Toshiaki Saida

Tumor regression by combined immunotherapy and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma

Immunotherapy (IT) has become an accepted therapeutic modality. We previously reported that intracellular hyperthermia (IH) using magnetic nanoparticles induces antitumor immunity. We undertook these studies in order to study the combined effects of IT and IH on melanoma. Magnetite cationic liposomes (MCLs) have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs were injected into a B16 melanoma nodule in C57BL/6 mice, which were subjected to AMF for 30 min. The temperature at the tumor reached 43°C and was maintained by controlling the magnetic field intensity. At 24 h after IH, interleukin‐2 (IL‐2) or granulocyte macrophage‐colony stimulating factor (GM‐CSF) was injected directly into the melanoma. Mice were divided into six groups: group I (control), group II (IH), group III (IL‐2), group IV (GM‐CSF), group V (IH+IL‐2), and group VI (IH+GM‐CSF). Complete regression of tumors was observed in mice of groups V and VI (75% (6/ 8) and 40% (4/10) of the mice, respectively), while no tumor regression was observed in mice of the other groups. This study supports the combined use of IT and IH using MCLs in patients with advanced malignancies. (Cancer Sci 2003; 94: 308–313)

Intratumoral injection of immature dendritic cells enhances antitumor effect of hyperthermia using magnetic nanoparticles

Dendritic cells (DCs) are potent antigen‐presenting cells that play a pivotal role in regulating immune responses in cancer and have recently been shown to be activated by heat shock proteins (HSPs). We previously reported that HSP70 expression after hyperthermia induces antitumor immunity. Our hyperthermia system using magnetite cationic liposomes (MCLs) induced necrotic cell death that was correlated with HSP70 release. In the present study, we investigated the therapeutic effects of DC therapy combined with MCL‐induced hyperthermia on mouse melanoma. In an in vitro study, when immature DCs were pulsed with mouse B16 melanoma cells heated at 43°C, major histocompatibility complex (MHC) class I/II, costimulatory molecules CD80/CD86 and CCR7 in the DCs were upregulated, thus resulting in DC maturation. C57BL/6 mice bearing a melanoma nodule were subjected to combination therapy using hyperthermia and DC immunotherapy in vivo by means of tumor‐specific hyperthermia using MCLs and directly injected immature DCs. Mice were divided into 4 groups: group I (control), group II (hyperthermia), group III (DC therapy) and group IV (hyperthermia + DC therapy). Complete regression of tumors was observed in 60% of mice in group IV, while no tumor regression was seen among mice in the other groups. Increased cytotoxic T lymphocyte (CTL) and natural killer (NK) activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of B16 melanoma cells. This study has important implications for the application of MCL‐induced hyperthermia plus DC therapy in patients with advanced malignancies as a novel cancer therapy.

Early Acral Melanoma In Situ: Correlation Between the Parallel Ridge Pattern on Dermoscopy and Microscopic Features

In non-white populations, acral skin is the most prevalent site of malignant melanoma. Early melanomas of this anatomic site are often misdiagnosed as melanocytic nevi, which are not uncommon on acral skin. In fact, clinical and/or histopathological features of melanocytic nevi occasionally mimic those of early acral melanoma and vice versa, and thus differentiation of early acral melanoma from melanocytic nevus is sometimes very difficult for clinicians as well as for histopathologists. Our dermoscopic investigation has revealed that the parallel ridge pattern, a band-like pigmentation on the ridges of the skin markings, is highly specific to malignant melanoma in situ on acral volar skin. In the present study, we reviewed 22 acral melanocytic lesions that showed the parallel ridge pattern on dermoscopy but had very subtle clinical and/or histopathological presentations. We diagnosed 20 of them as early melanoma in situ by careful histopathological examination, which revealed histopathological features very similar to those seen in macular portions of overt acral melanoma, but fundamentally different from features found in melanocytic nevi on acral skin. In correspondence with their dermoscopic pattern, in these early lesions of acral melanomas, proliferation of solitary arranged melanocytes was mainly detected in the crista profunda intermedia, the epidermal rete ridge underlying the ridge of the skin marking. The two remaining lesions were diagnosed as possible cases of acquired melanocytic nevus because of the formation of well-demarcated nests of melanocytes in the epidermal rete ridges. We propose that a finding of preferential proliferation of solitary arranged melanocytes in the crista profunda intermedia is an important clue for the histopathological diagnosis of early phases of acral melanoma.

A New Melanoma Antigen Fatty Acid-Binding Protein 7, Involved in Proliferation and Invasion, Is a Potential Target for Immunotherapy and Molecular Target Therapy

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

The place of lymphatic mapping and sentinel node biopsy in oncology.

The techniques of sentinel lymphatic mapping (LM) and sentinel lymph node biopsy (SLNB) have become almost routine for the staging of clinically node-negative patients with high-risk cutaneous melanoma. The techniques are also widely applied to staging of the axilla in breast cancer. Investigations of the use of LM and SLNB for other solid malignancies have also shown promise. LM/SLNB requires a multidisciplinary effort involving experienced surgeons, nuclear medicine physicians, and surgical pathologists. The techniques require a learning curve for all involved personnel, requiring experience with at least 30 cases followed by complete nodal dissection after SLNB to achieve full competency. Surgical pathologists play a pivotal role in determining optimum sentinel node analysis. The techniques have lower morbidity and greater accuracy than traditional complete regional node dissection. Pathologists are receiving increasing numbers of SLN specimens and are expected to evaluate the results of the application of the LM/SLNB techniques to a range of solid tumors. We have reviewed LM/SLNB in regard to melanoma and breast cancer and other types of malignancies. The techniques have much to offer, but despite their seeming simplicity, need considerable technical skill and clinical judgment if they are to be effectively applied. They also provide unique opportunities for basic and translational research.

