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Protein kinase Cγ antibodies and paraneoplastic cerebellar degeneration
Journal of Neuroimmunology 256 (2013) 91–93
Contents lists available at SciVerse ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Protein kinase Cγ antibodies and paraneoplastic cerebellar degeneration Romana Höftberger a, b, 1, Gabor G. Kovacs c, 1, Lidia Sabater a, b, Peter Nagy d, Gergely Racz d, Rosa Miquel e, Josep Dalmau f, g, Francesc Graus a, b,⁎ a
Service of Neurology, Hospital Clínic, Universitat de Barcelona, Barcelona, Spain Institut d´Investigació Biomèdica August Pi i Suyer (IDIBAPS), Barcelona, Spain Institute of Neurology, Medical University of Vienna, Austria d First Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary e Pathology Department, Hospital Clínic, IDIBAPS, Barcelona, Spain f Institució Catalana de Recerca i Estudis Avançats (ICREA), IDIBAPS, Hospital Clínic, Barcelona, Spain g Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA b c
a r t i c l e
i n f o
Article history: Received 19 October 2012 Received in revised form 3 December 2012 Accepted 6 December 2012 Keywords: PKC gamma Paraneoplastic cerebellar degeneration Onconeural antibodies Adenocarcinoma
a b s t r a c t Onconeural antibodies are diagnostic markers for paraneoplastic neurological syndromes and indicate the underlying tumor type. Recently, a new antibody against protein-kinase Cγ (PKCγ) was detected in a patient with paraneoplastic cerebellar degeneration (PCD). We report here a second patient. A 70-year-old woman presented with cerebellar ataxia, dysdiadochokinesia, and dysarthria. Her serum showed immunoreactivity against the cytoplasm, dendrites and axons of Purkinje cells in tissue-based screening, later confirmed by immunoblot and phage plaques as anti-PKCγ. Tumor search revealed an adenocarcinoma of hepatobiliary origin. Detection of PKCγ antibodies should be considered in PCD and adenocarcinoma without typical onconeural antibodies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The cerebellum is a frequent target of tumor-induced autoimmunity. The pathology of paraneoplastic cerebellar degeneration (PCD) is characterized by Purkinje cell loss, cortical degeneration of the cerebellum, and, sometimes, inflammatory infiltrates that are mostly composed of T cells (Albert et al., 1998; Aboul-Enein et al., 2008). Symptoms include subacute cerebellar gait and limb ataxia, nystagmus, and dysarthria. Usually, PCD is associated with small cell lung cancer (SCLC) or gynecological tumors and well-characterized onconeural antibodies such as Hu or Yo in serum and CSF of the patients (Graus et al., 2010). However, PCD can also be observed in patients with many different tumor types, either with or without well-characterized onconeural antibodies (Hammack et al., 1990; Aboul-Enein et al., 2008). A previous systematic analysis of clinical and immunological findings in patients with PCD and non-small cell lung cancer (NSCLC) revealed one patient with a novel antibody directed against protein kinase Cγ (PKCγ), a protein which is highly expressed in the cytoplasm of Purkinje cells (Sabater et al., 2006). The study suggested that NSCLC can associate with PCD and antibodies different from those associated with SCLC, however,
⁎ Corresponding author at: Service of Neurology, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. Tel.: +34 932275414; fax: +34 932275783. E-mail address: [email protected] (F. Graus). 1 Equal contribution. 0165-5728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2012.12.002
additional patients are necessary to confirm the link between antiPKCγ and PCD.
