DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Continued Examination Under 37 CFR 1.114
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on February 20, 2026 has been entered.
Claims 1, 5, 9-17, 19, and 20 are pending and being examined. Claim 19 contains a minor amendment to correct grammar.
It is noted that the terms p16INK4, P16INK4a, CDKN2, and p16 all refer to the same protein. It is noted that Ki67 and Ki-67 refer to the same protein.
Maintained Rejections
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
2. Claims 1, 5, 9-17 remain rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over US Patent 7,452,727, Hennig et al, published 2002; in view of EP1628135, Ridder et al, published August 20, 2004; Yin et al (Human Pathology, 2008, 39:527-535; Epub Jan 2008), Mulder et al (Journal of Pathology, 1992, 166:37-43); Santos et al (Int. J. Cancer, 2003, 105:267-272); Toma et al (World J Urol. 2004, 22:145-149); Golijanin et al (The Journal of Urology, 2000, 164:1922-1925); Furth et al (Neoplasia, 2006, 8:429-436); van der Loos (Journal of Histochemistry & Cytochemistry, April 2008, 56:313-328); and WO 2004038418, Ridder et al, published May 2004.
Hennig teaches a method comprising:
(a) obtaining a biological cell sample from a subject diagnosed with cancer or a tumor, wherein the sample can be urine (col. 5, lines 15-22); and
(b) identifying the presence of cancer cells in the sample by staining cells in the sample in an immunocytochemical assay for the presence of both p16 and Ki67 to visualize simultaneous expression of p16 (p16INK4a) and Ki67 in the cells;
wherein p16 and Ki67 expression in the cells are measured simultaneously by performing a chromogenic-based immunocytochemical reaction on the sample comprising contacting the sample with primary anti-p16 and anti-Ki67 monoclonal antibodies,
wherein the reaction results in deposition of DAB in proximity to one primary antibody and deposition of Fast Red in proximity to the other primary antibody bound to the sample;
generating a first detectable signal comprising generation of a colored precipitate using HRP for enzymatic conversion of a first chromogen (DAB) and generation of a second detectable signal comprising generation of a colored precipitate using an alkaline phosphatase enzyme for enzymatic conversion of a second chromogen (Fast Red) which are distinguishable when co-localized so as to enable detecting a single cell co-expressing the proteins by visualization; (abstract; col, 3, line 45 to col. 6, line 30; Examples; claims 1-8);
wherein the method of sample preparation, staining, and image analysis is automated (col. 2, line 61 to col. 3, line 45; col. 4, line 34-49; col. 6, lines 10-26; claims 1-8);
wherein the method is used for early diagnosis of cancer and precancerous stages (col. 1, lines 15; col. 4, lines 34-49; col. 5, lines 22-29; claim 1).
Hennig teaches known secondary antibody compositions for detection of primary rabbit and mouse antibodies, including anti-mouse immunoglobulin and anti-rabbit immunoglobulin antibodies (col. 7, lines 13-30).
Hennig teaches detecting Ki67 and p16 expression as proteins known to be expressed in an altered manner in cancer and play a role in the genesis of cancer, wherein Ki67 is a known proliferation gene associated with growth control of cells, and p16 is a known oncogene, (col. 1, lines 30-45).
Assaying urine samples from bladder tumor patients for Ki67/p16
Although Hennig teaches detecting cancer and precancerous cells in cell samples, including urine, from patients with tumors by dual staining of cells for p16 and Ki67, Hennig does not teach the patients have bladder tumors or were previously diagnosed as having bladder cancer.
EP1628135 Ridder teaches an immunoassay for detecting p16 and/or pINK4a protein expression in cells of a liquid-based cytology (LBC) sample, such as a urine sample, from cancer patients, such as bladder cancer patients ([55]; Example 5; Table 7). In Example 5 (Table 7), Ridder demonstrates conducting an immunoassay for p16INK4a, p14AEF and HER2 protein expression on the cells of LBC samples, including urine samples from patients diagnosed with bladder tumors, wherein the immunoassay was ELISA-based detection utilizing plates coated with commercially available mouse monoclonal anti-p16INK4a antibody E6H4. Ridder compared the LBC ELISA for protein expression with that of immunocytochemical analysis and found good correlation between results ([215-217]). Ridder teaches analyzing LBC samples for protein expression in order to detect the presence or absence of a protein marker relevant to a condition or disease such as cancer, including cancers of the urinary system (claims 1-5, 17-20, 36, 47-50). Ridder suggest multiple biomarkers can be assessed by the LBC immunoassay including cyclin-dependent kinase inhibitor p16INK4a and cell proliferation maintenance protein Ki67 ([94]; Table 3; claims 9-11, 24-26, 39-41, 53-55), and suggest assessing overexpression of medically relevant protein biomarkers in an LBC sample (claims 32-36).
Yin established that increased/strong expression levels or staining of p16INK4a in bladder cancer tissue samples correlates with increased invasiveness of bladder cancer. p16INK4a overexpression occurred in invasive bladder cancer, with strong p16INK4a staining present in both cytoplasm and nucleus. Normal bladder tissue did not overexpress p16INK4a, and only expressed weak levels of p16INK4a in the cytoplasm (Table 1; Figures 2 and 3; p. 530-533). Yin utilized commercially available mouse anti-human p16INK4a antibody and immunolabeling with horseradish peroxidase (HRP) mouse antibody to visualize staining of p16INK4a at the cellular level (section 2.2). Yin teaches:
“The risk factors for progression and recurrence of UC are associated with tumor size, tumor multifocality, depth of invasion, and presence of urothelial carcinoma in situ (CIS) [2]. In addition, tumor extension beyond the bladder, lymph node metastases, and presence of systemic dissemination are associated with a poor prognosis [3]. Therefore, the importance of early and accurate detection of CIS, a precursor of the invasive carcinoma, can never be overemphasized. Diagnostically, distinction of urothelial CIS from reactive atypia on the basis of morphology alone may be difficult in some cases [4,5]. Thus, improved methods for definitive early detection should identify the majority of de novo invasive lesions at superficial and highly treatable points in their natural history and could improve diagnostic accuracy over morphology alone.” (p. 528, col. 1 in section 1)
“Abnormalities of the p16 pathway have been previously reported in cases of UC of the bladder [15,16]. These changes are also seen in CIS. Some studies assessing panels of immunohistochemical markers to distinguish nonneoplastic from neoplastic urothelium have been reported, but no specific marker with high sensitivity and specificity has been identified [4,5]. p16INK4 has been shown to be an excellent marker of cervical dysplasia in squamous neoplastic lesions of the cervix [17-19]. The aims of this study were (1) to analyze and characterize the expression of p16INK4 protein in
normal urothelium, atypical urothelium, and urothelial neoplasms of different grades and stages, (2) to assess its possible utility in distinguishing reactive atypia from CIS,
and (3) to assess the concurrent chromosomal 9 abnormalities in urothelial tumorigenesis.” (p. 528, col. 1 in section 1)
“Consistent overexpression of p16INK4 was identified in both de novo CIS and CIS with concurrent high-grade papillary or invasive UCs. The strong nuclear and/or
cytoplasmic immunoreactivity of neoplastic cells in the CIS lesions clearly distinguishes them from the normal or reactive urothelial cells. In contrast, the bladders with reactive atypia did not show significant changes in p16INK4 expression except for a small percentage of biopsy cases exhibiting loss of p16INK4. Therefore, the finding of the present study indicates that p16INK4 immunohistochemical stain can be used as a marker to distinguish CIS foci from reactive atypia (100% sensitivity), with the CIS cells characterized by strong nuclear staining. None of the nonneoplastic urothelium
demonstrated p16INK4 overexpression (100% specificity for CIS detection). In the scoring system of this study, both nuclear and cytoplasm staining are included, which was based on both the extent and intensity of immunopositivity. However, it was noted that all the CIS lesions showed both nuclear and cytoplasmic expression of p16INK4 and that most of the other urothelial lesions demonstrated cytoplasmic positivity with variable nuclear staining. It was also observed that the intensity of immunoreactivity correlated well with the combined scores of both the intensity and extent of positive
staining; thus, intensity alone can be used as a simpler scoring method in the diagnostic setting.” (p. 533, Discussion section 4)
“The data from the current study show perfect sensitivity and specificity when properly identifying normal or overexpression of p16INK4. In addition, the mutation of INK4a is one of the early and often critical events in urothelial carcinogenesis and tumor
progression [6-8]. p16INK4 evaluation is potentially useful in predicting the aggressive behavior and increased recurrence rate in both high- and low-grade urothelial lesions
[16,24,25]. Unlike CK20, detection of p16INK4 alterations offers prognostic value.” (p. 534, col. 1)
Yin tested urothelial carcinoma in situ (CIS) which is a precursor lesion of high-grade invasive carcinoma (abstract; section 2.1).
