**4. Prospect of using Kank family genes in genetic diagnosis and gene therapy for renal tumors**

### **4.1. Future diagnostics for RCC**

The lack of clinical impact of the current diagnostic markers for RCC apparently requires progress in methodology, biology and pathology (Stewart et al., 2011). The progress in methodology needs the quality of the methods to satisfy the specificity, stability and biological relevance of the markers for diagnosis. For this, sufficient numbers, tens to thousands, of markers would be needed and such markers could be obtained only through cellular signaling analyses. There are quite a number of potential protein and genetic markers for diagnosis and therapeutic targets of RCC based on the information of signal transduction (see Section 2), and more information would be added in the future. While sampling is easier for DNA and RNAbased assays, protein assays such as immunohistochemistry and more advanced massspectrometry techniques have problems of contamination and degradation/modification at sampling and processing. In immunohistochemisty, protein cross-linking at the preparation steps disturbs antibody binding. Sampling of homogenously expressed proteins is crucial for the stability of assays, but would not be possible for most sampling cases as the tissue itself is not homogenous. However, diagnosis even for such cases could be possible with markers sufficiently distinguishing heterogenously expressed proteins in different parts of the diseased tissue. In all cases, a statistical significance analysis should be included as a standard evaluation step for quality control of multi-marker systems such as DNA microarrays (Shi et al., 2010).

Biologically relevant markers will be made available in the future based on the analysis of signal transduction, because, as shown in Fig. 1 (Section 2), there are a number of markers available even within a single signaling pathway and there are sufficient numbers of different pathways affected by the disease, which will contribute to the stability of assays. As discussed, the VHL and mTOR pathways have drawn much attentions to prognosis/diagnosis and therapeutic targets for RCC, but there are more pathways such as the Myc and FLCN pathways and pathways related to VEGF, PDGF and TGFα, and some are specific to subtypes of RCC (Linehan et al., 2010; Allory et al., 2011).

Meanwhile, pathologically relevant markers will also be made available in the future, although the situation is different from other technologies due to the technical limit in the number of markers to examine simultaneously.

### **4.2. A new fluorescence-based immunohistochemical technique**

investigation of this pathway will contribute to a new molecular targeting therapy for RCC (Suwaki et al., 2011). The difference in *VHL* mutations among the RCC histological subtypes suggests a difference in carcinogenesis for each histological subtype, though the origin of the

Given that the alteration of *Kank1* expression occurred at the early stage of carcinogenesis, our findings that *Kank1* expression differed among the histological subtypes of RCC might reflect a difference in cancer development (Kim et al., 2005). In clear cell RCC, the loss of *Kank1* expression occurred at a high rate in the lower grade tumors, and the expression was reoc‐ curred as the malignant grade increased. Although the reason for this is not clear, it is presumed that epigenetic modifications such as methylation might have been removed when the malignant grade increased, and consequently, the expression reoccurred (Kisseljova and Kisseljov, 2005). There was no difference in *Kank1* expression between the samples obtained from the groups of patients who survived or not (Table 2). This may reflect the fact that histological grade does not necessarily contribute to clinical outcome, but clinical stage (i.e. the presence of metastasis) is more crucial to obtaining a good prognosis (RCC patients diagnosed at the early stage have more than a 90% five year survival rate) (Lane and Kattan, 2008). The discordance of T stage (tumor size) and the malignant grade on *Kank1* expression could also be supposed for the same reason. A similar result was found for the expression of *CDKN2A* encoding a growth suppressor protein, which is located at 9p21 and close to *Kank1* (9p24) (unpublished data). Although the loss of *Kank1* expression resulted in increased proliferation and poor differentiation in *in vitro* study (Sarkar et al., 2002), our results about the *in vivo* expression of *Kank1* in clinical cases proved that reduced expression does not necessarily reflect a high grade malignancy or poor clinical outcome. These contradictory experimental and clinical results are very interesting, because they suggest that malignant transformation of a normal renal tubular cell has many genetic alterations and clinical outcome

**4. Prospect of using Kank family genes in genetic diagnosis and gene**

The lack of clinical impact of the current diagnostic markers for RCC apparently requires progress in methodology, biology and pathology (Stewart et al., 2011). The progress in methodology needs the quality of the methods to satisfy the specificity, stability and biological relevance of the markers for diagnosis. For this, sufficient numbers, tens to thousands, of markers would be needed and such markers could be obtained only through cellular signaling analyses. There are quite a number of potential protein and genetic markers for diagnosis and therapeutic targets of RCC based on the information of signal transduction (see Section 2), and more information would be added in the future. While sampling is easier for DNA and RNAbased assays, protein assays such as immunohistochemistry and more advanced massspectrometry techniques have problems of contamination and degradation/modification at

cancer is always a renal tubular cell.

20 Renal Tumor

is contributed to by many factors in RCC.