2. Case report We report a 70-year-old woman who presented with vertigo and vomiting in January 2003. The neurological examination showed gait and limb ataxia, dysdiadochokinesia, and dysarthria. There was no evidence of pyramidal or extrapyramidal symptoms such as chorea, dyskinesia, or dystonia, or autonomic instability. The cognitive status was normal. CSF was unremarkable. After seven months of illness, in July 2003, the patient's serum was examined for onconeural antibodies. A novel pattern of reactivity was identified on tissue-based screening with rat cerebellum, which was not compatible with that of well-characterized onconeural antibodies. The screening for cancer revealed a tumor in the gall bladder that infiltrated the liver. Histopathology showed a papillary adenocarcinoma of hepatobiliary origin. Cranial CT excluded brain metastases. The patient was lost after cancer diagnosis. Nine years later, the archived serum, stored at −20 °C, was reevaluated for onconeural antibodies. The tissue-based screening on rat cerebellum showed a strong staining of Purkinje cell dendrites, axons and synaptic terminals in the cerebellar nuclei, identical to the pattern recently described for PKCγ antibodies (Sabater et al., 2006) (Fig. 1). No antibodies against neuronal surface antigens (NMDAR, AMPAR, GABABR, mGluR1/5, LGI1, VGCC, and Caspr2) and onconeural antigens (Hu, Yo, Ri, CV2, Tr, amphiphysin, Ma2) were identified. To further characterize the antibody,
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R. Höftberger et al. / Journal of Neuroimmunology 256 (2013) 91–93
Fig. 1. Immunohistochemistry of rat cerebellum and tumor biopsy. (A, B) PKCγ is strongly positive in Purkinje cell cytoplasm, axons (arrows, A), and dendrites (arrowhead, B) of rat cerebellum (rectangle in A enlarged in B). (C) Neuron of the cerebellar nucleus showing intensive labeling of synaptic terminals (arrows). (D, E) Tumor biopsy reveals a papillary adenocarcinoma with expression of CK20 (D) and PKCγ (E).
an immunoblot with electrophoretically separated protein extracts of rat cerebellum was incubated either with patient's serum, commercial PKCγ antibody (Santa Cruz Biotechnology Inc., CA, USA), or normal human serum. The patient's serum recognized a single band of ~80 kDa. An identical band was detected using side by side the commercial PKCγ antibody whereas the control serum remained negative (Fig. 2A). To confirm the identity of the antigen as PKCγ, phage plaques expressing PKCγ were used as previously reported (Sabater et al., 2006). Briefly, filters with purified phage plaques either expressing PKCγ or irrelevant Escherichia coli proteins were incubated with patient's serum, the
commercial PKCγ antibody, or normal human serum. The PKCγ expressing phage plaques showed strong reactivity with the patient's serum and the commercial antibody, whereas normal human serum remained negative (Fig. 2B). As the previously published patient with anti-PKCγ antibodies suffered from a NSCLC, we wanted to confirm the hepatobiliary tumor origin in our patient with an expanded immunohistochemical investigation. Tumor cells revealed prominent positivity for cytokeratin 20 (CK20), a histodiagnostic marker confined to gastric and intestinal epithelia (Fig. 1D). TTF1alpha, a specific marker for adenocarcinoma of the lung, remained negative, ruling out the possibility of a
Fig. 2. (A) Immunoblot of protein extracts of rat cerebellum demonstrated a band of 80 kDa with a commercial PKCγ antibody (lane 1) and the patient's serum (lane 2), whereas the serum from a healthy individual was negative (lane 3). (B) Filters with purified phage plaques expressing PKCγ specifically reacted with a commercial PKCγ antibody (1), and the patient's serum (2) but not with serum from a healthy individual (3).