Mulder establishes that increased percentage of Ki67 positive-stained cells in bladder tumor tissue samples correlates with increased tumor stage and increased grade (Figures 5 and 6; Table II), wherein Table II demonstrates poor prognosis for patients having increased percentage of Ki67 positive cells, including disease progression, death, metastases, and muscle invasion (Discussion). Mulder concludes that progressive disease was confined to cancers with 10% or more Ki67-positive cells (p. 42, col. 1). Mulder suggests utilizing Ki67 as a marker to aid in determining grade and assessing prognosis of bladder cancer (p. 43, col. 2). Mulder recognizes Ki67 as a known cell proliferation marker, wherein the fraction of Ki67-positive cells in samples correlated with the growth fraction of the tumor and with infiltrating tumors (Discussion, p. 41, col. 2 to p. 43, col. 2; Table III). Mulder utilized a commercially available mouse anti-Ki67 antibody to detect Ki67, labeled the detection antibody with rabbit anti-mouse antibody conjugated to HRP, and visualized bound Ki67 with diaminobenzidine (DAB) (“immunohistochemistry” p. 39). Mulder detected Ki67 as bound to tumor cell nuclei (p. 39, “Counting Ki-67 labeled cells”).
Santos establishes that an increased percentage of cell nuclei staining for Ki67 of bladder tumor tissue samples, or a high Ki67 index, significantly correlates with a high risk of recurrence and progression (abstract; Tables I and II; Figure 2). Santos used commercially available primary and secondary antibodies to detect Ki67 expression, wherein the secondary antibody was labeled with HRP and Ki67 was visualized with DAB (“Immunohistochemistry” p. 268).
Santos teaches that Ki67 is a known cell proliferation marker present in cell nuclei of proliferating cells and is absent in resting cells (p. 267, col. 2):
“Proliferation abnormalities that result from disruption of cell cycle regulators have been observed and studied as prognostic factors in urothelial carcinomas.16,17. One of the most common markers used is Ki-67 labeling index.18 Since Ki-67 protein is absent in resting cells, the monoclonal antibody anti-Ki-67 reacts exclusively with the nuclei of the proliferating cells.19,20 Tsuji et al.21 showed that high Ki-67 index correlates with p53 nuclear accumulation in urothelial bladder carcinoma. Asakura et al.22 found Ki-67 as a prognostic factor for recurrence and progression in superficial bladder carcinoma.”
Santos teaches (p. 270, col. 1-2):
“Our findings show that the most reliable biological prognostic factor in the superficial bladder tumors is Ki-67 labeling index… in agreement with the present report, several authors showed this marker to be related to low recurrence-free and progression-free survival in superficial (Ta and T1) bladder carcinoma as well as an independent prognostic factor.39 – 45 Recently Pich et al.46 observed that Ki-67
immunopositivity was an independent prognostic factor, predicting recurrence in Ta/G1 bladder carcinomas and in low malignant potential neoplasm, and Yan et al.36 observed that Ki-67 could be used to identify patients at high risk for a first recurrence in USPC.”
Santos tested urothelial superficial papillary carcinomas (USPC) Ta/T1 which are early cancer precursor lesions (“Discussion”, first sentence; “Patients And Methods”).
Toma teaches urinary cytology can be improved by immunostaining with monoclonal antibodies against tumor-associated antigens in order to detect bladder cancer (abstract; Discussion; ImmunoCyt/Ucyt+ test). Toma demonstrates successfully collecting urine samples from bladder tumor patients, incubating the cells in the sample with a cocktail of three primary monoclonal antibodies, each binding a different tumor-associated antigen and each containing a different color fluorescent label, and detecting fluorescence in the cells (commercially available ImmunoCyt/Ucyt+ assay; Materials and Methods). Toma teaches utilizing urine to detect cells expressing tumor biomarkers is a non-invasive way to diagnose bladder cancer and to follow up on bladder carcinoma after diagnosis (abstract; p. 146, col. 1; p. 149, col. 1).
Golijanin teaches successfully collecting urine samples from bladder tumor patients that have primary or recurrent bladder cancer, and conducting immunocytology on the cells from the samples by contacting the cells with a primary antibody that binds to a tumor associated antigen, contacting the cells with a secondary HRP labeled antibody, and microscopically visualizing brown staining in the cells (Patients and Methods, p. 1922-1923; Figure 2; Table 1 and 2). Golijanin summarizes the known Immunocyt assay taught by Toma (p. 1922, col. 1; p. 1924, col. 1-2). Golijanin teaches that the identification of tumor antigens in bladder cancer has led to the development of various methods of non-invasive detection of bladder tumors using immunocytology, immunochemistry or molecular assays on voided urine samples (p. 1922, col. 1), and when detecting cancer-specific markers, immunocytology has the advantage of much better specificity for patients with concomitant urological disorders (p. 1924, col. 2).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to assay urine from bladder tumor patients for Ki67 and p16 in the method of Hennig. One would have been motivated to because: (1) Hennig suggests the cell sample assayed is urine and from patients with tumors; (2) Hennig teaches simultaneously assaying known proliferation marker Ki67 and oncogene marker p16 for early detection of cancer or precancerous cells; (3) EP1628135 Ridder suggests conducting an immunoassay for p16 or Ki67 expression in cells of a liquid-based cytology (LBC) sample, such as a urine sample, from cancer patients, including bladder cancer patients; (4) Yin demonstrates p16 is a known, expressed biomarker of cancer in bladder tumor cells and precursor lesions; (5) Mulder establishes that Ki67 positive-stained cells are increased in bladder tumor tissue samples and correlated with increased tumor stage and increased grade; (6) Santos teaches that Ki67 is a known cell proliferation marker present in cell nuclei of proliferating cells and is absent in resting cells, and demonstrates Ki67 expression is increased in bladder tumor cells; (7) Toma teaches analyzing urine samples for bladder cancer cells expressing multiple tumor biomarkers is a non-invasive method for bladder cancer diagnosis/surveillance, and is useful to supplement urinary cytology; and (8) Golijanin teaches immunocytology of cells in the urine of bladder tumor patients is non-invasive and can provide better specificity for patients with concomitant urological disorders. One would have a reasonable expectation of success to conduct the method of Hennig on urine samples from bladder tumor patients given: (1) EP1628135 Ridder demonstrates successfully conducting an immunoassay for p16INK4a protein expression on the cells of LBC samples, including urine samples from patients diagnosed with bladder tumors, wherein the immunoassay utilized commercially available mouse monoclonal anti-p16INK4a antibodies; (2) Yin, Mulder, and Santos demonstrate successfully detecting p16 or Ki67 expression in bladder tumor cells utilizing commercially available monoclonal antibodies and labeled secondary antibodies, and establish they are both biomarkers associated with bladder cancer; (3) Toma demonstrates successfully simultaneously assaying multiple tumor biomarker expression in cells of urine samples from bladder tumor patients, utilizing a commercial assay comprising antibodies that bind to the biomarker proteins, and Toma demonstrates the urine immunocytology assay successfully supplements urinary cytology to aid in cancer diagnosis and detecting the presence of bladder cancer cells during surveillance; and (4) Golijanin demonstrates successfully conducting immunocytology with primary and secondary antibody staining of cells from bladder tumor patient urine to detect tumor antigen expression and the presence of cancer cells.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to assay urine from bladder tumor patients previously diagnosed with bladder cancer. One would have been motivated to, and have a reasonable expectation of success to, given (1) Yin, Mulder, and Santos demonstrate successfully detecting p16 or Ki67 expression in bladder tumor cells, and establish they are biomarkers associated with the presence of bladder cancer, progression, and prognosis in patients already diagnosed with bladder cancer; (2) Toma teaches analyzing bladder tumor cells in urine for simultaneous tumor biomarker expression functions in diagnosis and surveillance of disease or follow-up after bladder cancer diagnosis; and (3) Golijanin teaches assaying urine by immunocytology from both primary and recurrent bladder cancer patients previously diagnosed.
Antibodies, their associated chromogens, and staining procedure:
Although Hennig teaches, in detail, the necessary steps and reagents to practice a method for staining and detecting Ki67 and p16 simultaneously with antibodies and DAB and Fast Red first and second chromogens, Hennig does not specifically assign the DAB chromogen reporter system to detection of bound anti-p16 antibody and assign the Fast Red chromogen reporter system to detection of bound Ki67 antibody in the sample, resulting in brown stained cells (DAB) with red stained nuclei (Fast Red). Hennig does not delineate the steps for DAB and Fast Red reporter systems as recited in instant claim 10. Hennig does not specifically teach that the anti-Ki67 antibody is a rabbit monoclonal antibody or that the anti-p16INK4a antibody is a mouse monoclonal antibody and detecting them with the respective anti-rabbit and anti-mouse secondary antibodies.
Furth teaches and demonstrate successful dual fluorescence detection of Ki67 and p16INK4a expression on a patient sample utilizing a primary anti-p16INK4a mouse monoclonal antibody and a commercially available primary anti-Ki67 rabbit antibody, as well as secondary anti-mouse and anti-rabbit antibodies conjugated to Cy2 or Cy3 fluorescent labels for detection of the primary antibodies bound to the sample (p. 430, col. 1-2).
van der Loos teaches known methods for dual detection/double staining of proteins utilizing primary mouse and primary rabbit antibodies, secondary anti-mouse and anti-rabbit antibodies, and explain detection of alkaline phosphatase (AP) activity (Fast Red) and HRP activity (DAB+) utilizing the labeled antibodies. Van der Loos teach the red-brown color combination ensures two sensitive/efficient enzymatic visualization procedures both with crisp microscopical appearance (abstract; Figure 1; p. 317, col. 2 to p. 318).
