when tissue sampled by LCM was examined, DNA recovery after hematoxylin staining was equivalent to the recovery after methyl green staining. We conclude that DNA recovery from LCM-sampled tissue is independent of the histological stain chosen to highlight nuclear detail.

Receptor extracellular domains may contain trafficking information. Studies of the 300-kDa mannose 6-phosphate receptor.

The 300-kDa mannose 6-phosphate receptor cycles between the trans Golgi network and late endosomes, and between the plasma membrane and early endosomes, to deliver lysosomal enzymes to prelysosomes. Mannose 6-phosphate receptor trafficking requires structural determinants present in the cytoplasmic domain. However, when this domain was joined with the extracellular and transmembrane domains of the epidermal growth factor receptor, it was not sufficient to direct this chimera to late endosomes and the trans Golgi network (Dintzis, S. M., and Pfeffer, S. R. (1990) EMBO J. 9, 77-84). These findings suggested a role for extracellular and/or transmembrane domains in mannose 6-phosphate receptor trafficking. We describe here the construction and expression of chimeric receptors comprised of mannose 6-phosphate receptor extracellular and transmembrane sequences joined with cytoplasmic domain sequences derived from the human epidermal growth factor receptor or the human low density lipoprotein receptor. The chimeras were stable proteins which were efficiently endocytosed and competent to bind a mannose 6-phosphate-containing ligand. Antibody binding assays and indirect immunofluorescence showed that the chimeras containing the mannose 6-phosphate receptor extracellular domain colocalized with mannose 6-phosphate receptors in intracellular compartments. These experiments suggest that the presence of the mannose 6-phosphate receptor extracellular domain may interfere with the rapid recycling of receptors from early endosomes to the cell surface and detain receptors within endosomes.

The mannose 6-phosphate receptor cytoplasmic domain is not sufficient to alter the cellular distribution of a chimeric EGF receptor.

Unlike most receptors, 300 kd mannose 6-phosphate receptors (MPRs) are localized primarily in the trans-Golgi network (TGN) and endosomes, and they cycle constitutively between these compartments. Yet, when present at the cell surface, MPRs are internalized together with other cell surface receptors in clathrin-coated vesicles. We constructed a chimeric receptor, comprised of human EGF receptor extracellular and transmembrane domains joined to the bovine MPR cytoplasmic domain, to test whether the MPR cytoplasmic domain contained sufficient information to direct a cell surface receptor into both of these transport pathways. The expressed protein was stable, bound EGF with high affinity, and was efficiently endocytosed and recycled back to the cell surface, in the presence or absence of EGF. If the cytoplasmic domain alone is responsible for sorting native MPRs, chimeric receptors might have been expected to be located primarily in the TGN and in endosomes at steady state. Surprisingly, under conditions in which essentially all endogenous MPRs were intracellular, greater than 85% of the chimeric receptors were located at the cell surface. These experiments demonstrate that the MPR cytoplasmic domain is not sufficient to alter the distribution of the EGF receptor, and suggest a role for extracellular and transmembrane domains in MPR routing.