R. Höftberger et al. / Journal of Neuroimmunology 256 (2013) 91–93
metastasis from an occult NSCLC (data not shown). Finally we stained the tumor tissue with the same commercial anti-PKCγ antibody that we used for the immunoblot and phage plaques experiments. This anti-PKCγ antibody is applicable on formalin-fixed and paraffin embedded tissue and provides an excellent marker for Purkinje cell bodies and processes (data not shown) (Sabater et al., 2006). Staining of the adenocarcinoma identified a substantial number of tumor cells expressing PKCγ (Fig. 1E). 3. Discussion PCD is a classical paraneoplastic neurological syndrome (PNS) and typically related to well-characterized onconeural antibodies (Graus et al., 2010). Onconeural antibodies are helpful for the diagnosis and indicate the underlying tumor type. Unfortunately, in a considerable amount of patients with PNS, onconeural antibodies are never identifiable. Whether this is due to technical limitations or the patients actually do not have antibodies related to their neurological syndrome remains unknown. In the last years, an improvement of screening methods led to an enormous increase of newly diagnosed antibodies that could be associated with a specific spectrum of neurological diseases and a restricted subgroup of tumors. One of these antibodies was PKCγ that was found in a patient with PCD and non-small-cell lung cancer (NSCLC) (Sabater et al., 2006). PKC is a family of cytosolic enzymes that are recruited to the plasma membrane upon calcium and phospholipid dependent activation. They are involved in multiple cellular signaling pathways and depending on the isoform contribute to cancer formation and progression (Way et al., 2000). PKCγ is the neuron-specific isotype of the classical PKC family and thus exclusively found in the central nervous system but not in other tissues (Hug and Sarre, 1993). PKCγ is strongly expressed in the Purkinje cells of the cerebellum and involved in modulation of synaptic plasticity for long term potentiation and depression (Saito and Shirai, 2002). In contrast to other PKC isoforms, PKCγ seems to be less involved in tumor formation, however, some effect in malignant transformation in vivo is proposed (Mazzoni et al., 2003; Martiny-Baron and Fabbro, 2007). Here we report the second patient with anti-PKCγ antibodies and PCD and in the current case the underlying tumor was an adenocarcinoma of hepatobiliary origin. Until now, PKCγ could be associated with different types of adenocarcinomas in vitro and in vivo, including mammary carcinoma, colon adenocarcinoma, lung adenocarcinoma, and pancreatic carcinoma (Kuranami et al., 1995; Franz et al., 1996; Lin et al., 2002; Mazzoni et al., 2003; Sabater et al., 2006). Moreover, PKCγ expression was found in a limited number of Burkitt lymphomas (Kamimura et al., 2004). Thus a higher variability of underlying tumor types than originally assumed could associate with anti-PKCγ antibodies. Our patient and the one previously published showed a strong expression of PKCγ in the carcinoma cells. Together with the occurrence of anti-PKCγ antibodies in the serum of both patients these findings give more evidence that the PKCγ expression in the tumor might have been involved in initiating the immune attack against the cerebellum. As our case is the second patient, we confirm the relation of anti-PKCγ antibodies with PCD. Although PKCγ is an intracellular protein and the antibodies are unlikely to be pathogenic, they can serve as specific diagnostic marker for the clinical syndrome and the underlying tumor. We detected anti-PKCγ antibodies on routinely performed tissue-based screening (Höftberger et al., 2012). The staining pattern was unique and specific and made it easily distinguishable from staining patterns of other onconeural antibodies. However, anti-PKCγ immunoreactivity is quite similar to that of anti-carbonic anhydrase-related protein VIII, an antibody found in a patient with PCD and melanoma (Bataller et al., 2004). Another antibody to consider in the differential diagnosis of the immunohistochemical pattern of anti-PKCγ was described in a patient with subacute cerebellar ataxia without cancer and target the cytoplasmic antigen called Rho GTPase activating protein 26 (ARHGAP26) (Jarius et
93