WO 2004038418 Ridder discloses that p16INK4a overexpression is a biomarker for cancerous cells and teaches combining p16INK4a detection of expression in cells with detection of Ki67 cell proliferation biomarker in a simultaneous immunoassay. Ridder teaches that dysplastic, cancerous cells, can be discriminated from normal cells by simultaneous detection of overexpression of p16INK4a protein and a known marker characteristic for cell proliferation, such as Ki67 (p. 5-7, 12; claims 1-6, 23). Ridder teaches conducting immunocytochemical imaging to measure and detect simultaneous expression of p16INK4a with a proliferation marker such as Ki67, utilizing primary monoclonal antibodies that bind to the marker proteins and secondary antibodies that bind to the primary antibodies, and employing a cytochemical staining procedure such as chromogenic staining of cells in order to visualize protein expression (p. 12-15). The biological samples assayed encompass any samples that contain cells, including body fluids and cytological samples (p. 15; claim 23). Ridder exemplify successfully simultaneously staining p16INK4a and Ki67 in cervical tissue samples by utilizing commercially available mouse anti-human p16INK4a monoclonal primary antibody (MTM); goat anti-mouse secondary antibody (Peroxidase/DakoCytomation), and DAB visualization, and utilizing commercially available rabbit anti-human Ki67 antibody, goat anti-rabbit secondary antibody (Alkaline phosphatase labeled / Dako Cytomation), and visualized with FastRed (p. 25-26; Example 6). Double staining of Ki67 + p16INK4a renders brownish cell staining with red nuclei (Figure 7; p. 9; Example 6). Ridder teaches the dysplastic cells are double stained for Ki67 + p16INK4a, whereas non-dysplastic cells lack double staining (p. 26).
In Example 7, Ridder continues to exemplify the successful simultaneous staining of liquid-based cytology (LBC) samples from cervix for p16INK4a and Ki67 using the same methods described above in Example 6. Ridder demonstrates that only dysplastic cells double stained for overexpression of p16INK4a and Ki67, wherein the non-dysplastic cells did not stain for Ki67 (Figure 8).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to assign the DAB chromogen reporter system to detection of bound anti-p16 antibody and assign the Fast Red chromogen reporter system to detection of bound Ki67 antibody in the sample in the method of Hennig. One would have been motivated to in order to simultaneously detect Ki67 and p16 expression within single cells as taught, claimed, and suggested by Hennig. One of ordinary skill in the art would have a reasonable expectation of success, given the detailed protocol to practice the method provided by Hennig in the Examples and patented claims, and given WO 2004038418 Ridder exemplifies successfully simultaneously staining p16INK4a and Ki67 in cell samples by utilizing commercially available mouse anti-human p16INK4a monoclonal primary antibody (MTM); goat anti-mouse secondary antibody (Peroxidase/DakoCytomation), and DAB visualization, and utilizing commercially available rabbit anti-human Ki67 antibody, goat anti-rabbit secondary antibody (Alkaline phosphatase labeled / Dako Cytomation), and visualized with FastRed, resulting in brown stained cells with red stained nuclei.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to utilize a rabbit monoclonal anti-Ki67 antibody and a mouse monoclonal anti-p16INK4a antibody and corresponding anti-mouse and anti-rabbit secondary antibodies for chromogenic-based detection in the method of Hennig. One would have been motivated to: (1) in order to provide antibodies to simultaneously and specifically detect Ki67 and p16 expression within single cells as taught, claimed, and suggested by Hennig; (2) because Hennig, Furth, van der Loos, and WO 2004038418 Ridder recognize and teach the known use of secondary antibodies specific to the primary antibodies for detection; (3) because van der Loos explain the known and established method for dual staining and visualization of proteins utilizing primary and secondary antibodies with HRP/DAB and AP/Fast Red; and (4) because rabbit monoclonal anti-Ki67 antibody and mouse monoclonal anti-p16INK4a antibody are commercially, readily available for immunoassays. One of ordinary skill in the art would have a reasonable expectation of success given the cited art demonstrates rabbit monoclonal anti-Ki67 antibody and mouse monoclonal anti-p16INK4a antibody successfully detect p16INK4a and Ki67 proteins in patient cell samples, and given methods for dual staining and visualization of proteins utilizing mouse/rabbit primary antibodies and secondary antibodies with HRP/DAB and AP/Fast Red signaling are established.
In summary, the cited prior art both teaches and exemplifies the known immunoassay for staining cancer cells simultaneously with commercially available anti-p16, anti-Ki67, secondary antibodies, and DAB/FastRed in order to visualize brown stained cells with red stained nuclei. This assay is known in the prior art and successfully practiced on cancer cells. The cited prior art also teaches that p16 and Ki67 are known cancer cell biomarkers across different cancer cell types, including bladder cancers, and teach simultaneously detecting both markers (p16 as the oncogene marker and Ki67 as the proliferation marker to detecting proliferating cancer cells) in order to enhance cancer diagnostics with dual biomarker detection as opposed to single biomarker detection. The cited prior art also teaches routine practice of utilizing urine as a non-invasive cytological sample to stain bladder cancer cells simultaneously for cancer biomarkers in order to detect cancer cells. Given: (1) the demonstrated success of the p16/Ki67 DAB/FastRed chromogenic immunoassay and commercially available reagents to practice it, including on liquid cytology samples, (2) the known application of the immunoassay to successfully detect cancer cells expressing known cancer biomarkers p16 and Ki67, and (3) the known application of immunoassays to non-invasive urine cytology samples for bladder cancer cell detection, including multiple biomarker detection, it is well within the level of the ordinary skilled artisan to apply the p16/Ki67 DAB/FastRed chromogenic immunoassay to urine samples to detect p16/Ki67-expressing cancer cells, and with a reasonable expectation of success.
3. Claims 19 and 20 remain rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over US Patent 7,452,727, Hennig et al, published 2002; in view of EP1628135, Ridder et al, published August 20, 2004; Yin et al (Human Pathology, 2008, 39:527-535; Epub Jan 2008), Mulder et al (Journal of Pathology, 1992, 166:37-43); Santos et al (Int. J. Cancer, 2003, 105:267-272); Toma et al (World J Urol. 2004, 22:145-149); Golijanin et al (The Journal of Urology, 2000, 164:1922-1925); Furth et al (Neoplasia, 2006, 8:429-436); van der Loos (Journal of Histochemistry & Cytochemistry, April 2008, 56:313-328); and WO 2004038418, Ridder et al, published May 2004; as applied to claims 1, 5, 9-17 above, and further in view of US Patent Application Publication 2003/0190602, Pressman et al; and Samuelsson et al (INFECTION AND IMMUNITY, June 2004, 72: p. 3179–3186).
Hennig, EP1628135 Ridder, Yin, Mulder, Santos, Toma, Golijanin, Furth, van der Loos, and WO 2004038418 Ridder (the combined references) teach an automated method comprising:
reacting bladder cells within a urine sample derived form a subject having a bladder tumor and previously diagnosed as having bladder cancer, with an anti-human p16 mouse monoclonal antibody under conditions sufficient to specifically bind the anti-human p16 monoclonal antibody to human p16 protein included within the bladder cells in the urine sample;
reacting the bladder cells within the urine sample with a peroxidase enzyme under conditions sufficient to specifically bind the peroxidase enzyme to the anti-human p16 monoclonal antibody bound to the bladder cells within the urine sample;
reacting the peroxidase enzyme bound to the anti-human p16 monoclonal antibody with a first set of agents comprising 3,3’-diaminobenzidine (DAB) under conditions sufficient to precipitate the DAB onto the bladder cells in the urine sample that express the human p16 protein;
reacting the bladder cells in the urine sample with an anti-human Ki67 rabbit monoclonal antibody under conditions sufficient to specifically bind the anti-human Ki67 monoclonal antibody to human Ki67 protein included within the bladder cells in the urine sample;
reacting the bladder cells in the urine sample with an alkaline phosphatase enzyme under conditions sufficient to specifically bind the alkaline phosphatase enzyme to the anti-human Ki67 monoclonal antibody bound to the bladder cells in the urine sample;
reacting the alkaline phosphatase enzyme bound to the anti-human Ki67 monoclonal antibody with a second set of agents comprising a FastRed chromogen under conditions sufficient to precipitate the FastRed chromogen onto the bladder cells in the urine sample that express the human Ki67 protein; and
wherein the DAB and the FastRed are precipitated on the bladder cells at levels sufficient to visualize coexpression of the p16and Ki67 in a single bladder cell within the urine sample.
Hennig further suggests using flow cytometry to analyze protein expression in the cells as an automated detection method (col. 5, lines 30-41).
The combined references do not demonstrate a step of performing automated flow cytometry to analyze the bladder cells.
Pressman teaches methods of diagnosing bladder cancer by collecting a cell sample from a bladder tumor patient, such as urine or bladder washings ([18]; [77]; [104]; [106]; [872-875]; [878]; [881]; [938]; [945-947]; [949]); contacting the cell sample with multiple antibody probes binding to cancer biomarkers wherein the antibodies are detectably labeled with chromogenic or colored tags to visualize protein expression ([34]; [58]; [60]; [263]). Pressman exemplifies detecting protein expression in cells using commercially available HRP-mouse or -rabbit antibodies and reacting them with DAB to visualize brown color precipitate at the site of reaction ([257]; Table 7; [260]; Table 9).
