YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.

Lysophagy protects against propagation of α-synuclein aggregation through ruptured lysosomal vesicles.

The neuron-to-neuron propagation of misfolded α-synuclein (αSyn) aggregates is thought to be key to the pathogenesis of synucleinopathies. Recent studies have shown that extracellular αSyn aggregates taken up by the endosomal-lysosomal system can rupture the lysosomal vesicular membrane; however, it remains unclear whether lysosomal rupture leads to the transmission of αSyn aggregation. Here, we applied cell-based αSyn propagation models to show that ruptured lysosomes are the pathway through which exogenous αSyn aggregates transmit aggregation, and furthermore, this process was prevented by lysophagy, i.e., selective autophagy of damaged lysosomes. αSyn aggregates accumulated predominantly in lysosomes, causing their rupture, and seeded the aggregation of endogenous αSyn, initially around damaged lysosomes. Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes. This LC3 accumulation was not observed in cells in which a key regulator of autophagy, RB1CC1/FIP200, was knocked out and was confirmed as lysophagy by transmission electron microscopy. Importantly, RB1CC1/FIP200-deficient cells treated with αSyn aggregates had increased numbers of ruptured lysosomes and enhanced propagation of αSyn aggregation. Furthermore, various types of lysosomal damage induced using lysosomotropic reagents, depletion of lysosomal enzymes, or more toxic species of αSyn fibrils also exacerbated the propagation of αSyn aggregation, and impaired lysophagy and lysosomal membrane damage synergistically enhanced propagation. These results indicate that lysophagy prevents exogenous αSyn aggregates from escaping the endosomal-lysosomal system and transmitting aggregation to endogenous cytosolic αSyn via ruptured lysosomal vesicles. Our findings suggest that the progression and severity of synucleinopathies are associated with damage to lysosomal membranes and impaired lysophagy.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging.

Lysosomes are degradative organelles and signaling hubs that maintain cell and tissue homeostasis, and lysosomal dysfunction is implicated in aging and reduced longevity. Lysosomes are frequently damaged, but their repair mechanisms remain unclear. Here, we demonstrate that damaged lysosomal membranes are repaired by microautophagy (a process termed "microlysophagy") and identify key regulators of the first and last steps. We reveal the AGC kinase STK38 as a novel microlysophagy regulator. Through phosphorylation of the scaffold protein DOK1, STK38 is specifically required for the lysosomal recruitment of the AAA+ ATPase VPS4, which terminates microlysophagy by promoting the disassembly of ESCRT components. By contrast, microlysophagy initiation involves non-canonical lipidation of ATG8s, especially the GABARAP subfamily, which is required for ESCRT assembly through interaction with ALIX. Depletion of STK38 and GABARAPs accelerates DNA damage-induced cellular senescence in human cells and curtails lifespan in C. elegans, respectively. Thus, microlysophagy is regulated by STK38 and GABARAPs and could be essential for maintaining lysosomal integrity and preventing aging.

Remarkable efficacy of temozolomide for relapsed spinal myxopapillary ependymoma with multiple recurrence and cerebrospinal dissemination: a case report and literature review.

PURPOSE: Myxopapillary ependymomas are intradural tumors which grow from the terminal filum of the spinal cord. Although they are classified as WHO grade I, they sometimes cause cerebrospinal fluid dissemination or local recurrence. In this report, we describe a case in that temozolomide (TMZ) showed remarkable efficacy on a recurrent spinal myxopapillary ependymoma. CASE REPORT: A 26-year-old female underwent resection of an intradural myxopapillary ependymoma at L5 initially. Although an en bloc total resection, including the capsule, could be achieved, she needed two additional tumor resection surgeries with postoperative radiotherapy at L4 and at L3 (2 and 6 years after the initial surgery, respectively). Moreover, 4 years after the initial surgery, a disseminated metastatic tumor occurred at T11/12 and local radiotherapy was not effective. After the third surgery, an aggressive adjuvant therapy was necessary because there was a high risk of another recurrence. Therefore, TMZ was administered for 1 year. After 6 months of TMZ treatment, remarkably, the disseminated metastatic tumor at T11/12 had disappeared completely. Presently, 6 years after finishing the TMZ treatment, the follow-up MRI has shown no recurrence in the brain and whole spine. CONCLUSIONS: TMZ is usually used in the treatment of glioblastoma and, recently, it has been reported to be effective for the lower grade spinal gliomas including spinal intramedullary ependymomas. However, for myxopapillary ependymomas, there has been no report that TMZ is effective. According to our results, TMZ could be one of the possible candidates for adjuvant therapy in multiple recurrent myxopapillary ependymomas.