al., 2010). To confirm the identity of the antigen we used an immunoblot with electrophoretically separated protein extracts of rat cerebellum and phage plaques expressing PKCγ. All these techniques provided reliable tools that could detect the antibody even in a serum that was stored for 9 years at −20 °C. In summary, our report confirms the association of anti-PKCγ antibodies with PCD, which is important since more patients could be affected by this syndrome. Analysis for this antibody should be considered in patients with PCD and adenocarcinoma without typical onconeural antibodies. Conflict of interest Dr. Dalmau has a research grant from Euroimmun, and receives royalties from patents for the use of Ma2 and NMDAR as autoantibody tests. None of the other contributors has any conflict of interest. Acknowledgments The authors thank Mercè Albà for technical assistance. This study was supported in part by grant PS09/0193 Fondo de Investigaciones Sanitarias, Madrid, Spain. RH was funded by the Fonds zur Förderung der wissenschaftlichen Forschung, Austria, project J3230. We are grateful for Dr. Zsuzsanna Simon for providing the clinical information and Dr. Ellen Gelpi for helpful comments. References Aboul-Enein, F., Höftberger, R., Buxhofer-Ausch, V., Drlicek, M., Lassmann, H., Budka, H., Kristoferitsch, W., 2008. Neocortical neurones may be targeted by immune attack in anti-Yo paraneoplastic syndrome. Neuropathol. Appl. Neurobiol. 34, 248–252. Albert, M.L., Darnell, J.C., Bender, A., Francisco, L.M., Bhardwaj, N., Darnell, R.B., 1998. Tumor-specific killer cells in paraneoplastic cerebellar degeneration. Nat. Med. 4, 1321–1324. Bataller, L., Sabater, L., Saiz, A., Serra, C., Claramonte, B., Graus, F., 2004. Carbonic anhydrase-related protein VIII: autoantigen in paraneoplastic cerebellar degeneration. Ann. Neurol. 56, 575–579. Franz, M.G., Norman, J.G., Fabri, P.J., Gower Jr., W.R., 1996. Differentiation of pancreatic ductal carcinoma cells associated with selective expression of protein kinase C isoforms. Ann. Surg. Oncol. 3, 564–569. Graus, F., Saiz, A., Dalmau, J., 2010. Antibodies and neuronal autoimmune disorders of the CNS. J. Neurol. 257, 509–517. Hammack, J.E., Kimmel, D.W., O'Neill, B.P., Lennon, V.A., 1990. Paraneoplastic cerebellar degeneration: a clinical comparison of patients with and without Purkinje cell cytoplasmic antibodies. Mayo Clin. Proc. 65, 1423–1431. Höftberger, R., Dalmau, J., Graus, F., 2012. Clinical neuropathology practice guide 5-2012: updated guideline for the diagnosis of antineuronal antibodies. Clin. Neuropathol. 31, 337–341. Hug, H., Sarre, T.F., 1993. Protein kinase C isoenzymes: divergence in signal transduction? Biochem. J. 291 (Pt 2), 329–343. Jarius, S., Wandinger, K.P., Horn, S., Heuer, H., Wildemann, B., 2010. A new Purkinje cell antibody (anti-Ca) associated with subacute cerebellar ataxia: immunological characterization. J. Neuroinflammation 7, 21. Kamimura, K., Hojo, H., Abe, M., 2004. Characterization of expression of protein kinase C isozymes in human B-cell lymphoma: relationship between its expression and prognosis. Pathol. Int. 54, 224–230. Kuranami, M., Powell, C.T., Hug, H., Zeng, Z., Cohen, A.M., Guillem, J.G., 1995. Differential expression of protein kinase C isoforms in human colorectal cancers. J. Surg. Res. 58, 233–239. Lin, S.Y., Liang, Y.C., Ho, Y.S., Tsai, S.H., Pan, S., Lee, W.S., 2002. Involvement of both extracellular signal-regulated kinase and c-jun N-terminal kinase pathways in the 12-O-tetradecanoylphorbol-13-acetate-induced upregulation of p21(Cip1) in colon cancer cells. Mol. Carcinog. 35, 21–28. Martiny-Baron, G., Fabbro, D., 2007. Classical PKC isoforms in cancer. Pharmacol. Res. 55, 477–486. Mazzoni, E., Adam, A., Bal de Kier Joffe, E., Aguirre-Ghiso, J.A., 2003. Immortalized mammary epithelial cells overexpressing protein kinase C gamma acquire a malignant phenotype and become tumorigenic in vivo. Mol. Cancer Res. 1, 776–787. Sabater, L., Bataller, L., Carpentier, A.F., Aguirre-Cruz, M.L., Saiz, A., Benyahia, B., Dalmau, J., Graus, F., 2006. Protein kinase Cgamma autoimmunity in paraneoplastic cerebellar degeneration and non-small-cell lung cancer. J. Neurol. Neurosurg. Psychiatry 77, 1359–1362. Saito, N., Shirai, Y., 2002. Protein kinase C gamma (PKC gamma): function of neuron specific isotype. J. Biochem. 132, 683–687. Way, K.J., Chou, E., King, G.L., 2000. Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol. Sci. 21, 181–187.