Pressman teaches immunocytochemistry of urine samples is gaining popularity because it is noninvasive and has increased sensitivity compare to urine cytology, and mentions the known Immunocyt assay as a successful urinalysis example for the detection of bladder cancer cells, although it uses fluorescence detection ([876]; [947]; [949]).
Pressman teaches p16 as a known oncogene marker in bladder tumors ([940]) and Ki67 is a preferred biomarker for diagnosing bladder tumors (Figure 8a; [884]), and Ki67 is a known marker localized in the cell nucleus ([62]).
Pressman suggests their methods of detecting cells expressing biomarkers can utilize flow cytometry, wherein a probe (antibody) is attached to a detectable label such as chromogenic or enzymatic moieties, the label is visualized using reader instrumentation (equipment), and the visualized label allows for detection of the protein bound by the probe ([60]; [16]; [58]). Pressman teaches methods of flow cytometry are known ([10]).
Samuelsson demonstrates successfully applying flow cytometry to cells in a urine sample, wherein the cells from a urine sample were contacted with primary mouse monoclonal antibody and primary rabbit monoclonal antibody each binding to different antigens expressed by the cells in the urine sample; then contacted with secondary anti-mouse and anti-rabbit monoclonal antibodies labeled with different detectable labels; and finally analyzed in a commercial, automated flow cytometer (p. 3180, col. 2; “Analysis of TLR4 and CD14 on excreted urinary cells by flow cytometry”; Figure 2).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to add a step of flow cytometry to the method of the combined references. One would have been motivated, and have a reasonable expectation of success to, given Hennig and Pressman suggest automated flow cytometry methods can be used to detect multiple biomarker expression in cells, Pressman teaches such methods in the art are known, and Samuelsson demonstrates successfully detecting and analyzing cells from urine samples that are expressing two different biomarkers by using commercially available automated flow cytometry.
Response to Arguments
The cited art does not teach visualization of the co-expression of p16 and Ki67 in a single cell
4. Applicants argue that the cited art does not teach visualization of the co-expression of p16 and Ki67 in a single cell of a urine cytology sample, therefore there is no motivation or reasonable expectation of success to. Applicants argue that the cited art does not disclose or suggest that such co-expressing cells appear as brown-stained cells having red-stained nuclei, which is the claimed morphological staining pattern. Applicants argue the cited art does not teach or suggest a duplex chromogenic protocol that assigns DAB to p16 and Fast Red to Ki67 as claimed, and yields the required, reliably reasonable morphological staining pattern in single cells. Applicants argue that, instead, Hennig teaches an algorithmic or post hoc categorization of cells based on mixed or combined color values. Applicants argue that Hennig teaches an automated method of detecting tumor regions and is directed toward computational processing of staining-derived signals for automated classification, and does not teach duplex chromogenic visualization protocol that produces the claimed morphological staining pattern.
Applicants argue that the present claims require visualization of p16/Ki67 co-expression within a single urine cytology cell with a very specific morphological staining appearance. Applicants argue that Hennig does not disclose or suggest that result. Instead, Hennig describes a workflow in which acquired signals must (1) be detected in relative spatial proximity that is either signals in one cell or signals in a restricted area of the sample or tissue region, and (2) be above or below individually defined intensity/threshold values. Applicants argue that Hennig discloses detecting color mixtures of signals and describes detecting the secondary color of a mixture of individual colors within a cell or constituent region of a tissue section. Applicants argue that Hennig describes relating the secondary color detected to a threshold value, wherein a value of the secondary color detected above or below the threshold value is predetermined to be indicative of the presence of cancer cells. Applicants argue this disclosure is part of Hennig’s automated method to allow for a quantitative assessment in the form of an area value obtained for particular color mixtures. Applicants argue that Hennig does not teach detecting discrete signals from individual detectable reagents in their automated method, but rather, Hennig teaches detecting the combined, accredited and threshold secondary colors that are observed and used to determine whether a sample includes healthy or abnormal cells. Applicants argue that Hennig does not teach observing the co-expression of p16 and Ki67 based on visualizing two different colors in a single cell, therefore Hennig does not teach the claimed invention.
Applicants argue that Hennig’s method is automated or automatable, which makes it fundamentally different from the claimed invention. Applicants reiterate that Hennig does not teach visualizing the two separate colors within a single cell, but rather, teaches detecting a mix of the two colors that is translated to a combined signal intensity value for comparison to a threshold value. Applicants argue that Hennig does not teach a Fast Red/DAB chromogenic assay that would result in the claimed morphological staining pattern of a brown-stained cell with red nucleus. Applicants argue that Hennig’s method of dual staining of cells would not necessarily result in the morphological staining pattern of brown cells with red nuclei.
Applicants argue the claims require a single-cell visualization requirement.
5. The arguments have been considered but are not persuasive. Arguments that Hennig teaches their method is automatable to detect a combined color signal value are not persuasive. Hennig’s disclosure of automation of their method of dual biomarker detection in single cells to identify cancerous cells does not teach away from the instant invention, nor do the instant claims exclude the automation of Hennig’s dual detection method, nor do the claimed methods exclude additional steps of using a machine to combine and read the color signals into a single signal intensity for comparison to threshold values.
Hennig suggests their methods can be automated, or “automatable” (see abstract; col. 3, lines 45-54; col. 6, lines 3-4; claim 1), which does not teach away from the instantly claimed invention. Hennig defines the phrase “automatable detection” and teaches that some steps in the method can be automated, while others are still manually performed by human labor (col. 5, lines 30-41). The suggestion by Hennig to automate their method does not teach away from the instantly claimed invention. Currently, there is no limitation recited in the rejected claims that would exclude an automated method or an automation of any part of the method, and the instant claims reasonably encompass the method of Hennig that is automated or automatable for visualization. Further, instant claim 19 recites a step of “performing an automated flow-cytometric analysis”, thereby claiming a method with an automated step.
With regard to the result of dual staining, the instantly claimed limitation of defining how the DAB and Fast Red appear in a single cell after dual staining does not exclude the method of dual staining taught by of Hennig. The dual staining method taught by Hennig, including by DAB and Fast Red, would produce the same dual staining of a single cell and produce the same individual color signals for each biomarker within a cell as instantly claimed. The instant claims recite:
wherein single cells within the urine sample that co-express both the p16 protein and the Ki67 protein appear as brown-stained cells having red-stained nucleic (claim 1).
immunoenzymatically staining bladder cells within the urine sample…such that individual cells of the urine sample that co-express both human p16 and Ki67 appear as brown-stained bladder cells having red-stained nuclei (claim 9);
wherein the DAB and Fast Red are precipitated on the bladder cells at levels sufficient to visualize co-expression of the p16 and Ki67 proteins in a single bladder cell within the urine sample, wherein singles cells within the urine sample that co-express both the p16 and Ki67 proteins appear as brown-stained cells having red-stained nuclei (claims 19).
These claim limitations do not exclude staining methods of Hennig, and Examiner maintains the staining methods of Hennig necessarily result in dual staining of single cells as claimed, emitting individual color signals for each biomarker. Hennig teaches dual staining of single cells, and Hennig teaches the two stains produce detectable individual color signals, therefore the two stains would necessarily “appear” as two different staining colors, as required by the claims. There is no step recited in the claims for imaging/visualizing the dual staining that would exclude the staining or imaging methods described by Hennig. Applicants have not persuasively argued that the dual staining method of Hennig would not result in the appearance of dual staining of proteins within a single cell, wherein each stain emits its own color signal.
Arguments that Hennig teaches a method of imaging the stained cells by measuring the intensity of the mixed signals rather than measuring the intensity of individual signals, are not persuasive. As stated above, there is no claim limitation excluding Hennig’s method of measuring the distinct color signals after applying the dual stain. Hennig teaches conducting the dual stain in order to provide two measurable signals for different biomarkers, including within a single cell. Processing the two separate color signals by combining them into a single signal measurement requires measuring a mixture of two separate protein staining signals simultaneously in a cell, and this method still necessitates each protein/chromogen signal to be individually detectable. The claims do not exclude further imaging the dually stained cancer cells utilizing automated methods or by producing a mixed signal intensity representative of the co-expression of the two stained proteins. Applicants have not persuasively argued that the method of Hennig would not result in the dual staining of a single cell, where each stain is visibly distinct within the cell.
With regards to assigning the DAB chromogen to p16 and the Fast Red staining to Ki67, specifically, in order to arrive at morphological staining of brown cells with red nuclei, it is the combination of secondary references with Hennig that render obvious this specific combination and morphological staining pattern for the reasons stated in the rejection. In particular, WO 2004038418 Ridder teaches and successfully demonstrates double staining of Ki67 + p16 in liquid cancer cell samples with Fast Red and DAB produces the morphological staining pattern of brown cells with red nuclei recited in the claims and argued by Applicants. As stated in the rejection:
Antibodies, their associated chromogens, and staining procedure:
Although Hennig teaches, in detail, the necessary steps and reagents to practice a method for staining and detecting Ki67 and p16 simultaneously with antibodies and DAB and Fast Red first and second chromogens, Hennig does not specifically assign the DAB chromogen reporter system to detection of bound anti-p16 antibody and assign the Fast Red chromogen reporter system to detection of bound Ki67 antibody in the sample….
van der Loos teaches known methods for dual detection/double staining of proteins utilizing primary mouse and primary rabbit antibodies, secondary anti-mouse and anti-rabbit antibodies, and explain detection of alkaline phosphatase (AP) activity (Fast Red) and HRP activity (DAB+) utilizing the labeled antibodies. Van der Loos teach the red-brown color combination ensures two sensitive/efficient enzymatic visualization procedures both with crisp microscopical appearance (abstract; Figure 1; p. 317, col. 2 to p. 318).
