INTRAEPITHELIAL NEOPLASIA RISK STRATIFICATION

Cross-Reference to Related Applications

This application claims priority to U.S. Provisional Patent Application No. 62/252,388 filed on November 6, 2015, entitled "HIGH-GRADE PROSTATIC INTRAEPITHELIAL NEOPLASIA RISK STRATIFICATION," the disclosure of which is incorporated herein by reference.

Field of the Invention

Embodiments of the invention relate to high-grade prostatic intraepithelial neoplasia

(HGPIN), and to cancer risk stratifications related thereto.

Background to the Invention

Prostate cancer is one of the most common types of cancer that men contract, second only to skin cancer, and is one of the leading causes of cancer death in men. The American Cancer Society estimates that there are approximately 180,000 new cases of prostate cancer per year.

Early detection can allow for treatment of prostate cancer when the cancer is easier to treat. There are various methods that allow for early detection of prostate cancer, from a prostate-specific antigen (PSA) blood test, to a digital rectal exam (DRE), to the detection of specific precursors to cancer. Prostatic intraepithelial neoplasia (PIN) has been identified as a precursor to prostate cancer that can allow for early detection.

PIN is a condition in which some prostate cells look and behave differently from typical prostate cells. These cells are typically located in the lining of acini or in the lining of the ducts that carry fluid to the main ejaculatory duct. With PIN, the lining remains intact even though the cells are abnormal, whereas when prostate cancer develops, the epithelial lining is ruptured. PIN can be diagnosed as either low-grade (i.e., LGPIN) or high-grade (i.e., HGPIN). In HGPIN, there is a higher degree of abnormality in the cells. Furthermore, it tends to develop in the peripheral zone of the prostate, which is where most cases of prostate cancer develop. Summary of the Invention

In one aspect of the invention, a method of determining a cancer risk profile stratification for a subject is disclosed. The method involves determining an expression level of ERG oncoprotein in high grade prostatic intraepithelial neoplasia (HGPIN) prostate tissue isolated from the subject compared to a control sample, and determining the presence or absence of at least one morphological variable compared to a second control sample, whereby when the expression level of ERG oncoprotein is high compared to the control sample and the at least one morphological variable is present compared to the second control sample, a high cancer risk profile stratification is present, and when the expression level of ERG oncoprotein is low compared to the control sample and the at least one morphological variable is absent compared to the second control sample, a low cancer risk profile stratification is present.

The at least one morphological variable may include, without limitation, bilaterality, multifocality, greater than 1 core involvement, or cribriform morphology, or any combination thereof. The method may further involve isolating the prostate tissue prior to determining the expression level of ERG as described herein. The method may further involve conducting a prostate biopsy from the subject when a high cancer risk profile stratification is present. Optionally, the method may further involve treating the subject with a prostate cancer treatment regimen when a high cancer risk profile stratification is present. The expression level of ERG oncoprotein may be determined by immunostaining with a non-human antibody. The non-human antibody may be derived from a rabbit. The non-human antibody may be a rabbit monoclonal antibody. In another aspect, certain steps of the methods disclosed herein may be carried out ex vivo.

In another aspect, a method of treating prostate cancer in a subject is disclosed. The method involves determining an expression level of ERG oncoprotein in HGPIN prostate tissue isolated from the subject compared to a control sample. The method further involves determining the presence or absence of at least one morphological variable compared to a second control sample, and treating the subject with any known prostate cancer treatment regimen when the expression level of ERG oncoprotein is high compared to the control sample and the at least one morphological variable is present compared to the second control sample. The at least one morphological variable may include, without limitation, bilaterality, multifocality, greater than 1 core involvement, or cribriform morphology, or any combination thereof. The method may involve isolating the prostate tissue prior to determining the expression level of ERG as described herein. The method may involve determining the expression level of ERG oncoprotein by immunostaining with a non-human antibody. The non-human antibody may be derived from a rabbit. The non-human antibody may be a rabbit monoclonal antibody.