Contents lists available at SciVerse ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Protein kinase Cγ antibodies and paraneoplastic cerebellar degeneration Romana Höftberger a, b, 1, Gabor G. Kovacs c, 1, Lidia Sabater a, b, Peter Nagy d, Gergely Racz d, Rosa Miquel e, Josep Dalmau f, g, Francesc Graus a, b,⁎ a
Service of Neurology, Hospital Clínic, Universitat de Barcelona, Barcelona, Spain Institut d´Investigació Biomèdica August Pi i Suyer (IDIBAPS), Barcelona, Spain Institute of Neurology, Medical University of Vienna, Austria d First Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary e Pathology Department, Hospital Clínic, IDIBAPS, Barcelona, Spain f Institució Catalana de Recerca i Estudis Avançats (ICREA), IDIBAPS, Hospital Clínic, Barcelona, Spain g Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA b c
a r t i c l e
i n f o
Article history: Received 19 October 2012 Received in revised form 3 December 2012 Accepted 6 December 2012 Keywords: PKC gamma Paraneoplastic cerebellar degeneration Onconeural antibodies Adenocarcinoma
a b s t r a c t Onconeural antibodies are diagnostic markers for paraneoplastic neurological syndromes and indicate the underlying tumor type. Recently, a new antibody against protein-kinase Cγ (PKCγ) was detected in a patient with paraneoplastic cerebellar degeneration (PCD). We report here a second patient. A 70-year-old woman presented with cerebellar ataxia, dysdiadochokinesia, and dysarthria. Her serum showed immunoreactivity against the cytoplasm, dendrites and axons of Purkinje cells in tissue-based screening, later confirmed by immunoblot and phage plaques as anti-PKCγ. Tumor search revealed an adenocarcinoma of hepatobiliary origin. Detection of PKCγ antibodies should be considered in PCD and adenocarcinoma without typical onconeural antibodies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The cerebellum is a frequent target of tumor-induced autoimmunity. The pathology of paraneoplastic cerebellar degeneration (PCD) is characterized by Purkinje cell loss, cortical degeneration of the cerebellum, and, sometimes, inflammatory infiltrates that are mostly composed of T cells (Albert et al., 1998; Aboul-Enein et al., 2008). Symptoms include subacute cerebellar gait and limb ataxia, nystagmus, and dysarthria. Usually, PCD is associated with small cell lung cancer (SCLC) or gynecological tumors and well-characterized onconeural antibodies such as Hu or Yo in serum and CSF of the patients (Graus et al., 2010). However, PCD can also be observed in patients with many different tumor types, either with or without well-characterized onconeural antibodies (Hammack et al., 1990; Aboul-Enein et al., 2008). A previous systematic analysis of clinical and immunological findings in patients with PCD and non-small cell lung cancer (NSCLC) revealed one patient with a novel antibody directed against protein kinase Cγ (PKCγ), a protein which is highly expressed in the cytoplasm of Purkinje cells (Sabater et al., 2006). The study suggested that NSCLC can associate with PCD and antibodies different from those associated with SCLC, however,
⁎ Corresponding author at: Service of Neurology, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. Tel.: +34 932275414; fax: +34 932275783. E-mail address: [email protected] (F. Graus). 1 Equal contribution. 0165-5728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2012.12.002
additional patients are necessary to confirm the link between antiPKCγ and PCD.