WO 2004038418 Ridder discloses that p16INK4a overexpression is a biomarker for cancerous cells and teaches combining p16INK4a detection of expression in cells with detection of Ki67 cell proliferation biomarker in a simultaneous immunoassay. Ridder teaches that dysplastic, cancerous cells, can be discriminated from normal cells by simultaneous detection of overexpression of p16INK4a protein and a known marker characteristic for cell proliferation, such as Ki67 (p. 5-7, 12; claims 1-6, 23). Ridder teaches conducting immunocytochemical imaging to measure and detect simultaneous expression of p16INK4a with a proliferation marker such as Ki67, utilizing primary monoclonal antibodies that bind to the marker proteins and secondary antibodies that bind to the primary antibodies, and employing a cytochemical staining procedure such as chromogenic staining of cells in order to visualize protein expression (p. 12-15). The biological samples assayed encompass any samples that contain cells, including body fluids and cytological samples (p. 15; claim 23). Ridder exemplify successfully simultaneously staining p16INK4a and Ki67 in cervical tissue samples by utilizing commercially available mouse anti-human p16INK4a monoclonal primary antibody (MTM); goat anti-mouse secondary antibody (Peroxidase/DakoCytomation), and DAB visualization, and utilizing commercially available rabbit anti-human Ki67 antibody, goat anti-rabbit secondary antibody (Alkaline phosphatase labeled / Dako Cytomation), and visualized with FastRed (p. 25-26; Example 6). Double staining of Ki67 + p16INK4a renders brownish cell staining with red nuclei (Figure 7; p. 9; Example 6). Ridder teaches the dysplastic cells are double stained for Ki67 + p16INK4a, whereas non-dysplastic cells lack double staining (p. 26).
In Example 7, Ridder continues to exemplify the successful simultaneous staining of liquid-based cytology (LBC) samples from cervix for p16INK4a and Ki67 using the same methods described above in Example 6. Ridder demonstrates that only dysplastic cells double stained for overexpression of p16INK4a and Ki67, wherein the non-dysplastic cells did not stain for Ki67 (Figure 8).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to assign the DAB chromogen reporter system to detection of bound anti-p16 antibody and assign the Fast Red chromogen reporter system to detection of bound Ki67 antibody in the sample in the method of Hennig. One would have been motivated to in order to simultaneously detect Ki67 and p16 expression within single cells as taught, claimed, and suggested by Hennig. One of ordinary skill in the art would have a reasonable expectation of success, given the detailed protocol to practice the method provided by Hennig in the Examples and patented claims, and given WO 2004038418 Ridder exemplifies successfully simultaneously staining p16INK4a and Ki67 in cell samples by utilizing commercially available mouse anti-human p16INK4a monoclonal primary antibody (MTM); goat anti-mouse secondary antibody (Peroxidase/DakoCytomation), and DAB visualization, and utilizing commercially available rabbit anti-human Ki67 antibody, goat anti-rabbit secondary antibody (Alkaline phosphatase labeled / Dako Cytomation), and visualized with FastRed.
Contrary to arguments, the cited combined references do obviate visualizing a single cell stained with DAB/FastRed for p16/Ki67 detection, for the reasons stated above and of record.
6. Applicants argue that Furth and Van der Loos fail to remedy the deficiency of Hennig. Applicants argue Furth used a fluorescent label for simultaneous dual staining of Ki67 and p16 in cells and does not teach DAB/FastRed. Applicants argue that Van der Loos teaches away from using DAB and FastRed together because they do not contrast well visually and DAB can clog at high staining intensities producing a dark brown deposit that can be missed. Applicants argue that dark DAB deposition would predictably reduce contrast and/or obscure nuclear detail, so there is no reasonable expectation of success that co-expressing cells would be present as brown-stained cells having red-stained nuclei. Applicants point to Yin and argue that Yin teaches p16INK4a overexpression occurred in invasive bladder cancer, with strong p16INK4a staining present in both cytoplasm and nucleus. Applicants argue that because Yin teaches strong staining of p16, it is expected bladder cancer cells would have the problem of reduced contrast and/or obscured nuclear detail taught by van der Loos. Applicants argue that evidence of strong p16 staining in bladder cancer cells taught by Yin does not provide evidence of reasonable expectation of success to produce the claimed morphological staining pattern of a brown-stained cell with red nucleus.
7. The arguments have been considered but are not persuasive. Furth demonstrates that simultaneous detection of p16 and Ki67 utilizing primary anti-p16INK4a mouse monoclonal antibody and a commercially available primary anti-Ki67 rabbit antibody, as well as secondary anti-mouse and anti-rabbit antibodies conjugated to different color labels is successfully practiced. It is noted that Hennig recognizes dual staining of cells can be used with either fluorescent or chromogenic labels (see patented claim 3).
van der Loos teaches known methods for dual detection/double staining of proteins utilizing primary mouse and primary rabbit antibodies, secondary anti-mouse and anti-rabbit antibodies, and explains detection of alkaline phosphatase (AP) activity (Fast Red) and HRP activity (DAB+) utilizing the labeled antibodies. Contrary to arguments, van der Loos does not teach away from utilizing DAB and FastRed together. Van der Loos teaches the red-brown color combination ensures two sensitive/efficient enzymatic visualization procedures both with crisp microscopical appearance (abstract; Figure 1; p. 317, col. 2 to p. 318).
Contrary to arguments, Yin does not provide evidence that dual staining of DAB and FastRed together would not work as expected. As stated previously on record, Van der Loos provides a solution for the problem noted for strong DAB staining by diluting the primary antibody. Yin was provided as a reference that exemplifies successfully using commercially available mouse anti-human p16 antibody and immunolabeling with horseradish peroxidase (HRP) mouse antibody to visualize staining of p16 at the cellular level in bladder cancer cells. Yin established p16 as a biomarker of bladder cancer cells, where increased p16 expression in bladder cancer tissue samples correlates with increased invasiveness of bladder cancer, with p16 staining present in both cytoplasm and nucleus, while normal tissues lacked p16 staining or stained weakly in the cytoplasm. Thus, Yin provides motivation and reasonable expectation of success to use commercially available antibodies and to detect p16 specifically in bladder cancer cells. Further, the rejection of record points to WO 2004038418 Ridder that exemplified successfully simultaneously staining p16INK4a and Ki67 in cell samples by utilizing commercially available mouse anti-human p16INK4a monoclonal primary antibody (MTM); goat anti-mouse secondary antibody (Peroxidase/DakoCytomation), and DAB visualization, and utilizing commercially available rabbit anti-human Ki67 antibody, goat anti-rabbit secondary antibody (Alkaline phosphatase labeled / Dako Cytomation), and visualized with FastRed. Therefore, WO 2004038418 Ridder provides additional reasonable expectation of success to conduct dual stain of cancer cells with p16 and Ki67 with DAB and Fast Red. Nowhere, do Van der Loos, Yin, or any cited references teach away from the success of dual staining cells with DAB and Fast Red or teach away from staining p16 as a biomarker for bladder cancer cells.
The cited art does not supply the rationale or reasonable expectation of success or a skilled artisan to select p16 and ki67 for duplex staining of bladder-tumor urine cytology
8. Applicants argue that Hennig does not correlate markers to specimen types, and provides no selection guidance for bladder-tumor urine cytology. Applicants argue that Hennig does not supply a rationale to select p16 and Ki67 biomarkers for detection in bladder cancer cells. Applicants argue that Hennig does not disclose or suggest urine cytology samples derived from bladder tumor subjects for detecting urothelial cancer. Applicants then argue that Hennig does teach detecting the biomarker pair combination of p16/Ki67 specifically, but that Hennig also teaches detecting other biomarker pair combinations, and does not teach selecting p16/Ki67 specifically for staining a urine cytology sample from a bladder tumor patient and does not teach a correlation of p16/Ki67 with bladder tumor cells. Applicants argue that Hennig teaches testing various biological samples in addition to urine, but does not teach testing urine samples for bladder tumor cells and for p16/Ki67 biomarkers specifically. Applicants argue that Hennig provides lists of samples, cancers, and biomarker combinations but does not provide selection guidance. Applicants argue that Hennig provides an invitation to experiment and is unpredictable. Applicants argue that listing possible cancers, biological samples, and biomarker pairs does not supply the rationale to select p16/Ki67 detection in urine samples form bladder tumor patients and does not supply a reasonable expectation of success to.
