Pleural recurrence after transthoracic needle lung biopsy in stage I lung cancer: a systematic review and individual patient-level meta-analysis.

INTRODUCTION: Conflicting results exist regarding whether preoperative transthoracic biopsy increases the risk of pleural recurrence in early lung cancer. We conducted a systematic, patient-level meta-analysis to evaluate the risk of pleural recurrence in stage I lung cancer after percutaneous transthoracic lung biopsy. METHODS: A systematic search of OVID-MEDLINE, Embase and the Cochrane Database of Systematic Reviews was performed through October 2018. Eligible studies were original articles on the risk of pleural recurrence in stage I lung cancer after transthoracic biopsy. We contacted the corresponding authors of eligible studies to obtain individual patient-level data. We used the Fine-Gray model for time to recurrence and lung cancer-specific survival and a Cox proportional hazards model for overall survival. RESULTS: We analysed 2394 individual patient data from 6 out of 10 eligible studies. Compared with other diagnostic procedures, transthoracic biopsy was associated with a higher risk for ipsilateral pleural recurrence, which manifested solely (subdistribution HR (sHR), 2.58; 95% CI 1.15 to 5.78) and concomitantly with other metastases (sHR 1.99; 95% CI 1.14 to 3.48). In the analysis of secondary outcomes considering a significant interaction between diagnostic procedures and age groups, reductions of time to recurrence (sHR, 2.01; 95% CI 1.11 to 3.64), lung cancer-specific survival (sHR 2.53; 95% CI 1.06 to 6.05) and overall survival (HR 2.08; 95% CI 1.12 to 3.87) were observed in patients younger than 55 years, whereas such associations were not observed in other age groups. DISCUSSION: Preoperative transthoracic lung biopsy was associated with increased pleural recurrence in stage I lung cancer and reduced survival in patients younger than 55 years.

Optimized magnitude of cryosurgery facilitating anti-tumor immunoreaction in a mouse model of Lewis lung cancer.

BACKGROUND: Cryosurgery has reemerged as a less invasive local treatment with possible immune-regulatory effects. However, the optimal magnitude of cryosurgery for achieving immune-regulatory responses at abscopal tumor sites remains unclear. We aimed to investigate appropriate magnitude of cryosurgery for this goal using a mouse model. METHODS: C57BL/6J mice were inoculated with Lewis lung carcinoma cells or B16 melanoma cells in bilateral flanks. The left-sided tumor was cryoablated with repeated freeze/thaw cycles either once, twice, or thrice. The peritumoral injections of LPS were performed. Abscopal tumor volumes were measured, immunohistochemistry was performed for CD4, CD8, Foxp3, and Ki-67, and proinflammatory cytokines were measured in lavage fluid of cryoablated tumor. RESULTS: The growth rate of the abscopal tumor was slowest in the Cryosurgery ×2 group among the five experimental groups. The proportions of CD4(+) T cells and CD8(+) T cells in the abscopal tumor were also significantly higher in the Cryosurgery ×2 group. The levels of IL-1ß, IL-2, IL-6, IL-12ß, IFN-γ, and TNF-α in the peritumoral lavage fluid in Cryosurgery ×2 + LPS group were significantly increased compared with the other groups. CONCLUSIONS: This study suggested that achievement of approximately 73 % damaged area in the cryoablated tumor by two cycles of cryosurgery generates the most favorable immune-regulatory response for abscopal tumors via activation of anti-tumor immune cells as well as increased secretion of proinflammatory cytokines.

Coadministration of erlotinib and curcumin augmentatively reduces cell viability in lung cancer cells.

Resistance to erlotinib in lung cancer cases includes T790M mutant epidermal growth factor receptor and c-Met gene amplification, but other unknown mechanisms account for about 30% of the resistance. Activation of the nuclear factor kappa B (NFkappaB)-related pathways in association with the reduction in ikappaB level may be one of such potential mechanisms. It is known that curcumin inhibits the inducible activation of NFkappaB at least in part by sustaining ikappaB expression level. Therefore, we evaluated the effects of coadministration of erlotinib and curcumin on lung cancer cells. We found that erlotinib and curcumin augmentatively reduced cell viability. Studies in PC9 cells showed that induction of apoptosis was involved. Expression of ikappaB was elevated in PC9 cells by curcumin administration, and pretreatment with siRNAs for ikappaB significantly attenuated the reduction in cell viability after coadministration of erlotinib and curcumin. Furthermore, coadministration of erlotinib and/or curcumin augmentatively attenuated the growth of PC9 tumors in mice. These results suggested the existence of an augmentative tumor growth inhibitory effect between erlotinib and curcumin, and this effect was at least in part mediated by the increase in the expression of ikappaB induced by curcumin.