2. Case report We report a 70-year-old woman who presented with vertigo and vomiting in January 2003. The neurological examination showed gait and limb ataxia, dysdiadochokinesia, and dysarthria. There was no evidence of pyramidal or extrapyramidal symptoms such as chorea, dyskinesia, or dystonia, or autonomic instability. The cognitive status was normal. CSF was unremarkable. After seven months of illness, in July 2003, the patient's serum was examined for onconeural antibodies. A novel pattern of reactivity was identified on tissue-based screening with rat cerebellum, which was not compatible with that of well-characterized onconeural antibodies. The screening for cancer revealed a tumor in the gall bladder that infiltrated the liver. Histopathology showed a papillary adenocarcinoma of hepatobiliary origin. Cranial CT excluded brain metastases. The patient was lost after cancer diagnosis. Nine years later, the archived serum, stored at −20 °C, was reevaluated for onconeural antibodies. The tissue-based screening on rat cerebellum showed a strong staining of Purkinje cell dendrites, axons and synaptic terminals in the cerebellar nuclei, identical to the pattern recently described for PKCγ antibodies (Sabater et al., 2006) (Fig. 1). No antibodies against neuronal surface antigens (NMDAR, AMPAR, GABABR, mGluR1/5, LGI1, VGCC, and Caspr2) and onconeural antigens (Hu, Yo, Ri, CV2, Tr, amphiphysin, Ma2) were identified. To further characterize the antibody,
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R. Höftberger et al. / Journal of Neuroimmunology 256 (2013) 91–93
Fig. 1. Immunohistochemistry of rat cerebellum and tumor biopsy. (A, B) PKCγ is strongly positive in Purkinje cell cytoplasm, axons (arrows, A), and dendrites (arrowhead, B) of rat cerebellum (rectangle in A enlarged in B). (C) Neuron of the cerebellar nucleus showing intensive labeling of synaptic terminals (arrows). (D, E) Tumor biopsy reveals a papillary adenocarcinoma with expression of CK20 (D) and PKCγ (E).
an immunoblot with electrophoretically separated protein extracts of rat cerebellum was incubated either with patient's serum, commercial PKCγ antibody (Santa Cruz Biotechnology Inc., CA, USA), or normal human serum. The patient's serum recognized a single band of ~80 kDa. An identical band was detected using side by side the commercial PKCγ antibody whereas the control serum remained negative (Fig. 2A). To confirm the identity of the antigen as PKCγ, phage plaques expressing PKCγ were used as previously reported (Sabater et al., 2006). Briefly, filters with purified phage plaques either expressing PKCγ or irrelevant Escherichia coli proteins were incubated with patient's serum, the
commercial PKCγ antibody, or normal human serum. The PKCγ expressing phage plaques showed strong reactivity with the patient's serum and the commercial antibody, whereas normal human serum remained negative (Fig. 2B). As the previously published patient with anti-PKCγ antibodies suffered from a NSCLC, we wanted to confirm the hepatobiliary tumor origin in our patient with an expanded immunohistochemical investigation. Tumor cells revealed prominent positivity for cytokeratin 20 (CK20), a histodiagnostic marker confined to gastric and intestinal epithelia (Fig. 1D). TTF1alpha, a specific marker for adenocarcinoma of the lung, remained negative, ruling out the possibility of a
Fig. 2. (A) Immunoblot of protein extracts of rat cerebellum demonstrated a band of 80 kDa with a commercial PKCγ antibody (lane 1) and the patient's serum (lane 2), whereas the serum from a healthy individual was negative (lane 3). (B) Filters with purified phage plaques expressing PKCγ specifically reacted with a commercial PKCγ antibody (1), and the patient's serum (2) but not with serum from a healthy individual (3).