9. The arguments have been considered but are not persuasive. Although Applicants argue that the claimed biomarkers and urine sample are disclosed by Hennig in a list with other species, such as other biomarker pairs and other specimen types, Examiner asserts the disclosure of additional species does not render the disclosure of species biomarker pair p16 and Ki-67 and urine samples any less anticipatory or less obvious. MPEP 2131.02 states: A reference that clearly names the claimed species anticipates the claim no matter how many other species are named. A genus does not always anticipate a claim to a species within the genus. However, when the species is clearly named, the species claim is anticipated no matter how many other species are additionally named. Ex parte A, 17 USPQ2d 1716 (Bd. Pat. App. & Inter. 1990) (The claimed compound was named in a reference which also disclosed 45 other compounds. The Board held that the comprehensiveness of the listing did not negate the fact that the compound claimed was specifically taught. The Board compared the facts to the situation in which the compound was found in the Merck Index, saying that “the tenth edition of the Merck Index lists ten thousand compounds. In our view, each and every one of those compounds is described’ as that term is used in 35 U.S.C. § 102(a), in that publication.”). Id. at 1718. See also In re Sivaramakrishnan, 673 F.2d 1383, 213 USPQ 441 (CCPA 1982) (The claims were directed to polycarbonate containing cadmium laurate as an additive. The court upheld the Board’s finding that a reference specifically naming cadmium laurate as an additive amongst a list of many suitable salts in polycarbonate resin anticipated the claims. The applicant had argued that cadmium laurate was only disclosed as representative of the salts and was expected to have the same properties as the other salts listed while, as shown in the application, cadmium laurate had unexpected properties. The court held that it did not matter that the salt was not disclosed as being preferred, the reference still anticipated the claims and because the claim was anticipated, the unexpected properties were immaterial.).
In the instant case, Hennig clearly names detecting the claimed species pair of biomarkers p16 and Ki-67 together, and names using the species of cells from urine samples, explicitly to identify the presence of cancer cells. Hennig both discloses and patented claims for their method to combine the p16 and Ki-67 markers for detecting cancerous cells. Hennig teaches that the pair of biomarkers Ki-67 and p16 are a preferred embodiment (col. 4, lines 30-33) and claims the pair for identification of cancer cells in claim 4. Contrary to arguments, the entire disclosure of Hennig is clear about using their methods to detect cancer cells and does not teach away from doing so and does not teach uncertainty for doing so.
The secondary references cited in the rejection of record provide motivation and reasonable expectation of success to select the Ki67 and p16 biomarker pair for testing in urine samples from bladder tumor patients. See the section in the rejection with the header “Assaying urine samples from bladder tumor patients for Ki67/p16” for analysis of motivation and success provided by the secondary references.
The cited combined references recognize that p16 and Ki67 are established biomarkers of proliferating cancer cells across many cancer types (as taught by Hennig (col. 1, lines 30-45); EP1628135 Ridder ([94]; Table 3); Furth (Discussion, Figure 3); WO 2004038418 Ridder (p. 5-7, 12; claims 1-6, 23); and Pressman ([940]; Figure 8a; [884]; [62]). The cited prior art already recognizes these two markers are established for the detection of proliferating cancer cells, providing motivation and reasonable expectation of success to select the p16/Ki67 combination suggested by Hennig for cancer cell detection. The secondary references cited under the rejection section of “Assaying urine samples from bladder tumor patients for Ki67/p16” provide motivation and reasonable expectation of success to specifically select and apply the Ki67/p16 combination detection to bladder cancer cells in a urine sample for the reasons of record.
10. Applicants argue that Ridder 1 (EP1628135 Ridder) does not remedy the deficiencies argued for Hennig for selecting Ki67 and p16 biomarker combination for detection in urine samples from bladder tumor patients. Applicants argue that Ridder I teaches a bladder cancer sample tested negative for p16 expression. Applicants argue that Ridder I teaches other body fluid samples for testing and that the samples can be tested for presence or absence of cell types including neoplastic/dysplastic cells, or pathogens such as viruses, and other cell types. Applicants argue that Ridder I at Example 5 demonstrates that p16 biomarker can be detected by either ELISA or immunocytological staining. Applicants argue that Ridder demonstrates detecting positive p16 expression in cervical samples by ELISA and immunocytological staining, and did not detect positive p16 expression in a urine sample derived from subjects having bladder cancer by ELISA and immunocytological staining. Applicants argue that because of this result, Ridder does not provide motivation or reasonable expectation of success to detect p16 expression in urine samples from bladder tumor patients. Applicants argue that Ridder I does not teach or suggest combining Ki67 detection with the p16 detection in urine samples from bladder tumor patients. Applicants argue that although Ridder 1 demonstrates one can attempt to assay urine LBC samples from bladder tumor patients for p16 expression, Applicants argue their results demonstrate no affirmation that p16 will be detected in bladder tumor urine samples. Applicants argue that Ridder 1’s demonstration of positive detection results for p16 in LBC cervical cancer samples does not provide a reasonable expectation of success to detect p16 or p16/Ki67 in bladder tumor cells.
11. The arguments have been considered but are not persuasive. Contrary to arguments, Ridder I demonstrates motivation and reasonable expectation of success to assay for p16 expression in urine samples of bladder tumor patients, even if the result was negative for both liquid-based cytology (LBC) immunocytochemical analysis and ELISA. Ridder I demonstrated the LBC assay was consistent with routine ELISA detection results. Ridder I also demonstrated that the result of immunocytology of LBC was congruent with ELISAs for p16 detection in cervical cancer patients, providing success and predictability for protein biomarker detection in LBC samples. Nowhere does Ridder teach away from testing for p16 expression in cells in urine from bladder tumor patients, even with their negative result. Nowhere does Ridder 1 teach the result indicates lack of predictable expression or detection of p16 in bladder tumor LBC samples. It is noted that the instant claims do not require determining a positive result by conducting the assay. Those of ordinary skill in the art recognize that testing biological samples for biomarker expression is not guaranteed 100% biomarker detection every sample, and this is why testing must be done. As stated in the rejection of record, Ridder provides motivation to assay LBC samples, including urine, for medically relevant protein biomarkers including p16 and Ki67: “Ridder teaches analyzing LBC samples for protein expression in order to detect the presence or absence of a protein marker relevant to a condition or disease such as cancer, including cancers of the urinary system (claims 1-5, 17-20, 36, 47-50). Ridder suggests multiple biomarkers can be assessed by the LBC immunoassay including cyclin-dependent kinase inhibitor p16INK4a and cell proliferation maintenance protein Ki67 ([94]; Table 3; claims 9-11, 24-26, 39-41, 53-55), and suggests assessing overexpression of medically relevant protein biomarkers in an LBC sample (claims 32-36).”
It is reiterated that the combination of cited references in the rejection provide motivation for selecting Ki67/p16 detection in urine samples of bladder tumor patients with a reasonable expectation of success for the reasons of record, and the obviousness rationale is not based solely on Ridder 1 or Hennig.
12. Applicants argue that Yin demonstrates detecting p16 expression in urothelial lesions by staining tissue sections with antibody. Applicants argue that Yin is directed to tissue section immunohistochemistry and not to urine cytology. Applicants argue Yin detected both increased and decreased p16 expression in high- and low-grade urothelial carcinoma and teaches p16 is of no value as a diagnostic marker for urothelial lesions other than for carcinoma in situ. Applicants argue Yin teaches p16 is presented as limited in diagnostic utility for urothelial lesions outside a narrow use case, which does not support the Patent Office’s assertion that a skilled artisan would select p16 for bladder tumor urine cytology with a reasonable expectation of success.
13. The arguments have been considered but are not persuasive. Yin demonstrates successfully using commercially available mouse anti-human p16INK4a antibody and immunolabeling with horseradish peroxidase (HRP) mouse antibody to visualize staining of p16INK4a at the cellular level. Yin established that p16 is a biomarker expressed by bladder cancer cells. Yin provides motivation and reasonable expectation of success to detect p16 as a biomarker of bladder cancer cells. Yin established that increased/strong expression levels or staining of p16INK4a in bladder cancer tissue samples correlates with increased invasiveness of bladder cancer. p16INK4a overexpression occurred in invasive bladder cancer, with strong p16INK4a staining present in both cytoplasm and nucleus. Normal bladder tissue did not overexpress p16INK4a, and only expressed weak levels of p16INK4a in the cytoplasm. As stated in the rejection, Yin teaches: “The data from the current study show perfect sensitivity and specificity when properly identifying normal or overexpression of p16INK4. In addition, the mutation of INK4a is one of the early and often critical events in urothelial carcinogenesis and tumor progression [6-8]. p16INK4 evaluation is potentially useful in predicting the aggressive behavior and increased recurrence rate in both high- and low-grade urothelial lesions [16,24,25]. Unlike CK20, detection of p16INK4 alterations offers prognostic value.” (p. 534, col. 1).
Therefore, contrary to arguments, Yin teaches both motivation and reasonable expectation of success to select and detect p16 as a biomarker in bladder tumor cells, and to utilize commercially available mouse anti-human p16INK4a antibody and immunolabeling with horseradish peroxidase (HRP) mouse antibody to visualize staining of p16INK4a at the cellular level.
It is reiterated that the combination of cited references in the rejection provide motivation for selecting Ki67/p16 detection in urine samples of bladder tumor patients with a reasonable expectation of success for the reasons of record, and the obviousness rationale is not based solely on Yin.
14. Applicants argue that Mulder teaches staining for Ki67 on tissue samples and not urine cytology samples, not p16 staining, and not duplex p16/Ki67 chromogenic visualization in bladder-tumor urine samples.