Airway administration of vascular endothelial growth factor siRNAs induces transient airspace enlargement in mice.

PURPOSE: Reduction in the level of vascular endothelial growth factor (VEGF) has been implicated in the pathogenesis of pulmonary emphysema. To this end, pharmacological VEGF receptor blockade, and the Cre-lox system models have been utilized to study the effects of VEGF depletion in the lung. These models generally reproduce air space enlargement resembling clinical emphysema. Here we report a potentially more readily available model of lung targeted VEGF depletion by airway administration of VEGF small inhibitory RNA oligonucleotides (siRNAs) in mice. METHODS: Airway administration of VEGF siRNAs were done in C57BL/6 mice. The lungs were removed for histology and protein analysis 2, and 4 days later. Airspace enlargement was evaluated by lung volume measurement, and histological analyses. VEGF levels were analyzed by western blot and immunohistochemistry. RESULTS: Airway administration of VEGF siRNAs induced transient air space enlargement in the mouse lung morphologically resembling the previously reported models of pulmonary emphysema. VEGF expression was significantly reduced in the lung, particularly in the alveolar septal cells. We also found that in this particular model, sequential airway administration of recombinant VEGF protein attenuated this air space enlargement. Additionally, we found that airway administration of DCI, a combination of dexamethasone, 3'-5'-cyclic adenosine monophosphate, and isobutylmethylxanthine attenuated the air space enlargement in this particular model, at least in part through the recovery of lung VEGF expression. CONCLUSIONS: The pathogenesis of pulmonary emphysema is likely to be multifaceted, but the present mouse model may be useful in dissecting the involvement of VEGF in pulmonary emphysema.

Computed tomographic appearance of lung tumors treated with percutaneous cryoablation.

PURPOSE: To describe the computed tomographic (CT) appearance of lung tumors treated with cryoablation to establish a reliable reference profile. MATERIALS AND METHODS: CT images of 56 patients who underwent follow-up CT for at least 1 year for treatment with cryoablation of 79 tumors from 2003 to 2010 were retrospectively reviewed. Patients had a follow-up CT scan immediately after the procedure; 1 day, 1 week (two-phase dynamic CT), and 1 month later; and then at 3-month intervals. The appearance of ablation zones on CT images was classified into five patterns, and bidimensional diameters and other imaging features were evaluated. RESULTS: Seventy-eight percent of ablation zones (62 of 79) showed transformation similar to the following: a consolidation or nodular pattern was seen within the 1-week follow-up, involution and a "stripe" pattern was shown at 1 month or later, and zones later became indistinct. Eighty percent of cases of local progression (eight of 10) arose from the stripe pattern on follow-up CT 6 months or later, after the ablation zones showed a transformation opposite the aforementioned pattern. Ice balls could not always be visualized exactly because of dense peritumoral hemorrhage. Internal and marginal enhancement of the ablation zone within the 3-month follow-up did not show a direct relationship with local progression. In total, cavitation and peritumoral ground-glass opacity were seen in 35% (n = 28) and 85% (n = 66) of ablation zones, respectively. CONCLUSIONS: The reference profile of CT appearance, which is mandatory for follow-up, has been established. No single indicator of complete ablation was proven throughout this study. Careful long-term follow-up with CT is indispensable.

Percutaneous cryoablation of lung tumors: feasibility and safety.