R. Höftberger et al. / Journal of Neuroimmunology 256 (2013) 91–93
metastasis from an occult NSCLC (data not shown). Finally we stained the tumor tissue with the same commercial anti-PKCγ antibody that we used for the immunoblot and phage plaques experiments. This anti-PKCγ antibody is applicable on formalin-fixed and paraffin embedded tissue and provides an excellent marker for Purkinje cell bodies and processes (data not shown) (Sabater et al., 2006). Staining of the adenocarcinoma identified a substantial number of tumor cells expressing PKCγ (Fig. 1E). 3. Discussion PCD is a classical paraneoplastic neurological syndrome (PNS) and typically related to well-characterized onconeural antibodies (Graus et al., 2010). Onconeural antibodies are helpful for the diagnosis and indicate the underlying tumor type. Unfortunately, in a considerable amount of patients with PNS, onconeural antibodies are never identifiable. Whether this is due to technical limitations or the patients actually do not have antibodies related to their neurological syndrome remains unknown. In the last years, an improvement of screening methods led to an enormous increase of newly diagnosed antibodies that could be associated with a specific spectrum of neurological diseases and a restricted subgroup of tumors. One of these antibodies was PKCγ that was found in a patient with PCD and non-small-cell lung cancer (NSCLC) (Sabater et al., 2006). PKC is a family of cytosolic enzymes that are recruited to the plasma membrane upon calcium and phospholipid dependent activation. They are involved in multiple cellular signaling pathways and depending on the isoform contribute to cancer formation and progression (Way et al., 2000). PKCγ is the neuron-specific isotype of the classical PKC family and thus exclusively found in the central nervous system but not in other tissues (Hug and Sarre, 1993). PKCγ is strongly expressed in the Purkinje cells of the cerebellum and involved in modulation of synaptic plasticity for long term potentiation and depression (Saito and Shirai, 2002). In contrast to other PKC isoforms, PKCγ seems to be less involved in tumor formation, however, some effect in malignant transformation in vivo is proposed (Mazzoni et al., 2003; Martiny-Baron and Fabbro, 2007). Here we report the second patient with anti-PKCγ antibodies and PCD and in the current case the underlying tumor was an adenocarcinoma of hepatobiliary origin. Until now, PKCγ could be associated with different types of adenocarcinomas in vitro and in vivo, including mammary carcinoma, colon adenocarcinoma, lung adenocarcinoma, and pancreatic carcinoma (Kuranami et al., 1995; Franz et al., 1996; Lin et al., 2002; Mazzoni et al., 2003; Sabater et al., 2006). Moreover, PKCγ expression was found in a limited number of Burkitt lymphomas (Kamimura et al., 2004). Thus a higher variability of underlying tumor types than originally assumed could associate with anti-PKCγ antibodies. Our patient and the one previously published showed a strong expression of PKCγ in the carcinoma cells. Together with the occurrence of anti-PKCγ antibodies in the serum of both patients these findings give more evidence that the PKCγ expression in the tumor might have been involved in initiating the immune attack against the cerebellum. As our case is the second patient, we confirm the relation of anti-PKCγ antibodies with PCD. Although PKCγ is an intracellular protein and the antibodies are unlikely to be pathogenic, they can serve as specific diagnostic marker for the clinical syndrome and the underlying tumor. We detected anti-PKCγ antibodies on routinely performed tissue-based screening (Höftberger et al., 2012). The staining pattern was unique and specific and made it easily distinguishable from staining patterns of other onconeural antibodies. However, anti-PKCγ immunoreactivity is quite similar to that of anti-carbonic anhydrase-related protein VIII, an antibody found in a patient with PCD and melanoma (Bataller et al., 2004). Another antibody to consider in the differential diagnosis of the immunohistochemical pattern of anti-PKCγ was described in a patient with subacute cerebellar ataxia without cancer and target the cytoplasmic antigen called Rho GTPase activating protein 26 (ARHGAP26) (Jarius et
93