15. The arguments have been considered but are not persuasive. The limitations argued by Applicants as not taught by Mulder were addressed by the combination of cited references in the rejection of record. Mulder recognizes Ki67 as a known cell proliferation marker, and established Ki67 is a biomarker of bladder cancer cells. Mulder utilized a commercially available mouse anti-Ki67 antibody to detect Ki67, labeled the detection antibody with rabbit anti-mouse antibody conjugated to HRP, and visualized bound Ki67 with diaminobenzidine (DAB), successfully detecting Ki67 as bound to tumor cell nuclei. Therefore, Mulder established that primary and secondary antibody reagents for detection of Ki67 and with DAB staining are commercially available and successfully used. Mulder teaches Ki67 is confirmed as a biomarker of bladder cancer cells, and provides motivation and reasonable expectation of success to detect Ki67 expression bladder cancer cells.
It is reiterated that the combination of cited references in the rejection provide motivation for selecting Ki67/p16 detection in urine samples of bladder tumor patients with a reasonable expectation of success for the reasons of record, and the obviousness rationale is not based solely on Mulder.
16. Applicants argue that Santos teaches evaluating p16 and Ki67 expression in fixed tissue samples by immunohistochemistry. Applicants argue that Santos does not teach staining urine cytology samples or single cell co-expression and visualization of p16/Ki67 yielding brown-stained cells with red nuclei.
17. The arguments have been considered but are not persuasive. The limitations argued by Applicants as not taught by Santos were addressed by the combination of cited references in the rejection of record. Santos teaches it is known and established that Ki67 is a cell proliferation marker present in cell nuclei of proliferating cells and is absent in resting cells (also taught by Hennig, Mulder, and WO 2004038418, “Ridder 2”). Santos established that primary and secondary antibody reagents for detection of Ki67 and with DAB staining are commercially available and successfully used. Santos teaches Ki67 is confirmed as a biomarker of bladder cancer cells.
It is reiterated that the combination of cited references in the rejection provide motivation for selecting Ki67/p16 detection in urine samples of bladder tumor patients and assigning DAB/FastRed to p16/Ki67 detection with a reasonable expectation of success for the reasons of record, and the obviousness rationale is not based solely on Santos.
18. Applicants argue that Golijanin is directed to immunostaining urine samples for CK20 and does not disclose p16 or Ki67 combination staining, or duplex chromogenic urine-cytology method yielding the brown-stained cells with red nuclei.
19. The arguments have been considered but are not persuasive. The limitations argued by Applicants as not taught by Golijanin were addressed by the combination of cited references in the rejection of record. Golijanin teaches immunocytology of cells in the urine of bladder tumor patients is non-invasive and can provide better specificity for patients with concomitant urological disorders. Golijanin demonstrates successfully conducting immunocytology with primary and secondary antibody staining of cells from bladder tumor patient urine to detect tumor antigen expression and the presence of cancer cells. Golijanin teaches assaying urine by immunocytology from both primary and recurrent bladder cancer patients previously diagnosed.
It is reiterated that the combination of cited references in the rejection provide motivation for selecting Ki67/p16 detection in urine samples of bladder tumor patients and assigning DAB/FastRed to p16/Ki67 detection with a reasonable expectation of success for the reasons of record, and the obviousness rationale is not based solely on Golijanin.
20. Applicants argue that Toma teaches assaying urine samples using commercially available Immunocyt/Uct+ test that utilized monoclonal antibodies against tumor-associated antigens, however Toma does not teach using antibodies against p16 an dKi67 alone or combination, or teach duplex chromogenic urine-cytology method yielding the brown-stained cells with red nuclei.
21. The arguments have been considered but are not persuasive. The limitations argued by Applicants as not taught by Toma were addressed by the combination of cited references in the rejection of record. Toma teaches analyzing urine samples for bladder cancer cells expressing multiple tumor biomarkers is a non-invasive method for bladder cancer diagnosis/surveillance, and is useful to supplement urinary cytology; Toma demonstrates successfully simultaneously assaying multiple tumor biomarker expression in cells of urine samples from bladder tumor patients, utilizing a commercial assay comprising antibodies that bind to the biomarker proteins; and Toma demonstrates the urine immunocytology assay successfully supplements urinary cytology to aid in cancer diagnosis and detecting the presence of bladder cancer cells during surveillance.
It is reiterated that the combination of cited references in the rejection provide motivation for selecting Ki67/p16 detection in urine samples of bladder tumor patients and assigning DAB/FastRed to p16/Ki67 detection with a reasonable expectation of success for the reasons of record, and the obviousness rationale is not based solely on Toma.
22. Applicants argue that Yin and Santos disclose staining p16 and Ki67 on tissue samples and not urine cytology samples, and one would not reasonably or predictably extrapolate the detection of p16 and Ki67 from bladder cancer tissue to detection to detection in urine cytology from bladder cancer patients. Applicants argue that the fact p16 and Ki67 are stained in bladder cancer tissues does not support the conclusion they can be reliably stained in a urine cytology sample or by dual-staining to visualize co-expression in a single cell. Applicants argue that the cited references demonstrating immunohistochemical staining of p16 or Ki67 in bladder cancer cells does not predictably extrapolate to successfully staining for p16 and Ki67 in bladder cancer cells in a urine/cytology sample, or simultaneously staining p16 and Ki67 in a single cell.
23. The arguments have been considered but are not persuasive. The cited references provide numerous examples of successfully staining for p16 and/or Ki67 individually or simultaneously in cancer cells and bladder cancer cells using commercially available anti-p16, anti-Ki67, and secondary antibodies, as well as demonstrating successful dual staining of single cells with commercially available antibodies and DAB/FastRed, providing a reasonable expectation of success to arrive at the claimed invention. Applicants have provided no reason or evidence why the commercially available antibodies and staining reagents would not function the same to bind and stain the same proteins in the same cancer cells that are located in either a tissue sample or a urine/cytology sample. The cited references also demonstrated successfully simultaneously staining urine cytology samples for multiple antigens with multiple antibodies, as well as successfully simultaneously staining cancer cells in a liquid cytology sample for p16 and Ki67 with DAB/FastRed to yield brown-stained cells with red-stained nuclei. The detecting reagents are commercially and readily available, and their functions in immunoassays/ protein detection together or separately in cancer cells, regardless of cell sample source, are well established and predictable. WO 2004038418 Ridder alone demonstrates the predictable success of staining cancer cells simultaneously with commercially available anti-p16, anti-Ki67, secondary antibodies, and DAB/FastRed in order to visualize brown stained cells with red stained nuclei. Therefore, contrary to arguments, the cited prior art provides ample evidence for a reasonable expectation of success to conduct the p16/Ki67 DAB/FastRed dual stain assay on urine cytology samples containing bladder cancer cells.
24. Applicants continue to reiterate several arguments in their remarks on pages 18-21. All of these arguments were addressed in the responses above. With regard to Ridder 2 (WO 2004038418 Ridder), Applicants argue that Ridder 2 discloses detecting p16 and Ki67 in cervical cancer tissue and cytology samples but does not each assaying urine cytology samples from bladder tumor patients. Applicants argue that Ridder 2 does not provide motivation to select assaying the combination of p16 and Ki67 together and in urine samples from bladder tumor patients.
25. The arguments have been considered but are not persuasive. Ridder 2 demonstrates successfully practicing the claimed dual-stain immunoassay for p16/Ki67 proteins using commercially available anti-p16 antibody, anti-Ki67 antibody, secondary antibodies, and DAB/FastRed, and visualizing brown-stained cells with red-stained nuclei, and on a liquid biological cytology sample. Therefore, the success of practicing this immunoassay on liquid cytology cancer cell samples and visualizing brown stained cells with red stained nucleic is known and established. The combined cited references provide motivation and reasonable expectation of success to practice this same immunoassay on urine cytology samples from bladder cancer patients for the reasons of record, and the obviousness rationale is not based solely on Ridder 2.
26. Applicants argue that Pressman does not teach detecting p16 as a biomarker for bladder cancer, let alone detecting combined p16/Ki67 using DAB/FastRed for visualization in a single cell as claimed. Applicants argue Samuelsson does not teach chromogenic immnocytochemcial assay, dual-format chromogenic assay in urine for p16/Ki67 co-expression in a single cell, or the claimed morphological staining pattern of brown stained cells with red nuclei. Applicants argue Samuelsson’s disclosure of flow cytometry for detecting unrelated markers does not render obvious these deficiencies.
27. The arguments have been considered but are not persuasive. The limitations argued by Applicants as not taught by Pressman and Samuelsson were addressed by the combination of cited references in the rejection of record. Pressman and Samuelsson provide motivation and reasonable expectation of success to add a step of flow cytometry to the method of the combined references for the reasons of record.
New Rejection
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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28. Claims 1, 5, 9-17, 19, and 20 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-5 of U.S. Patent No. 8,728,745 in view of US Patent 7,452,727, Hennig et al, published 2002; EP1628135, Ridder et al, published August 20, 2004; Yin et al (Human Pathology, 2008, 39:527-535; Epub Jan 2008), Mulder et al (Journal of Pathology, 1992, 166:37-43); Santos et al (Int. J. Cancer, 2003, 105:267-272); Toma et al (World J Urol. 2004, 22:145-149); Golijanin et al (The Journal of Urology, 2000, 164:1922-1925); Furth et al (Neoplasia, 2006, 8:429-436); van der Loos (Journal of Histochemistry & Cytochemistry, April 2008, 56:313-328); and WO 2004038418, Ridder et al, published May 2004; US Patent Application Publication 2003/0190602, Pressman et al; and Samuelsson et al (INFECTION AND IMMUNITY, June 2004, 72: p. 3179–3186).