In another aspect, a use of an anti-ERG antibody is disclosed to determine a cancer risk profile stratification in a subject. When the expression level of ERG oncoprotein is high in HGPIN prostate tissue of the subject compared to a control sample, a high cancer risk profile stratification is present, and when the expression level of ERG oncoprotein is low in the HGPIN prostate tissue compared to the control sample, a low cancer risk profile stratification is present. The use may further involve the use of at least one morphological variable in combination with the anti-ERG antibody to determine the cancer risk profile stratification, wherein when the expression level of ERG oncoprotein is high in the HGPIN prostate tissue of the subject compared to a control sample and the at least one morphological variable is present compared to a second control sample, a high cancer risk profile stratification is present, and when the expression level of ERG oncoprotein is low compared to the control sample and the at least one morphological variable is absent compared to the second control sample, a low cancer risk profile stratification is present.

The at least one morphological variable may include, without limitation, bilaterality, multifocality, greater than 1 core involvement, or cribriform morphology, or any combination thereof. The expression level of ERG oncoprotein may be determined by immunostaining with a non-human antibody. The non-human antibody may be derived from a rabbit. The non-human antibody may be a rabbit monoclonal antibody.

Brief Description of the Drawings

The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure, and together with the description serve to explain the principles of the disclosure, wherein:

FIG. 1 depicts a prostate biopsy as described in further detail herein.

FIG. 2 depicts luminal epithelial cells as described in further detail herein.

FIG. 3 depicts a multivariable analysis as described herein.

FIG. 4 depicts a multivariable analysis as described herein.

Detailed Description of the Invention

Aspects of the invention detailed herein involve methods of determining a risk profile stratification related to HGPIN and prostate cancer. In one aspect of the invention, a method of determining a cancer risk profile stratification for a subject is disclosed. The subject may include, without limitation, a mammal. The mammal may include, without limitation, a human. In particular, the human subject may include a human male subject. The method involves determining an expression level of ERG oncoprotein in prostate cancer, including without limitation, high grade prostatic intraepithelial neoplasia (HGPIN) prostate tissue isolated from the subject compared to a control sample. ERG expression may be determined at a protein level or, as a non-limiting example, through monitoring mRNA expression. Protein level expression can be monitored, for example, by immunostaining or other methods for determining the level of protein, including, without limitation, Western-blot or Western transfer, ELISA (enzyme-linked immunosorbent assay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochernical and immunohistochemical techniques, techniques based on the use of protein biochips or microarrays which include specific antibodies or assay s based on colloidal precipitation in formats such as dipsticks.

When determining ERG oncoprotein expression by way of immvmohistochemistry, the presence or absence of ERG oncoprotein can be determined by way of counting the number of cells that stain positive wherein a positive sample contains 1 positive cell out of approximately 1000 cells; or 1 positive cell out of approximately 900 cells: or 1 positive cell out of approximately 800 cells; or 1 positive cell out of approximately 700 cells; or 1 positive ceil out of approximately 600 cells; or 1 positive cell out of approximately 500 cells; or 1 positive cell out of approximately 400 cells; or 1 positive cell out of approximately 300 cells; or 1 positive cell out of approximately 200 cells; or 1 positive cell out of approximately 175 ceils; or 1 positive cell out of approximately 150 cells; or 1 positive cell out of approximately 125 ceils; or 1 positive cell out of approximately 100 cells; or 1 positive cell out of approximately 90 cells; or 1 positive cell out of approximately 80 cells; or 1 positive cell out of approximately 70 cells; or 1 positive ceil out of approximately 60 ceils; or 1 positive cell out of approximately 50 cells; or 1 positive cell out of approximately 40 cells; or 1 positive ceil out of approximately 30 ceils; or 1 positive cell out of approximately 20 cells; or 1 positive cell out of approximately 10 cells, or less. Further, ERG oncoprotein expression can be determined through the use of image analysis software.

Monitoring niRNA expression can be carried out, for example, by Northern blotting, RT-PCR, or gene chip technology and the like.

The method further involves determining the presence or absence of at least one morphological variable compared to a second control sample, whereby when the expression level of ERG oncoprotein is high compared to the control sample and the at least one morphological variable is present compared to the second control sample, a high cancer risk profile stratification is present, and when the expression level of ERG oncoprotein is low compared to the control sample and the at least one morphological variable is absent compared to the second control sample, a low cancer risk profile stratification is present. The predictability with which a high or low cancer risk profile stratification is made may be based on the associated P values disclosed herein.