al., 2010). To confirm the identity of the antigen we used an immunoblot with electrophoretically separated protein extracts of rat cerebellum and phage plaques expressing PKCγ. All these techniques provided reliable tools that could detect the antibody even in a serum that was stored for 9 years at −20 °C. In summary, our report confirms the association of anti-PKCγ antibodies with PCD, which is important since more patients could be affected by this syndrome. Analysis for this antibody should be considered in patients with PCD and adenocarcinoma without typical onconeural antibodies. Conflict of interest Dr. Dalmau has a research grant from Euroimmun, and receives royalties from patents for the use of Ma2 and NMDAR as autoantibody tests. None of the other contributors has any conflict of interest. Acknowledgments The authors thank Mercè Albà for technical assistance. This study was supported in part by grant PS09/0193 Fondo de Investigaciones Sanitarias, Madrid, Spain. RH was funded by the Fonds zur Förderung der wissenschaftlichen Forschung, Austria, project J3230. We are grateful for Dr. Zsuzsanna Simon for providing the clinical information and Dr. Ellen Gelpi for helpful comments. References Aboul-Enein, F., Höftberger, R., Buxhofer-Ausch, V., Drlicek, M., Lassmann, H., Budka, H., Kristoferitsch, W., 2008. Neocortical neurones may be targeted by immune attack in anti-Yo paraneoplastic syndrome. Neuropathol. Appl. Neurobiol. 34, 248–252. Albert, M.L., Darnell, J.C., Bender, A., Francisco, L.M., Bhardwaj, N., Darnell, R.B., 1998. Tumor-specific killer cells in paraneoplastic cerebellar degeneration. Nat. Med. 4, 1321–1324. Bataller, L., Sabater, L., Saiz, A., Serra, C., Claramonte, B., Graus, F., 2004. Carbonic anhydrase-related protein VIII: autoantigen in paraneoplastic cerebellar degeneration. Ann. Neurol. 56, 575–579. Franz, M.G., Norman, J.G., Fabri, P.J., Gower Jr., W.R., 1996. Differentiation of pancreatic ductal carcinoma cells associated with selective expression of protein kinase C isoforms. Ann. Surg. Oncol. 3, 564–569. Graus, F., Saiz, A., Dalmau, J., 2010. Antibodies and neuronal autoimmune disorders of the CNS. J. Neurol. 257, 509–517. Hammack, J.E., Kimmel, D.W., O'Neill, B.P., Lennon, V.A., 1990. Paraneoplastic cerebellar degeneration: a clinical comparison of patients with and without Purkinje cell cytoplasmic antibodies. Mayo Clin. Proc. 65, 1423–1431. Höftberger, R., Dalmau, J., Graus, F., 2012. Clinical neuropathology practice guide 5-2012: updated guideline for the diagnosis of antineuronal antibodies. Clin. Neuropathol. 31, 337–341. Hug, H., Sarre, T.F., 1993. Protein kinase C isoenzymes: divergence in signal transduction? Biochem. J. 291 (Pt 2), 329–343. Jarius, S., Wandinger, K.P., Horn, S., Heuer, H., Wildemann, B., 2010. A new Purkinje cell antibody (anti-Ca) associated with subacute cerebellar ataxia: immunological characterization. J. Neuroinflammation 7, 21. Kamimura, K., Hojo, H., Abe, M., 2004. Characterization of expression of protein kinase C isozymes in human B-cell lymphoma: relationship between its expression and prognosis. Pathol. Int. 54, 224–230. Kuranami, M., Powell, C.T., Hug, H., Zeng, Z., Cohen, A.M., Guillem, J.G., 1995. Differential expression of protein kinase C isoforms in human colorectal cancers. J. Surg. Res. 58, 233–239. Lin, S.Y., Liang, Y.C., Ho, Y.S., Tsai, S.H., Pan, S., Lee, W.S., 2002. Involvement of both extracellular signal-regulated kinase and c-jun N-terminal kinase pathways in the 12-O-tetradecanoylphorbol-13-acetate-induced upregulation of p21(Cip1) in colon cancer cells. Mol. Carcinog. 35, 21–28. Martiny-Baron, G., Fabbro, D., 2007. Classical PKC isoforms in cancer. Pharmacol. Res. 55, 477–486. Mazzoni, E., Adam, A., Bal de Kier Joffe, E., Aguirre-Ghiso, J.A., 2003. Immortalized mammary epithelial cells overexpressing protein kinase C gamma acquire a malignant phenotype and become tumorigenic in vivo. Mol. Cancer Res. 1, 776–787. Sabater, L., Bataller, L., Carpentier, A.F., Aguirre-Cruz, M.L., Saiz, A., Benyahia, B., Dalmau, J., Graus, F., 2006. Protein kinase Cgamma autoimmunity in paraneoplastic cerebellar degeneration and non-small-cell lung cancer. J. Neurol. Neurosurg. Psychiatry 77, 1359–1362. Saito, N., Shirai, Y., 2002. Protein kinase C gamma (PKC gamma): function of neuron specific isotype. J. Biochem. 132, 683–687. Way, K.J., Chou, E., King, G.L., 2000. Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol. Sci. 21, 181–187.