The US Patent claims:
1. A method for predicting a potential for tumors of the urinary system for aggressive growth and/or a risk to progress to high grade cancer from low grade cancer, comprising:
obtaining a tumor sample from the urinary system of a subject;
measuring by a cell based detection procedure whether at least one single cell of the sample simultaneously overexpresses p16INK4a and expresses Ki67; and
determining that the tumors have a potential for aggressive growth and/or a risk to progress to high grade cancer when there is at least one single cell in the sample having the simultaneous presence of overexpression of p16INK4a and the expression of Ki67.
2. The method of claim 1, wherein the cell based detection procedure is immunohistochemistry or immunocytochemistry.
3. The method of claim 1, wherein the tumor is bladder cancer.
4. The method of claim 1, said measuring uses antibodies against p16INK4a and Ki67.
5. The method of claim 1, wherein the sample is a tissue sample or a cell sample.
Assaying urine samples from bladder tumor patients for Ki67/p16
Although the US Patent claims assaying tumors from the urinary system for simultaneous overexpression of p16 and Ki67 in single cells to assess aggressive growth and/or risk to progression to high grade cancer, and wherein the tumor is bladder cancer, the US Patent does not claim assaying cancer cells in urine samples.
Hennig teaches as set forth above, suggesting assaying urine samples.
EP1628135 Ridder teaches as set forth above.
Yin teaches as set forth above.
Mulder teaches as set forth above.
Santos teaches as set forth above.
Toma teaches as set forth above.
Golijanin teaches as set forth above.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to assay urine from bladder tumor patients for cells simultaneously expressing Ki67 and p16 in the method of the US Patent. One would have been motivated to because: (1) the US Patent claims assessing bladder tumor cell biomarker p16/Ki67 expression to determine cancer aggressiveness or grade progression; (2) Hennig suggests cell samples assayed for cancer biomarkers p16 and Ki67 include urine from patients with tumors; (3) EP1628135 Ridder suggests conducting an immunoassay for p16 or Ki67 expression in cells of a liquid-based cytology (LBC) sample, such as a urine sample, from cancer patients, including bladder cancer patients; (4) Yin demonstrates p16 is a known, expressed biomarker of cancer in bladder tumor cells and precursor lesions; (5) Mulder establishes that Ki67 positive-stained cells are increased in bladder tumor tissue samples and correlated with increased tumor stage and increased grade; (6) Santos teaches that Ki67 is a known cell proliferation marker present in cell nuclei of proliferating cells and is absent in resting cells, and demonstrates Ki67 expression is increased in bladder tumor cells; (7) Toma teaches analyzing urine samples for bladder cancer cells expressing multiple tumor biomarkers is a non-invasive method for bladder cancer diagnosis/surveillance, and is useful to supplement urinary cytology; and (8) Golijanin teaches immunocytology of cells in the urine of bladder tumor patients is non-invasive and can provide better specificity for patients with concomitant urological disorders. One would have a reasonable expectation of success to conduct the method of the US Patent on urine samples from bladder tumor patients given: (1) EP1628135 Ridder demonstrates successfully conducting an immunoassay for p16INK4a protein expression on the cells of LBC samples, including urine samples from patients diagnosed with bladder tumors, wherein the immunoassay utilized commercially available mouse monoclonal anti-p16INK4a antibodies; (2) Yin, Mulder, and Santos demonstrate successfully detecting p16 or Ki67 expression in bladder tumor cells utilizing commercially available monoclonal antibodies and labeled secondary antibodies, and establish they are both biomarkers associated with bladder cancer; (3) Toma demonstrates successfully simultaneously assaying multiple tumor biomarker expression in cells of urine samples from bladder tumor patients, utilizing a commercial assay comprising antibodies that bind to the biomarker proteins, and Toma demonstrates the urine immunocytology assay successfully supplements urinary cytology to aid in cancer diagnosis and detecting the presence of bladder cancer cells during surveillance; and (4) Golijanin demonstrates successfully conducting immunocytology with primary and secondary antibody staining of cells from bladder tumor patient urine to detect tumor antigen expression and the presence of cancer cells.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to assay urine from bladder tumor patients previously diagnosed with bladder cancer. One would have been motivated to, and have a reasonable expectation of success to, given: (1) the US Patent claims the subject tested has a bladder cancer tumor and claims assessing p16/Ki67 expression to determine cancer aggressiveness or grade progression; (2) Yin, Mulder, and Santos demonstrate successfully detecting p16 or Ki67 expression in bladder tumor cells, and establish they are biomarkers associated with the presence of bladder cancer, progression, and prognosis in patients already diagnosed with bladder cancer; (3) Toma teaches analyzing bladder tumor cells in urine for simultaneous tumor biomarker expression functions in diagnosis and surveillance of disease or follow-up after bladder cancer diagnosis; and (4) Golijanin teaches assaying urine by immunocytology from both primary and recurrent bladder cancer patients previously diagnosed.
Antibodies, their associated chromogens, and staining procedure:
The US Patent does not claim the immunocytochemistry assay to assess simultaneous presence of overexpression of p16INK4a and the expression of Ki67 in a single cell uses DAB and Fast Red stains, does not assign the DAB chromogen reporter system to detection of bound anti-p16 antibody and assign the Fast Red chromogen reporter system to detection of bound Ki67 antibody in the sample, resulting in brown stained cells (DAB) with red stained nuclei (Fast Red). The US Patent does not claim the anti-Ki67 antibody is a rabbit monoclonal antibody or the anti-p16INK4a antibody that is a mouse monoclonal antibody and detecting them with the respective anti-rabbit and anti-mouse secondary antibodies.
Hennig teaches, in detail, the necessary steps and reagents to practice a method for staining and detecting Ki67 and p16 simultaneously with antibodies and DAB and Fast Red first and second chromogens, as set forth above.
Furth teaches as set forth above.
van der Loos teaches as set forth above.
WO 2004038418 Ridder teaches successfully simultaneously detecting p16 and Ki67 expression in a single cancer cell to visualize brown-stained cells with red-stained nuclei in immunocytology, and using commercially available primary and secondary antibodies, as set forth above.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to utilize the DAB chromogen reporter system for detection of bound anti-p16 antibody and utilize the Fast Red chromogen reporter system for detection of bound Ki67 antibody to visualize simultaneous p16 and Ki67 expression in a single cell, in the immunoassay of the US Patent. One would have been motivated to in order to simultaneously detect Ki67 and p16 expression within single cells as claimed by the US Patent. One of ordinary skill in the art would have a reasonable expectation of success, given the detailed protocol to practice the method provided by Hennig in the Examples and patented claims, and given WO 2004038418 Ridder exemplifies successfully simultaneously staining p16INK4a and Ki67 in cell samples by utilizing commercially available mouse anti-human p16INK4a monoclonal primary antibody (MTM); goat anti-mouse secondary antibody (Peroxidase/DakoCytomation), and DAB visualization, and utilizing commercially available rabbit anti-human Ki67 antibody, goat anti-rabbit secondary antibody (Alkaline phosphatase labeled / Dako Cytomation), and visualized with FastRed, resulting in brown stained cells with red stained nuclei.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to utilize a rabbit monoclonal anti-Ki67 antibody and a mouse monoclonal anti-p16INK4a antibody and corresponding anti-mouse and anti-rabbit secondary antibodies for dual protein detection in the method of the US Patent. One would have been motivated to: (1) because the US Patent claims using anti-p16 and anti-Ki67 antibodies in their detection assay; (2) because Hennig, Furth, van der Loos, and WO 2004038418 Ridder recognize and teach the known use of secondary antibodies specific to the primary antibodies for detection; (3) because van der Loos explain the known and established method for dual staining and visualization of proteins utilizing primary and secondary antibodies with HRP/DAB and AP/Fast Red; and (4) because rabbit monoclonal anti-Ki67 antibody and mouse monoclonal anti-p16INK4a antibody are commercially, readily available for immunoassays. One of ordinary skill in the art would have a reasonable expectation of success given the cited art demonstrates rabbit monoclonal anti-Ki67 antibody and mouse monoclonal anti-p16INK4a antibody successfully detect p16INK4a and Ki67 proteins in patient cell samples, and given methods for dual staining and visualization of proteins utilizing mouse/rabbit primary antibodies and secondary antibodies with HRP/DAB and AP/Fast Red signaling are established.
Automated flow cytometry
The US Patent does not claim and combined references cited above do not disclose additionally performing automated flow cytometry to analyze the bladder tumor cells (with regard to instant claims 19 and 20).
Pressman teaches as set forth above.
Samuelsson teaches as set forth above.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to add a step of flow cytometry to the method of the US Patent and combined references. One would have been motivated, and have a reasonable expectation of success to, given Hennig and Pressman suggest automated flow cytometry methods can be used to detect multiple biomarker expression in cells, Pressman teaches such methods in the art are known, and Samuelsson demonstrates successfully detecting and analyzing cells from urine samples that are expressing two different biomarkers by using commercially available automated flow cytometry.
29. Conclusion: No claim is allowed.
30. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642