Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-07
2003-06-03
Horlick, Kenneth R. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100
Reexamination Certificate
active
06573043
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of tissue analysis. Specifically, the invention combines morphological staining and/or immunohistochemistry (IHC) with fluorescence in situ hybridization (FISH) within the same section of a tissue sample thereby allowing for accurate and simplified prognostic, diagnostic, or research applications on a subject's tissue sample. In addition, the invention provides kits for analysis of a tissue sample utilizing the present methods.
BACKGROUND OF THE INVENTION
Advancements in the understanding of genetics and developments in technology and epidemiology have allowed for the correlation of genetic abnormalities with certain malignancies as well as risk assessment of an individual for developing certain malignancies. However, most of the methodologies available for evaluation of tissue for the presence of genes associated with or predisposing an individual to a malignancy have well-known drawbacks. For example, methods that require disaggregation of the tissue, such as Southern, Northern, or Western blot analysis, are rendered less accurate by dilution of the malignant cells by the normal or otherwise non-malignant cells that are present in the same tissue. Furthermore, the resulting loss of tissue architecture precludes the ability to correlate malignant cells with the presence of genetic abnormalities in a context that allows morphological specificity. This issue is particularly problematic in tissue types known to be heterogeneous, such as in human breast carcinoma, where a significant percentage of the cells present in any area may be non-malignant.
Fluorescence in situ hybridization (FISH) is a recently developed method for directly assessing the presence of genes in intact cells. FISH is an attractive means of evaluating paraffin-embedded tissue for the presence of malignancy because it provides for cell specificity, yet overcomes the cross-linking problems and other protein-altering effects caused by formalin fixation. FISH has historically been combined with classical staining methodologies in an attempt to correlate genetic abnormalities with cellular morphology [see e.g., Anastasi et al.,
Blood
77:2456-2462 (1991); Anastasi et al.,
Blood
79:1796-1801 (1992); Anastasi et al.,
Blood
81:1580-1585 (1993); van Lom et al.,
Blood
82:884-888 (1992); Wolman et al.,
Diagnostic Molecular Pathology
1(3): 192-199 (1992); Zitzelberger,
Journal of Pathology
172:325-335 (1994)]. However, several of these studies address hematological disorders where genetic changes are assessed in freshly fixed smears from bone marrow aspirates or peripheral blood specimens. Of those two studies where paraffin-embedded tissue was analyzed, one involved evaluation of FISH and morphological staining on separate, serial sections. In the other study, both procedures were performed on the same section, but morphological staining was subsequent to evaluation by FISH. Use of serial sections in this type of analysis increases the probability of error, especially in heterogeneous tissue such as breast tissue.
Imnunohistochemical staining of tissue sections has been shown to be a reliable method of assessing alteration of proteins in a heterogeneous tissue. Immunohistochemistry (IHC) techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromagenic or fluorescent methods. This technique excels because it avoids the unwanted effects of disaggregation and allows for evaluation of individual cells in the context of morphology. In addition, the target protein is not altered by the freezing process.
The HER2
eu gene encodes a protein product, often identified as p185
HER2
. The native p185
HER2
protein is a membrane receptor-like molecule with homology to the epidermal growth factor receptor (EGFR). Amplification and overexpression of HER2 in human breast cancer has been correlated with shorter disease-free interval and shorter overall survival in some studies [van de Vijer et al.
New Eng. J. Med
. 317:1239(1988); Walker et al.
Br. J. Cancer
60:426(1989); Tandon et al.
J. Clin. Invest
. 7:1120 (1989); Wright et al.
Cancer Res
. 49:2087 (1989); McCann et al.
Cancer Res
51:3296 (1991); Paterson etal.
Cancer Res
. 51:556 (1991); and Winstanley et al.
Br. J. Cancer
63:447 (1991)] but not in others [Zhou et al.
Oncogene
4:105 (1989); Heintz et al.
Arch Path Lab Med
114:160 (1990); Kury et al.
Eur. J. Cancer
26:946 (1990); Clark et al.
Cancer Res
. 51:944 (1991); and Ravdin et al.
J. Clin. Oncol
. 12:467-74 (1994)].
In an initial evaluation of 103 patients with breast cancer, those having more than three tumor cell positive axillary lymph nodes (node positive) were more likely to overexpress HER2 protein than patients with less than three positive nodes [Slamon et al.
Science
235:177 (1987)]. In a subsequent evaluation of 86 node-positive patients with breast cancer, there was a significant correlation among the extent of gene amplification, early relapse, and short survival. HER2 overexpression was determined using Southern and Northern blotting which correlate with the HER2 oncoprotein expression evaluated by Western blotting and IHC [Slamon et al.
Science
235:177 (1987); Slamon et al.
Science
244:707 (1989)]. The median period of survival was found to be approximately 5-fold shorter in patients with more than five copies of the HER2 gene than in patients without gene amplification. This correlation was present even after correcting for nodal status and other prognostic factors in multivariate analyses. These studies were extended in 187 node-positive patients and indicated that gene amplification, increased amounts of mRNA (determined by Northern blotting), and increased protein expression (determined immunohistochemically) were also correlated with shortened survival time [Slamon et al.
Science
244:707 (1989)]. See also U.S. Pat. No. 4,968,603 to Slamon et al.
Nelson et al. have compared HER2
eu gene amplification using FISH with immunohistochemically determined overexpression in breast cancer [Nelson et al.
Modern Pathology
9 (1) 21A (1996)].
SUMMARY OF THE INVENTION
The present invention combines cellular morphological analysis with fluorescence in situ hybridization (FISH) to provide for a correlation of genetic abnormalities and cellular morphology within the same section of a subject's tissue sample. Accordingly, one may identify and score by FISH cancer cells (e.g. invasive ductal carcinoma cells) as distinct from other normal cells (e.g. stromal and inflammatory elements found in the biopsy). Alternatively, or additionally, the invention combines immunohistochemistry (IHC) with FISH to provide for a correlation of genetic abnormalities with protein expression in the same tissue section.
Morphologic assessment, or evaluation of protein expression, in a tissue prior to quantitative FISH analysis provides for accurate, specific evaluation of that tissue in a timely and cost-efficient manner. Thus, there is a need in research, prognostic, and diagnostic applications for a method that can allow for morphologic and/or protein expression analyses followed by FISH assessment in a single tissue sample section, particularly when testing a heterogeneous tissue. The invention described in this disclosure offers these features.
Accordingly, in a first aspect the invention provides a method of correlating cellular morphology with the presence of a cellular target nucleic acid sequence in a section of a tissue sample comprising the following steps:
(a) staining the section of tissue sample with a morphological stain;
(b) determining cellular morphology in the section of tissue sample;
(c) hybridizing a first fluorescently labeled nucleic acid probe to the target nucleic acid sequence in the same section of tissue sample;
(d) detecting the presence of the first nucleic acid probe in the section of tissue sample; and
(e) correlating step (b) with step (d).
In an alternative embodiment, the inventio
Cohen Robert L.
Gardiner Mary Beth
Sliwkowski Mark X.
Stelzer Gregory T.
Dreger Ginger R.
Genentech Inc.
Heller Ehrman White & McAuliffe LLP
Horlick Kenneth R.
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