The at least one morphological variable may include, without limitation, bilaterality, multifocality, greater than 1 core involvement, or cribriform morphology, or any combination of the foregoing. Determination of the at least one morphological variable can be made without the need for professional skill. For example, determination of the at least one morphological variable can be made by comparing the prostate tissue to well-known predetermined criteria in the second control sample. The method may further involve isolating the prostate tissue prior to determining the expression level of ERG as described herein. Such isolation can be carried out by a prostate biopsy. The method may further involve conducting a subsequent prostate biopsy from the subject when a high cancer risk profile stratification is present. The subsequent prostate biopsy may be used, for example, to diagnose prostate cancer. Optionally, the method may further involve treating the subject with a prostate cancer treatment regimen when a high cancer risk profile stratification is present. Further, and optionally, the method may involve treating the subject with a prostate cancer treatment regimen following a subsequent prostate biopsy or related diagnosis. Uniquely, the expression level of ERG oncoprotein may be determined by immunostaining with a non-human antibody. The non-human antibody may be derived from a number of species, including, but not limited to, a rabbit. Further, the non-human antibody may be a rabbit monoclonal antibody, as is disclosed in the Examples section detailed herein. In another aspect, certain steps disclosed herein or all of the steps of the methods disclosed herein may be carried out ex vivo.

In another aspect, a method of treating prostate cancer in a subject is disclosed. The method involves determining an expression level of ERG oncoprotein in HGPIN prostate tissue isolated from the subject compared to a control sample. The method further involves determining the presence or absence of at least one morphological variable compared to a second control sample, and treating the subject with a prostate cancer treatment regimen when the expression level of ERG oncoprotein is high compared to the control sample and the at least one morphological variable is present compared to the second control sample.

The at least one morphological variable may include, without limitation, bilaterality, multifocality, greater than 1 core involvement, or cribriform morphology, or any combination thereof. The method may involve isolating the prostate tissue prior to determining the expression level of ERG as described herein. The method may involve determining the expression level of ERG oncoprotein by immunostaining with a non-human antibody. The non-human antibody may be derived from a rabbit. The non-human antibody may be a rabbit monoclonal antibody. In another aspect, a use of an anti-ERG antibody is disclosed to determine a cancer risk profile stratification in a subject. When the expression level of ERG oncoprotein is high in HGPIN prostate tissue of the subject compared to a control sample, a high cancer risk profile stratification is present, and when the expression level of ERG oncoprotein is low in the HGPIN prostate tissue compared to the control sample, a low cancer risk profile stratification is present. The use may further involve the use of at least one morphological variable in combination with the anti-ERG antibody to determine the cancer risk profile stratification, wherein when the expression level of ERG oncoprotein is high in the HGPIN prostate tissue of the subject compared to a control sample and the at least one morphological variable is present compared to a second control sample, a high cancer risk profile stratification is present, and when the expression level of ERG oncoprotein is low compared to the control sample and the at least one morphological variable is absent compared to the second control sample, a low cancer risk profile stratification is present.

The at least one morphological variable may include, without limitation, bilaterality, multifocality, greater than 1 core involvement, or cribriform morphology, or any combination thereof. The expression level of ERG oncoprotein may be determined by immunostaining with a non-human antibody. The non-human antibody may be derived from a rabbit. The non-human antibody may be a rabbit monoclonal antibody.

Definitions

As used herein, the term "PCa" refers to prostate cancer.

As used herein, the term "ERG" refers to the ERG oncoprotein and natural and non- natural variants thereof.

As used herein, the term "one morphological variable" includes the clinicopathological parameters disclosed herein.

As used herein, the term "PSA" refers to prostate-specific antigen.

As used herein, the term "HGPIN" refers to high-grade prostatic intraepithelial neoplasia.