Carbonic Anhydrase II Is a Tumor Vessel Endothelium ^ Associated Antigen Targeted by Dendritic Cell Therapy

Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium–associated antigen in melanoma and other cancers, and elicitation of serum anti–CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.

Histogenesis of congenital and acquired melanocytic nevi: a unifying concept.

To the Editor: The histogenesis of melanocytic nevi has been debated since Unna proposed the theory of ‘‘Abtropfung’’ more than 100 years ago. In the midtwentieth century, Masson proposed the ‘‘dual theory’’ that the upper component of melanocytic nevi was derived from epidermal melanocytes, whereas the lower dermal component came from Schwann cells. Kawamura’s theory published in 1956 introduced the concept that embryonically misdirected cells of the neural crest lineage were the source of nevus cells; he believed the misdirected cells to have defective differentiation potential for both melanocytes and Schwann cells. Combining the theories proposed by Masson and by Kawamura, Mishima proposed a new theory of histogenesis of melanocytic nevi. He assumed 2 kinds of nevoblasts, namely melanocytic and schwannian nevoblasts, as precursor cells of various types of nevi. In the 1980s, Cramer proposed the concept of ‘‘Hochsteigerung’’ and insisted that melanocytic stem cells, located in the peripheral nerve sheath, migrate to the epidermis and produce the junctional component of melanocytic nevus. In 1990, Magana-Garcia and Ackerman voiced the opinion that nevus cells including neuroid cells in the deeper portion were nothing but melanocytes. In 1995, Happle proposed an important concept of nevogenesis. His definition of nevi is as follows: ‘‘Nevi are visible, circumscribed, long-lasting lesions of the skin and the neighboring mucosa, reflecting genetic mosaicism. With the exception of melanocytic nevi, they do not show neoplastic growth. They never show malignant neoplasia.’’ This concept seems to well explain various types of nevi; for example, the distribution along the Blaschko’s lines of mutated keratinocytes in fetal skin may produce linear, systematic lesions of epidermal nevi. However, melanocytic nevi, particularly acquired melanocytic nevi, challenge Happle’s concept. Acquired melanocytic nevi appear as solitary lesions anywhere on the body and develop even in late life. In addition, exogenous factors such as exposure to sunlight are reported to induce this type of nevus. These features are inconsistent with Happle’s concept of nevi. He tried to evade this problem by stating that ‘‘with the exception of melanocytic nevi’’ in his definition of nevus. But, why does his concept face trouble with melanocytic nevi? I simply think it is because a ‘‘melanocytic nevus’’ is not a nevus by his definition. I believe an acquired melanocytic nevus is not a nevus but a benign neoplasm of an epidermal melanocyte. The HUMARA (human androgen receptor) gene assay has revealed the clonality of nevus cells, supporting the neoplastic nature of acquired melanocytic nevi. v-raf murine sarcoma viral oncogene homolog B1 (BRAF) is a candidate gene responsible for the neoplastic growth of melanocytic nevus. Recent molecular investigations have revealed that V600E mutation in the BRAF gene is frequently detected in acquired melanocytic nevi (70% to 90%) and in malignant melanoma (53% to 80%). In our recent study, the BRAF mutation was frequently detected in acquired melanocytic nevi affecting acral skin and also in small congenital melanocytic nevi. BRAF serves as a key signal transducer in the RAS/RAF/MAPK signaling pathway, which regulates cell growth and differentiation. Growth of benign neoplasms is limited. By what mechanism does the proliferation of the BRAF-mutated melanocytes stop? The concept of oncogene-induced senescence could explain the limited growth. The proliferation of BRAF-mutated melanocytes is arrested after limited cycles of cell division if CDKN2A, which encodes p16 is intact in the cells. In fact, this type of senescence, which induces p16 and senescence-associated b-galactosidase, was demonstrated in the tissues of melanocytic nevi. In contrast, in malignant melanoma, oncogene-induced senescence may not occur since CDKN2A is often deficient in melanoma cells. According to Blewitt’s mathematical assumption, melanocytic nevi may be induced as a consequence of single spontaneous genetic mutations in a melanocyte-lineage cell (melanocyte or melanoblast). According to his calculation, a single stem cell of melanocyte doubles a total of 31 times to cover the entire epidermis of the whole body. He suggests that a mutation in melanocytes arises during the last 2 doubling cycles, which occur after birth. BRAF would be a candidate gene suggested in Blewitt’s assumption and could be responsible for the genesis of acquired melanocytic nevus. Interestingly, in our recent study, the rate of the BRAF mutation was lower in medium-sized congenital melanocytic nevi. However, in these larger congenital nevi, mutation of the neuroblastoma v-ras oncogene homolog gene was reported, which may be responsible for the proliferation of melanoblasts. Benign transformed melanocytes (nevus cells) become somewhat free from regulation by surrounding keratinocytes and initially proliferate as solitary units at the dermoepidermal junction, which produces a lentigo simplex. In due course, nests of melanocytes (nevus cells) form at the dermo-epidermal junction, and the lesion becomes a junctional nevus. In time, some of the melanocytes (nevus cells) enter the dermis, probably as a result of continuous proliferation and accumulation of melanocytes at the junction, thereby a compound nevus is established. Ultraviolet light may be the most potent stimulatory factor for the proliferation of melanocytes, and thus the proliferation rate depends on the anatomic site; there is a higher rate in the nevi on sun-exposed sites than that on sun-protected sites. The LETTER TO THE EDITOR