Treatment of locally advanced prostate cancer (Stage T3).

OBJECTIVE: Formerly, locally advanced prostate cancer exhibited poorly prognosis. In the late 1990s, new surgical and radiation technologies were introduced in combination with androgen deprivation. To evaluate respective strategies, outcomes were examined. PATIENTS AND METHODS: Between 2001 and 2010, 224 patients with T3N0M0 (10.9% of all prostate cancer cases) were treated with prostatectomy, external beam radiation therapy with/without androgen deprivation or hormone alone. Complete records were obtained by the end of 2015. RESULTS: Operation group first started without adjuvant treatment and prostate-specific antigen (PSA) relapse occurred in 39% of cases. Radiation therapy group was alternatively divided into two subgroups, that received either monotherapy or combination with androgen deprivation, and PSA relapse rates were 65 and 16%, respectively. High rates of PSA relapse in both the operation and radiation therapy groups were observed in patients without adjuvant therapy, but after relapse androgen deprivation proceeded favorable outcomes. In the radiation subgroups, PSA relapse rates were different, but both subsequent survival rates were the same. This may be due to the effect of androgen deprivation after relapse, indicating effect of delayed therapy. PSA relapse rate in the hormone therapy group was 25% and after relapse, patients applied to treatment with other hormonal and anticancer drugs. Overall survival rates were 91, 88 and 67% in the operation, radiation therapy and hormone therapy groups, respectively. CONCLUSION: Aggressive treatment with short-term androgen deprivation for locally advanced prostate cancer could be beneficial and not harmful when suitable candidates are selected. Delayed androgen deprivation was effective for no adjuvant patients after PSA relapse.

The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy.

Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in the autophagy field but also in other fields.

Management of men with a suspicion of prostate cancer after negative initial prostate biopsy results.

INTRODUCTION: For men with elevated prostate-specific antigen (PSA), appropriate management after negative prostate biopsy remains controversial. After determining PSA kinetics, subsequent follow-up was considered. PATIENTS AND METHODS: A total of 115 cases with negative repeat biopsy were followed by evaluating PSA kinetics and ratio of percent free PSA (F/T) and by performing second repeat biopsy. RESULTS: Eighteen cancer cases were diagnosed. Shorter PSA doubling times and faster velocities were found in cancer cases compared with cases without cancer. We observed a clear decrease in F/T among cancer cases. CONCLUSIONS: To avoid unnecessary repeat biopsies, cases with a suspicion of cancer after negative biopsy can be divided into two groups: one that requires additional biopsies and one with an average change in PSA of <1 ng/ml/year and no change in F/T, which is recommended for surveillance as stable disease without biopsy over a specified time period.

The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps.

Membrane rupture of lysosomes results in leakage of their contents, which is harmful to cells. Recent studies have reported that several systems contribute to the repair or elimination of damaged lysosomes. Lysophagy is a type of selective autophagy that plays a crucial role in the lysosomal damage response. Because multiple pathways are involved in this response, an assay that specifically evaluates lysophagy is needed. Here, we developed the TMEM192-mKeima probe to evaluate lysophagy. By comparing the use of this probe with the conventional galectin-3 assay, we showed that this probe is more specific to lysophagy. Using TMEM192-mKeima, we showed that TFEB and p62 are important for the lysosomal damage response but not for lysophagy, although they have previously been considered to be involved in lysophagy. We further investigated the initial steps in lysophagy and identified UBE2L3, UBE2N, TRIM10, 16, and 27 as factors involved in it. Our results demonstrate that the TMEM192-mKeima probe is a useful tool for investigating lysophagy.