Example

Case cohort

The study was approved by a responsible institutional review board. One hundred and fifty-five (155) consecutive prostate biopsies with an initial HGPIN diagnosis and at least one follow-up biopsy were retrieved from an institutional pathology database. Cases with prior or concomitant PCa or atypical glands suspicious for cancer (ATYP/ASAP) or atypical glands adjacent to HGPIN (PINATYP) categories were excluded. Atypical cribriform lesions containing basal cells with features indeterminate between intraductal carcinoma and HGPIN were also excluded. Cases in which representative HGPIN foci were not available for ERG immunostaining were excluded. A gland was defined as having high-grade PIN based on accepted criteria (see, for e.g. : Epstein et al. (1995) Am. J. Surg. Pathol. 19: 873.). Borderline PIN lesion not fitting HGPIN criteria were not included. Clinicopathological parameters

All cases were reviewed. Clinicopathological parameters analyzed for each case included patient's age, pre-biopsy serum PSA, number of cores sampled and timing of re- biopsy. Without the knowledge of the patient's repeat biopsy outcome, the foci of HGPIN on initial biopsy for each case was evaluated based on number of cores involved with HGPIN (1 vs. >1), extent of involvement (focal vs. multifocal), laterality (unilateral vs. bilateral), and morphological pattern (flat, tufting, micropapillary and cribriform and mixed). The presence of two or more separate foci of HGPIN present on the same core separated by benign prostate glands or 3 or more cores involved by HGPIN was defined as multifocal involvement. The pathologic findings of PCa detected at re-biopsy were recorded for Gleason score, number of cancer cores, and tumor volume.

ERG expression in HGPIN

All cores with HGPIN diagnosis in initial biopsy were immunostained for anti-ERG antibody. Briefly, paraffin-embedded formalin-fixed prostate biopsy tissues were sectioned at 4 μιτι. Immunohistochemical (IHC) staining was performed on a BenchMark XT automated stainer (Ventana Medical System Inc., Tuscon, AZ). The slides were stained utilizing the UltraView detection kits with the standard cell conditioning (CC1) antigen retrieval method for 60 minutes. CC1 is a pre-diluted tris based buffer with slightly basic pH. Slides were then incubated with anti-ERG rabbit monoclonal antibody (1 :50 dilution, clone ID: EPR3864; C- terminal antibody, Epitomics, San Diego, CA) for 32 minutes at 37 C temperature. Reaction of ERG antibody was visualized with a peroxidase-based brown detection system (UltraView Universal DAB Detection Kit, Ventana Medical System Inc., Tuscon, AZ). The slides were counterstained with hematoxylin I.

For interpretation of ERG IHC staining, only immunostains with ERG-positive normal endothelia (internal control) were considered valid, and only nuclear staining within secretory cells of HGPIN was considered as positive. Any nuclear staining of HGPIN cells was considered positive for ERG expression (see: Figures 1 and 2). Results were interpreted qualitatively as positive or negative.

With specific reference to Figure 1, a prostate biopsy is depicted which contains a focus ofHGPIN demonstrating enlarged nuclei and prominent nucleoli. With specific reference to Figure 2, luminal epithelia cells of HGPIN exhibit strong nuclear ERG over expression (see, for e.g. cells marked 20 in Figure 2). The retained basal cells are highlighted at the periphery of the gland (see, for e.g. cells marked 22 in Figure 2). Statistical analysis

Various clinicopathological features and ERG overexpression status for isolated HGPIN diagnosed in initial biopsy were compared between patients who did and did not have cancer in repeat biopsy using the Wilcoxon rank sum test or chi-square test. The odds ratio of risk of PCa development was calculated using logistic regression analysis. All tests were two sided and the results were considered significant at the level of a = 0.05.

Results

Clinicopathological Characteristics and ERG Overexpression Status of 155 Patients with Isolated HGPIN Diagnosis and Subsequent Prostate Cancer Risk

The mean age was 63 years (range, 46-83). A mean of 2.16 (range, 2 to 5) biopsies were performed with the mean follow up time of 7.5 months (range, 1-63). All 155 patients had at least one repeat biopsy. In addition 24 of 155 patients had more than one repeat biopsy. Means of 12 and 11.8 cores for initial and repeat biopsy were obtained (range, 8-24). Most rebiopsies (62%) were performed in < 6 months followed by 23% within 7-12 months and 15% > 1 year.