PURPOSE: To evaluate the safety and feasibility of cryoablation for lung tumors as well as the incidence of, and risk factors for, complications. MATERIALS AND METHODS: This study included 193 cryoablation sessions for 396 lung tumors in 117 consecutive patients. Univariate and multivariate analyses were performed to assess risk factors for common complications. Changes in laboratory values were analyzed the day after cryoablation. RESULTS: Pneumothorax, pleural effusion, and hemoptysis occurred after 119 (61.7%), 136 (70.5%), and 71 (36.8%) sessions, respectively. Phrenic nerve palsy, frostbite, and empyema occurred after one session each (0.52%). Proximal tumor implantation was observed in one of 471 punctures (0.20%). Of 119 sessions with pneumothorax, 21 (17.6%) required chest tube insertion and two (1.7%) required pleurodesis. Delayed and recurrent pneumothorax occurred in 15 of 193 sessions each (7.8%). A greater number of cryoprobes was a significant (P = .001) predictor of pneumothorax. Male sex (P = .047) and no history of ipsilateral surgery (P = .012) were predictors for the need for chest tube insertion, and no history of ipsilateral surgery (P = .021) was a predictor for delayed/recurrent pneumothorax. Greater number of cryoprobes (P = .001) and no history of ipsilateral surgery (P = .004) were predictors for pleural effusion. Greater number of cryoprobes (P < .001) and younger age (P = .034) were predictors for hemoptysis. Mean changes in white blood cell count, platelet count, hemoglobin level, and C-reactive protein level were 2,418/µL ± 2,260 (P < .001), -2.0 × 10(4)/µL ± 3.2 (P < .001), -0.77 mg/dL ± 0.89 (P < .001), and 3.0 mg/dL ± 2.9 (P < .001), respectively. CONCLUSIONS: Percutaneous cryoablation could be performed minimally invasively with acceptable rates of complications.

Curcumin induces autophagy in ACC-MESO-1 cells.

Malignant pleural mesothelioma is known to be widely resistant to therapy and new treatment strategies are needed. Curcumin, which has a long history as a dietary spice is known to suppress the growth of multiple cancer lines, but the effects on mesothelioma cells are not well defined. In the present study we examined the effects of curcumin on ACC-MESO-1, which is a human derived mesothelioma cell line. We found that curcumin dose-dependently reduced cell viability but did not induce apoptosis. Curcumin administration increased LC3B-II/LC3B-I expression, and induced the formation of autophagosomes on electron microscopy. These changes were attenuated by RNA silencing of atg5. From these findings it was speculated that induction of autophagy was at least in part involved in the reduction of cell viability by curcumin.

Pulmonary pleomorphic carcinoma producing granulocyte-macrophage colony-stimulating factor: report of a case.

We report a case of lung cancer producing granulocyte-macrophage colony-stimulating factor (GM-CSF). The patient, a 55-year-old woman, was found to have leukocytosis (leukocytes 28.8 × 10(3)/mm3) with eosinophilia (eosinophils 24.5%) without any evidence of infection or allergy. The serum concentration of GM-CSF was elevated to 44 pg/ml (normal range <2.0 pg/ml), which might have induced the leukocytosis and eosinophilia. We performed left pneumonectomy and diagnosed a pleomorphic carcinoma with p-T2bN0M0, based on histological examination of the resected tumor. Immunohistochemical examination revealed GM-CSF. The serum level of GM-CSF decreased to within the normal range 8 days after surgery. At the time of writing, 16 months after the surgery, she was alive without disease. To our knowledge, this represents the first case report of a GM-CSF-producing tumor effectively treated by surgical resection.

Innovative segmentectomy to remove the posterior segment of the lower lobe (S¹°) of the lung.

We describe our innovative technique for performing segmentectomy of the posterior segment of the lower lobe of the lung, being segment number 10 (S¹°). In segmentectomy of S¹°, it is difficult to identify A¹° from the interlobar fissure because the pulmonary artery to S¹° (A¹°) branches from A(9+10) and runs dorsally and deeply into the lung tissue. Moreover, to reach S¹° from the interlobar fissure, the lung tissue should be cut between S6 and S8, because S¹° is not located beside the interlobar fissure. However, it is difficult to identify the boundary between the S6 and S8 without a route marker. To solve these difficulties, we divided S6 and S¹° from each other at the beginning of the procedure, which enabled A¹° to be identified easily from the dorsal side. Because S6 and S(8-10) should be divided in S¹° segmentectomy at the end, the division between S6 and S(8-10) at the beginning of procedure is not only reasonable, but makes the procedure simple.

Airway administration of dexamethasone, 3'-5'-cyclic adenosine monophosphate, and isobutylmethylxanthine facilitates compensatory lung growth in adult mice.