Upfront Androgen Receptor-Axis-Targeted Therapies in Men with De Novo High-Volume Metastatic Hormone-Sensitive Prostate Cancer.

PURPOSE: The extent of effectiveness of upfront androgen receptor-axis-targeted therapies (ARAT) versus total androgen blockade (TAB) in improving prostate cancer-specific survival (CSS) and progression-free survival (PFS) in a real-world sample of Japanese patients with high-volume mHSPC remains unclear. We, therefore, investigated the efficacy and safety of upfront ARAT versus bicalutamide for de novo high-volume mHSPC in Japanese patients. MATERIAL AND METHODS: This was a multicenter retrospective study that analyzed CSS, clinical PFS, and adverse events (AEs) in 170 patients with newly diagnosed high-volume mHSPC. Fifty-six patients were treated with upfront ARAT, and 114 of them were prescribed bicalutamide in addition to ADT between January 2018 and March 2021. The primary and secondary endpoints were CSS and PFS, respectively. A 1:1 nearest neighbor propensity score matching (PSM) with a caliper of 0.2 was performed to match the ARAT group to TAB patients. RESULTS: During the follow-up for a median of 21.5 months, the median CSS was not reached and 37 months in the upfront ARAT and total androgen blockade (TAB) groups, respectively (log-rank test: P = 0.006) by propensity score matching (PSM). Moreover, while the PFS of ARAT was unreached, the median PFS of TAB was 9 months (log-rank test: P < 0.001). Nine patients discontinued ARAT owing to grade ≥ 3 AEs; one patient who was treated with TAB had a grade 3 AE. CONCLUSION: Upfront ARAT significantly prolonged the CSS and PFS of patients with high-volume mHSPC better than TAB, although ARAT was associated with a higher rate of grade ≥ 3 AEs. Upfront ARAT can be more beneficial for patients with de novo high-volume mHSPC than TAB.

Protocols to monitor TFEB activation following lysosomal damage in cultured cells using microscopy and immunoblotting.

Following lysosomal damage, activation and nuclear translocation of transcription factor EB (TFEB) is the key event to maintain lysosomal homeostasis. Here, we describe steps to induce lysosomal damage in HeLa cells. This can be followed by monitoring the changes in TFEB localization using widefield fluorescence microscopy. As a complementary approach, we describe the use of immunoblotting to follow the activation and localization of TFEB in cell lysates. These protocols enable quantitative analysis of TFEB. For complete details on the use and execution of this protocol, please refer to Nakamura et al. (2020).

Outcome of patients with localized prostate cancer treated by radiotherapy after confirming the absence of lymph node invasion.

OBJECTIVE: Management of lymph nodes in radiotherapy for prostate cancer is an issue for curative intent. To find the influence of lymph nodes, patients with T1-T3 prostate cancer and surgically confirmed negative nodes were treated with radiotherapy. METHODS: After lymphadenectomy, 118 patients received photon beam radiotherapy with 66 Gy to the prostate. No adjuvant treatment was performed until biochemical failure. After failure, hormone therapy was administered. Follow-up period was 57 months (mean). RESULTS: Biochemical failure occurred in 47 patients. Few failures were observed in patients with low (24%) and intermediate risks (14%). In contrast, 64% of high-risk patients experienced failure, 97% of whom showed until 36 months. Most patients with failure responded well to hormone therapy. After 15 months (mean), a second biochemical failure occurred in 21% of patients who had the first failure, most of them were high risk. Factors involving failure were high initial and nadir prostate-specific antigen, advanced stage, short prostate-specific antigen-doubling time and duration between radiation and first failure. Failure showed an insufficient reduction in prostate-specific antigen after radiotherapy. Factor for second failure was prostate-specific antigen-doubling time at first failure. CONCLUSIONS: Half of high-risk patients experienced biochemical failure, indicating one of the causes involves factors other than lymph nodes. Low-, intermediate- and the other half of high-risk patients did not need to take immediate hormone therapy after radiotherapy. After failure, delayed hormone therapy was effective. Prostate-specific antigen parameters were predictive factors for further outcome.

LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury.

Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.

PSA doubling time as a predictive factor on repeat biopsy for detection of prostate cancer.

OBJECTIVE: Detection of prostate cancer needs a biopsy of the prostate. Suspecting cancer from an increase in prostate-specific antigen (PSA) has a high negative rate at an initial prostate biopsy. Cases with negative initial biopsy may be the candidates of subsequent biopsy. For lowering unnecessary repeat biopsy, the use of predictive factors before a repeat biopsy is applied for indication. METHODS: Seventy-seven cases with negative initial prostate biopsy received a repeat biopsy and factors for the detection of cancer were examined. RESULTS: PSA doubling time distinguished a part of cancer cases. Its sensitivity of 30, 50 and 70 months was 36.6%, 30.4% and 10%, respectively. Cancer case did not show PSA doubling time of >100 months in general. Values of PSA transition zone density, %Free/total PSA and PSA velocity were similar between cancer and no cancer cases. CONCLUSIONS: PSA doubling time was one of the predictive factors for the detection of prostate cancer and was valuable for avoiding unnecessary repeat biopsy in some cases.

[Primary carcinoid tumor of the testis metastatic to the para-aortic lymph nodes in six years after the first operation: a case report].

This is an additional report of a case which Kinsui, et al. reported in 2000. A primary testicular tumor metastasized to the para-aortic lymph node in 6 years after the first high orichietectomy. In this case, we performed retroperitoneal lymph node dissection. After 4 months, the levels of 5-hydroxy indole acetic acid (5-HIAA), serotonin, urinary 5-HIAA were normalized without recurrence. This indicates testicular carcinoid needs long-term follow up.

COPII vesicles contribute to autophagosomal membranes.

A hallmark of autophagy is the de novo formation of double-membrane vesicles called autophagosomes, which sequester various cellular constituents for degradation in lysosomes or vacuoles. The membrane dynamics underlying the biogenesis of autophagosomes, including the origin of the autophagosomal membrane, are still elusive. Although previous studies suggested that COPII vesicles are closely associated with autophagosome biogenesis, it remains unclear whether these vesicles serve as a source of the autophagosomal membrane. Using a recently developed COPII vesicle-labeling system in fluorescence and immunoelectron microscopy in the budding yeast Saccharomyces cerevisiae, we show that the transmembrane cargo Axl2 is loaded into COPII vesicles in the ER. Axl2 is then transferred to autophagosome intermediates, ultimately becoming part of autophagosomal membranes. This study provides a definitive answer to a long-standing, fundamental question regarding the mechanisms of autophagosome formation by implicating COPII vesicles as a membrane source for autophagosomes.

Microscope and Fiberscope-assisted Subarachnoid-Subarachnoid (S-S) Bypass: A Novel Surgical Technique to Reestablish Cerebrospinal Fluid Flow in Treating Dorsal Spinal Arachnoid Webs, Diagnosed by Cine-MRI.