The clinicopathological characteristics and ERG expression status and subsequent outcome of 155 patients with an initial HGPIN diagnosis are summarized in Table 1 herein. PCa was found in subsequent biopsies in 39 of 155 patients (25%). If only one follow-up biopsy had been included, 34 of 155 (22%) men would have been found to have cancer. In total, 54% of detected cancers were found in < 6 months group, followed by 28% for 7-12 months group, and 18% for > 1 year group. The remaining patients had non-cancer diagnoses, including HGPIN in 38 (25%), ATYP in 1 (<1%) and benign prostate tissue in 77 (50%). ERG expression was present in 15 (9.6%) HGPIN patients. ERG expression was diffuse and strong and homogeneously positive in all (100%) of HGPIN cases (see: Figures 1 and 2). Patients with ERG expression in HGPIN were more likely to have PCa in repeat biopsy, with 9 (60%) of ERG-positive and 30 (21%) of ERG-negative patients having PCa (p=0.001). The presence of multifocal involvement (P =0.0001), cribriform morphology (P = 0.004) and bilaterality (P = 0.0075) of HGPIN were other significant clinicopathological risk factors for predicting PCa in a repeat biopsy at univariate analysis. In multivariable analysis, only ERG positivity and multifocal involvement of HGPIN on initial biopsy remained significant risk factors for PCa detection in repeat biopsy (see: Figure 3 herein). Table 1: Univariate analysis of ERG overexpression and various clinicopathological variables in 155 patients with initial HGPIN diagnosis and its predictability of finding prostate cancer at repeat biopsy

Relationship of Pathological Parameters with ERG Expression and Subsequent PCa Risk in Repeat Biopsy

Table 2 summarizes association of various morphological parameters and subsequent PCa detection in repeat biopsy in relation to ERG expression status. HGPIN positive for ERG and exhibiting bilaterality (83%), multifocality (100%), > 1 core involvement (64%) and cribriform morphology (80%) were associated with very high risk of finding PCa in subsequent biopsy. HGPIN negative for ERG but exhibiting multifocal involvement (45%) and cribriform morphology (67%) continued to show high risk for subsequent PCa detection. When controlled for other factors at multivariable analysis, in the setting of ERG negative HGPIN only the multifocal involvement continued to show significant risk for subsequent PCa detection at repeat biopsy (see: Figure 4 herein). Focal HGPIN involving only one core with ERG negativity had minimal (16%) PCa risk in the subsequent biopsy. In contrast the presence of focal HGPIN involving only one core with ERG positivity had high PCa risk (50%) in subsequent biopsy. A total of 46% of cases in this cohort had HGPIN involving one core and were ERG negative.

Table 2: Relationship of various morphological parameters with ERG expression and subsequent prostate cancer detection in repeat biopsy

Variable Prostate cancer in repeat biopsy

Yes [n, (%)] No [n, (%)]

Unilateral

ERG + 3 (38) 5(62)

ERG- 20(18) 92 (83)

Bilateral

ERG + 5 (83) 1(17)

ERG- 11 (30) 23 (70)

Focal

ERG + 3 (33) 6(67)

ERG- 25 (19) 108 (81)

Multifocal

ERG + 6 (100) 0(0)

ERG- 5(45) 6(55)

1 core involvement

ERG + 2(50) 2(50)

ERG- 13 (16) 67 (86)

>1 core involvement

ERG + 7(64) 4(36)

ERG- 18 (28) 47 (72)

Non-cribriform morphology

ERG + 5 (50) 5 (50)

ERG- 23 (19) 100 (81)

Cribriform morphology

ERG + 4 (80) 1(20)

ERG- 8(67) 14 (33) Characteristics of PCa detected in Subsequent Prostate Biopsy for ERG positive and ERG negative HGPIN

Table 3 summarizes characteristics of PCa detected in subsequent biopsies for men with HGPIN in reference to ERG overexpression status. The majority (67%) of men with ERG negative HGPIN had Gleason score 6 in repeat biopsies. In contrast, the majority (77%) of men with ERG positive HGPIN had Gleason score higher than 6 in repeat biopsies. Number of cancer cores and tumor volume characteristics were not significantly different between ERG positive vs. ERG negative PCa. Eight (89%) ERG positive HGPIN had PCa identified at identical site on subsequent biopsy, of which 5 (71%) were ERG positive. Two PCas were ERG negative and PCa focus was not available for ERG in one case.