The combination of dexamethasone, 8-bromo-3'-5'-cyclic adenosine monophosphate, and isobutylmethylxanthine, referred to as DCI, has been reported to optimally induce cell differentiation in fetal lung explants and type II epithelial cells. DCI administration is also known to modulate the expression levels of many genes known to be involved in the facilitation of lung growth. Recently, we found that RNA silencing of thyroid transcription factor 1 (TTF-1) delayed compensatory lung growth. DCI is also known to induce TTF-1 expression in pulmonary epithelial cells. From these findings, we hypothesized that DCI administration may facilitate compensatory lung growth. In the present study, using a postpneumonectomy lung growth model in 9-wk-old male mice, we found that compensatory lung growth was significantly facilitated by airway administration of DCI immediately following left pneumonectomy, as indicated by the increase in the residual right lung dry weight index. TTF-1 expression was significantly elevated by DCI administration, and transient knockdown of TTF-1 attenuated the facilitation of compensatory lung growth by DCI. These results suggested that DCI facilitated compensatory lung growth, at least in part, through the induction of TTF-1. Morphological analyses suggested that DCI administration increased the number of alveoli, made each of them smaller, and produced a net increase in the calculated surface area of the alveoli per volume of lung. The effect of a single administration was maintained during the observation period, which was 28 days. DCI with further modifications may provide the material to potentially augment residual lung function after resection.

Lovastatin and valproic acid additively attenuate cell invasion in ACC-MESO-1 cells.

Malignant pleural mesothelioma is known to be widely resistant to therapy and new treatment strategies are needed. Both statins and valproic acid are known to suppress the growth of multiple cancer lines, but the effects on mesothelioma cells are not well defined. In the present study we examined the effects of lovastatin and valproic acid on ACC-MESO-1, which is a human derived mesothelioma cell line. We found that lovastatin (2 µM) and/or valproic acid (5 mM) did not reduce cell viability nor induce apoptosis, but reduced cell invasion. The effect was additive when combined. Furthermore it was speculated that induction of autophagic changes was at least in part involved in this process.

The cytostatic effects of lovastatin on ACC-MESO-1 cells.

BACKGROUND: Malignant pleural mesothelioma is known to be widely resistant to therapy, and new treatment strategies are needed. Statins are small molecules that suppress the production of multiple hydrophobic substrates in the mevalonate pathway. Although still controversial, statins may decrease the risk of certain cancers such as colon cancer, lung cancer, and prostate cancer. Since the evaluations of the direct effect of statins on malignant mesothelioma are still few, the present study was done to evaluate the effects of lovastatin on ACC-MESO-1 cells in vivo and to investigate the potential mechanisms involved in vitro. MATERIALS AND METHODS: The in vivo effect of lovastatin was evaluated using an NOD/SCID/γnull (NOG) mouse model of human malignant mesothelioma using ACC-MESO-1 cells. Lovastatin was also applied to ACC-MESO-1 cells in vitro and the effects were observed. RESULTS: Lovastatin administration reduced primary tumor and metastasis in the NOG mouse model of human malignant mesothelioma. In vitro studies showed that lovastatin administration induced cytostatic effects as per reduced cell viability and cell migration in ACC-MESO-1 cells. These effects were suggested to be dependent on autophagic changes rather than apoptosis. Furthermore, induction of autophagic changes by lovastatin in ACC-MESO-1 cells was independent of mTOR, and was considered to be dependent at least in part on Rac/phospholipase C/ inositol 1,4,5-triphosphate axis. CONCLUSIONS: These results suggest that it may be possible to utilize statins, or other pharmacological agents that are known to induce mTOR-independent autophagy, as an adjunct to standard treatments in malignant mesothelioma.

Feasibility of CT-guided large-bore biopsy of lung tumors with the use of an outer sheath.

PURPOSE: To evaluate the feasibility, volume of biopsy specimens, and procedure-related tumor seeding of computed tomography (CT)-guided large-bore lung tumor biopsy through an outer sheath in advanced lung cancer. MATERIALS AND METHODS: Eighteen procedures were performed in 18 patients with established diagnosis of advanced or recurrent lung cancer. Biopsy specimens were obtained under local anesthesia and with fluoroscopic CT guidance. First, an outer sheath was placed in the tumor. Next, a semiautomatic cutting biopsy needle (14-gauge, n = 6; 12-gauge, n = 12) was inserted through the outer sheath, and the tumor biopsy specimen was obtained. After biopsy, the outer sheath was left in place for approximately 10 minutes, during which time hemostasis was achieved. The primary goal was feasibility. Secondary goals were volume of biopsy specimens and incidence of procedure-related tumor implantation. RESULTS: Bleeding volume during the procedure ranged from 0-50 g (median, 5 g; mean, 9 g) and stopped within 10 minutes in all patients. Pneumothorax occurred in two patients (11 %) and improved without chest tube insertion. Specimens could be obtained for the full needle length (2 cm) in 17 patients (94%), which enabled the chemosensitivity test to be performed in 14 patients (78%). Procedure-related tumor seeding was not apparent in any patient at a median of 16 months of follow-up after the procedure. CONCLUSIONS: The present study showed that lung tumor biopsy with a 12-gauge needle through an outer sheath was feasible and enabled acquisition of specimens adequate for chemosensitivity testing without apparent procedure-related tumor seeding.