**therapy for renal tumors**

**4.1. Future diagnostics for RCC**

One obstacle to improving immunohistochemistry is the availability of markers. Immu‐ nostaining is a relatively simple technique and thus can be used in unequipped laborato‐ ries and hospitals, because the preparation, storage and handling of samples are relatively simple. However, ordinary immunostaining is based on single-dye (or singlemarker) colorimetric techniques such as the alkaline phosphatase-based method. This is because of a lack of multi-dye (or multi-marker) colorimetric techniques due to expen‐ sive devices and, especially, inavailability of stable fluorescent dyes. Fluorescent dyes have been used in many technologies although this has not happened yet in immunohis‐ tochemistry because of the lack of their sufficient stability. Stable fluorescent dyes are thus needed for progress in immunohistochemistry.

We reported applications of a new fluorescent dye, Fluolid, for DNA microarray assays and immunohistochemistry (Zhu et al., 2011). Fluolid dyes, including Fluolid-Orange, show stability against heat and excess light compared with other dyes (Fig. 6) and thus can be stored for more than a year without losing fluorescence (data not shown). So, multi-color immuno‐ histochemistry with stable fluorescent dyes will change the pathological diagnostics in several ways: long-term storage of stained sections, simultaneous multi-marker detection and handling of fluorescently stained sections. Heat and light stable fluorescent dyes will enable us to store fluorescently stained sections at room temperature for a long time, which will be important for follow-up studies by microdissection of specific regions.

**Acknowledgements**

**Author details**

Ryoiti Kiyama1\*, Yun Zhu1

nology, Ibaraki, Japan

**References**

Sports, Science and Technology, Japan.

\*Address all correspondence to: kiyama.r@aist.go.jp

This research has been supported by a Special Coordination Fund for Promoting Science and Technology (Encouraging Development of Strategic Research Centers), a Knowledge Cluster Initiative program and a Grant-in-aid for Basic Areas from the Ministry of Education, Culture,

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 23

1 Biomedical Research Institute, National Institute of Advanced Industrial Science and Tech‐

[1] Allory, Y., Culine, S., and de la Taille, A. (2011). Kidney cancer pathology in the new

[2] Arai, E., and Kanai, Y. (2011). Genetic and epigenetic alterations during renal carcino‐

[3] Baldewijns, M.M., van Vlodrop, I.J., Schouten, L.J., Soetekouw, P.M., de Bruïne, A.P., and van Engeland, M. (2008). Genetics and epigenetics of renal cell cancer. Biochim.

[4] Beroukhim, R., Brunet, J.P., Di Napoli, A., Mertz, K.D., Seeley, A., Pires, M.M., Linhart, D., Worrell, R.A., Moch, H., Rubin, M.A., Sellers, W.R., Meyerson, M., Linehan, W.M., Kaelin, W.G. Jr., and Signoretti, S. (2009). Patterns of gene expression and copy-number alterations in von-hippel lindau disease-associated and sporadic clear cell carcinoma

[5] Bilim, V., Ougolkov, A., Yuuki, K., Naito, S., Kawazoe, H., Muto, A., Oya, M., Billadeau, D., Motoyama, T., and Tomita,Y. (2009). Glycogen synthase kinase-3: a new therapeutic

[6] Boggetti, B., Jasik, J., Takamiya, M., Strähle, U., Reugels, A.M., and Campos-Ortega, J.A. (2012). NBP, a zebrafish homolog of human Kank3, is a novel Numb interactor

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and Tei-ichiro Aoyagi2

2 Ibaraki Medical Center, Tokyo Medical University, Ibaraki, Japan

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**Figure 6.** Stability of fluorescently labeled IgG. (A) Photostability of Fluolid-Orange- or Cy3-labeled IgG under irradia‐ tion for up to 150 sec with a laser beam at 488 nm. (B) Heat stability. Fluolid-Orange- or Cy3-labeled IgG was left in an environment of 100ºC and fluorescence was measured every 30 min. For details, see Zhu et al. (2011).

#### **4.3. Future therapeutics**

As discussed in Section 4.1, future diagnosis will be based on sufficient numbers of protein markers possibly obtained from signal transduction pathways, which will give a statistically significant decision even for cases where no decisive markers, such as disease-causing mutations or constitutive active proteins, are available. In the case of future therapeutics, multiple targets will also be considered to be an effective strategy. Signal transduction-based targeted therapeutics have already been developed for some diseases and drugs such as imatinib or Gleevec/Glivec, a small molecule inhibitor against activated tyrosine kinase activity by the Bcr-Abl fusion gene used for the treatment of chronic myelogenous leukemia, are available (Radford, 2002). Other monoclonal antibody-based drugs such as trastuzumab or Herceptin, which blocks a growth factor receptor HER2/neu (c-erbB-2) to treat breast cancer, and panitumumab or Vectibix, which blocks HER1 to treat colorectal cancer, have been developed based on signal-transduction. Although these drugs are effective, continuous use will sometimes generate drug-resistant cancer (Schenone et al., 2011). So, treatment with multiple targeting drugs will be important in future therapeutics and the same is true for the matched diagnostics about multiple targets.