Table 3: Prostate cancer characteristics in repeat biopsy for patients with isolated HGPIN diagnosis stratified based on ERG status

In this study, PCa was found in subsequent repeat biopsies in 25% of patients. If only one follow-up biopsy had been performed, only 22% of men would have been found to have cancer, which is only slightly higher than risk of finding cancer after a benign diagnosis on needle biopsy. Although the cancer detection rate following the diagnosis of HGPIN has decreased in contemporary extended biopsy practice, its surveillance protocol is still debated with inconsistent opinions among experts.

In this study, it was demonstrated that the status of ERG expression in HGPIN along with other histological parameters can be utilized effectively to stratify patients into low or high risk groups for having a PCa in repeat biopsy. ERG expression was present in 15 (9.6%) HGPIN patients. Patients with ERG expression in HGPIN were significantly more likely to have PCa in repeat biopsy, with 9 (60%) of ERG-positive and 25 (18%) of ERG-negative patients having PCa (p=0.001). When controlled for other variables, ERG expressing HGPIN continued to be a significant risk factor for unsampled PCa in repeat biopsy at multivariable analysis. Overall, the current study strongly supports the conclusion that patients with ERG- positive HGPIN are more likely to harbor or develop PCa than those with ERG-negative HGPIN.

In addition to ERG positivity, several morphological parameters of HGPIN were useful in predicting higher risk of PCa in subsequent biopsy. The presence of bilaterality, multifocality, > 1 core involvement, cribriform morphology was significant at univariable analysis. However, for cases with no ERG expression, at multivariable analysis only the presence of multifocality remained significant histological factor. In this study for patients with HGPIN exhibiting both ERG positivity and multifocality, there was 100% risk of having PCa in repeat biopsy (see: Table 2). In this study, a difference was not determined in the risk of cancer with 1 vs. >1 core, however the risk proportionately was higher if further stratified by 3 (45%) or greater than 3 cores (60%).

As the majority of men with HGPIN in contemporary practice do not harbor or develop cancer in repeat biopsy, it is critical to determine which HGPIN group is at low-risk of having cancer at repeat biopsy so that they may be managed more conservatively. To this end, it has been determined how to demonstrate individual cancer risk for men with ERG negative HGPIN in reference to various histological parameters (see: Table 2). Most importantly, patients with focal HGPIN involving only one core with ERG negativity had minimal (16%) risk of PCa in a subsequent biopsy. In contrast, the presence of focal HGPIN involving only one core with ERG positivity had high PCa risk (50%) in subsequent biopsy. This finding is especially important as a significant proportion of HGPIN diagnosed in community practice would fall into this low risk group. In this cohort, 46% of men with HGPIN on initial biopsy presented with focal one core involvement and were ERG negative. Therefore these results demonstrate that ERG-negative HGPIN in the setting of certain histological parameters most notably non-cribriform morphology, focal, and with one or two cores involvement can be safely followed up delaying or potentially avoiding a repeat biopsy for patient with such a diagnosis. This study demonstrates the utility of ERG protein expression along with other histological risk factors in stratifying HGPIN into low and high risk groups for having PCa on repeat biopsy (see: summary Table 4 below). ERG overexpression and multifocality were independent risk factors for finding cancer on subsequent biopsies. A positive ERG expression especially in the setting of bilateral involvement, multifocality, >1 core involvement and cribriform morphology is associated with a very high risk of cancer detection on subsequent biopsy. In contrast, focal ERG negative HGPIN involving a single core or two cores can be safely followed up avoiding or delaying biopsy for patients with such a diagnosis. In further embodiments of the invention, a high risk score can be determined based on an extent of >3 cores or extensive involvement (separated by benign glands) even in the absence of ERG expression.

Table 4: Summary of correlation between risk score, extent, and ERG status based on disclosure herein.

It should be understood from the foregoing that, while particular implementations have been illustrated and described, various modifications may be made thereto and are contemplated herein. It is also not intended that the inventions described herein be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations and equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.