A "dorsal spinal arachnoid web" is the thickened arachnoid band on the surface of the spinal cord which disturbs the cerebrospinal fluid (CSF) flow, known as a rare cause of thoracic myelopathy. The ideal treatment is controversial because of the risk of readhesion after simple resection of the web. A subarachnoid-subarachnoid bypass is a method to reestablish CSF flow through a silicon tube between the cranial and caudal subarachnoid space. This method is reported to be useful for traumatic syringomyelia, adhesive arachnoiditis, etc. We applied this technique for arachnoid webs with the assistance of a microscope and fiberscope. After a dura incision, the thickened arachnoid web can be seen fluttering within the CSF flow inside the arachnoid space, which partitions the subarachnoid space into cranial and caudal parts. After opening the subarachnoid space and resection of the web under a microscope, the fiberscope is inserted toward the cranial and caudal directions to check for the presence of another arachnoid web. If another web is found, it is penetrated using a guiding wire. Then, a silicone tube is inserted into the cranial and caudal normal subarachnoid space. In this paper, we would like to introduce this technique.

Treatment strategy for metastatic prostate cancer with extremely high PSA level: reconsidering the value of vintage therapy.

The prognostic significance of initial prostate-specific antigen (PSA) level for metastatic prostate cancer remains uncertain. We investigated the differences in prognosis and response to hormonal therapies of metastatic prostate cancer patients according to initial PSA levels. We analyzed 184 patients diagnosed with metastatic prostate cancer and divided them into three PSA level groups as follows: low (<100 ng ml-1), intermediate (100-999 ng ml-1), and high (≥1000 ng ml-1). All patients received androgen deprivation therapy (ADT) immediately. We investigated PSA progression-free survival (PFS) for first-line ADT and overall survival (OS) within each of the three groups. Furthermore, we analyzed response to antiandrogen withdrawal (AW) and alternative antiandrogen (AA) therapies after development of castration-resistant prostate cancer (CRPC). No significant differences in OS were observed among the three groups (P = 0.654). Patients with high PSA levels had significantly short PFS for first-line ADT (P = 0.037). Conversely, patients in the high PSA level group had significantly longer PFS when treated with AW than those in the low PSA level group (P = 0.047). Furthermore, patients with high PSA levels had significantly longer PFS when provided with AA therapy (P = 0.049). PSA responders to AW and AA therapies had significantly longer survival after CRPC development than nonresponders (P = 0.011 and P < 0.001, respectively). Thus, extremely high PSA level predicted favorable response to vintage sequential ADT and AW. The current data suggest a novel aspect of extremely high PSA value as a favorable prognostic marker after development of CRPC.

Outcomes of patients older than 75 years with non-metastatic prostate cancer.

OBJECTIVE: Prostate cancer in elderly patients was formerly treated with androgen deprivation therapy. Since the latter of the 1990s new technologies were introduced into treatments, then strategies have varied. We aimed to observe the outcomes of elderly patients treated during transition period and compare each stage with others. METHODS: During 2008 and 2010, 255 patients with prostate cancer older than 75 years were sequentially treated. With exception of patients with bone and/or visceral metastasis, outcomes of 199 patients with localized and locally advanced stages were examined. Complete records were obtained by the end of 2015. RESULTS: In total, 122 (61%), 28 (14%), 37 (19%) and 12 (6%) of patients were in stages T1c-T2a, T2b-c, T3 and T4, respectively. Patients generally presented with abnormal screening or lower urinary tract symptom. Seventy-one percent of patients received androgen deprivation therapy as monotherapy and 22% of the radiation-treated patients added androgen deprivation therapy. Patients in stage T1c-T2a and T2b-c showed a favorable prognosis. Some cancer death appeared in patients with T3 and T4 during observation periods. Twenty-seven percent of patients died from prostate cancer-independent complications: pneumonia, heart disease, and brain vascular disease. Tendency is similar to that of Japanese elderly male population. No remarkable side effects from androgen deprivation therapy were noticed. CONCLUSION: Elderly patients with localized prostate cancer showed favorable prognosis by androgen deprivation therapy with/without radiation, thus efficacy of androgen deprivation therapy is suitable to elderly patients with applicable stages. Prognosis of patients with locally advanced stage is serious and remains to be improved.