KL-6 and CEA levels in epithelial lining fluid microsamples predict response to gefitinib in patients with advanced non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: Both Krebs von den Lungen-6 (KL-6) and carcinoembryonic antigen (CEA) are known to be tumour markers in non-small cell lung cancer (NSCLC). The aim of the present study was to assess whether or not intrabronchial epithelial lining fluid (ELF) levels of these markers predicted tumour response better than serum levels in patients with advanced NSCLC treated with gefitinib. METHODS: ELF samples were obtained both from near the tumour and from the contralateral lung using a bronchoscopic microsampling technique, before and 2 weeks after the start of gefitinib treatment. Serum samples were taken concurrently. Among the 22 patients enrolled in the study, 14 (64%) showed partial responses or stabilization of disease with gefitinib treatment (treatment responders), while 8 (36%) showed progression of disease (treatment non-responders), 4 weeks after the start of treatment. RESULTS: ELF KL-6 levels near the tumour decreased significantly after 2 weeks in the treatment responders group (P = 0.011), whereas there was a marginal increase in the treatment non-responders group (P = 0.049). ELF CEA levels near the tumour decreased significantly after 2 weeks in the treatment responders group (P = 0.004), whereas there was no significant change in the treatment non-responders group. For both markers, neither the serum levels nor the levels in contralateral ELF showed any significant changes in either group of patients. CONCLUSIONS: Both KL-6 and CEA levels in ELF near the tumour predicted tumour response in NSCLC patients treated with gefitinib, whereas serum levels did not.

Thyroid transcription factor-1 influences the early phase of compensatory lung growth in adult mice.

RATIONALE: Compensatory lung growth has been well described as a phenomenon in many animal models, but still little is known about the nature, extent, and modulation of such growth. We hypothesized that compensatory lung growth may at least in part recapitulate developmental lung growth, and factors known to be important during normal lung development, such as thyroid transcription factor 1 (TTF-1), may be reactivated during compensatory lung growth. OBJECTIVES: To investigate the role of TTF-1 in correlation with the morphological changes during compensatory lung growth. METHODS: Sequential changes in TTF-1 expression and morphology were examined in the residual right lung after left pneumonectomy in 9-week-old mice. The effect of temporary knockdown of TTF-1 on compensatory lung growth was also evaluated. MEASUREMENTS AND MAIN RESULTS: TTF-1 was transiently but significantly elevated at an early stage in compensatory lung growth. Morphologically, a process resembling septation in lung development may have been initiated during this period in the vicinity of the alveolar duct. furthermore, temporary knockdown of ttf-1 transiently but significantly delayed the early phase of compensatory lung growth. CONCLUSIONS: These results indicate the influential role of TTF-1 in modulating, and possibly initiating, the early phase of compensatory lung growth. Morphologically, compensatory lung growth may at least in part resemble developmental growth.

How many pathological T1N0M0 non-small cell lung cancers can be completely resected in one segment? Special reference to high-resolution computed tomography findings.

PURPOSE: Although segmentectomy is attempted for small non-small cell lung cancer (NSCLC) tumors, no reports have so far described how many of these tumors can be candidates for a successful resection by single segmentectomy. METHODS: In all, 135 patients with peripheral p-T1N0M0 NSCLC were examined. The tumors were classified into five groups divided by every 0.5-cm increase in size. Tumor locations were classified into two groups - limited to within one segment and extended beyond one segment - based on the identification of whether pulmonary vessels and the bronchi were involved in the tumors on high-resolution computed tomography. Differences in the proportion of tumors limited within one segment between tumors smaller and larger than each class of tumor size were assessed. RESULTS: The tumor sizes were 0-1.0 cm in 8 tumors, 1.1-1.5 cm in 27, 1.6-2.0 cm in 35, 2.1-2.5 cm in 34, and 2.6-3.0 cm in 31 tumors. Of these 135 tumors, 92 (65%) were limited to one segment, whereas 48 (35%) had extended beyond one segment. When the tumor size was less than 30 mm, the proportion of tumor limited within one segment did not show any significant difference depending on the size of the tumor. CONCLUSIONS: More than one-third of p-T1N0M0 NSCLC tumors extended beyond one segment, irrespective of size. It is therefore noteworthy that resection of up to two segments or lobectomy should be undertaken for prevention of local recurrence in patients with p-T1N0M0 peripheral NSCLC.

Sentinel nodes in lung cancer: review of our 10-year experience.

Sentinel node (SN) identification in patients with lung cancer is useful not only to minimize lymph node dissection, but also to target the best lymph nodes for intraoperative frozen section during segmentectomy. Since 2000, we have identified the SN in lung cancer patients using radioisotope (RI). This review presents our data on SN identification, describing the following: the procedure, using a radioisotope tracer; the flow of Tc-99 tin colloid after the injection; the characteristics of patients whose SNs could not be identified; ex vivo SN identification; reliability of in vivo SN identification; the algorithm for reducing mediastinal lymph node dissection; the differences in SN identification between large and small radioisotope particles; SNs at segmental lymph nodes; SN navigation segmentectomy for clinical stage IA non-small cell lung cancer; and small metastasis in the SN.

ADAM28 is a serological and histochemical marker for non-small-cell lung cancers.

ADAM28 (a disintegrin and metalloproteinase 28) is over-expressed in non-small-cell lung cancer (NSCLC) with correlation to cancer cell proliferation, tumor size and lymph node metastasis. The present study was aimed to develop an enzyme-linked immunosorbent assay (ELISA) system for diagnosis and monitoring of NSCLC. Our ELISA specifically measured ADAM28, showing negligible cross-reactivity with other metalloproteinases. The ADAM28 level in the NSCLC tissue was remarkably 36.9-fold higher than that in the non-neoplastic lung tissue (p < 0.001). The serum level was significantly 4.6-fold higher in the NSCLC patients (5.41 +/- 8.62 ng/ml; n = 102) than in the control subjects (1.17 +/- 0.93 ng/ml; n = 20) (p < 0.001), and increased with progress of tumor stage (p < 0.001). The level was also significantly higher in the patients with recurrent carcinoma than the control (p < 0.001) and in the patients with lymph node metastasis than those without metastasis (p < 0.001). The sensitivity, false-negative rate and AUC for ADAM28 were better than those for carcinoembryonic antigen. The combination of both assays improved the sensitivity, specificity, false-positive and false-negative rates for NSCLC. There was a positive correlation between the ADAM28 level measured by ELISA system and the degree of immunostaining in the lung adenocarcinomas with a size of

A non-invasive thymoma that occurred 29 years after complete resection of a non-invasive thymoma accompanied by a microthymoma.

A 55-year-old woman with a 7 cm non-invasive thymoma and myasthenia gravis had been treated by extended thymectomy via median sternotomy 29 years ago. A microscopic 0.15 cm thymoma (microthymoma) was incidentally found in the thymus during surgery. Twenty-nine years later, a 5 cm thymoma developed in the anterior mediastinum and was surgically treated. The non-invasive first thymoma, the microthymoma and the non-invasive third thymoma were all classified as type AB thymomas according to the World Health Organization (WHO) classification and showed extremely similar histological findings. We think the mechanism underlying the local recurrence of non-invasive thymomas would be intrathymic metastasis because of their clinical and pathological features.

On freeze-thaw sequence of vital organ of assuming the cryoablation for malignant lung tumors by using cryoprobe as heat source.

Regarding cryoablation for the malignant lung tumors, multiple trials of the freeze-thaw process have been made, and we considered it necessary to view and analyze the freeze-thaw process as a freeze-thaw sequence. We caused the sequence in a porcine lung in vivo by using an acicular, cylindrical stainless-steel probe as the heat source for the freeze-thaw sequence and cooling to -150 °C with super high-pressure argon gas by causing the Joule-Thomson effect phenomenon at the tip of the probe. In this experiment, we examined the sequence by measuring the temperature and using the isothermal curve and the freezing function. As a result, it was demonstrated that the freezing characteristics considerably differed in the first sequence and the second sequence from those of non-aerated organs such as liver and kidney. In our experiments on porcine lung, thermal properties were considered to change as the bleeding caused by the first thawing infiltrated in the lung parenchyma, and it was confirmed that the frozen area in the second cycle was dramatically enlarged as compared with the first cycle (when a similar sequence is continuously repeated, we say it as cycle). This paper provides these details.