Tumor Spread Through Air Spaces Identifies a Distinct Subgroup With Poor Prognosis in Surgically Resected Lung Pleomorphic Carcinoma

1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 28 29 3 31 32 33 34 35 36 37 38 39 41 42 43 44 45 46 47 48 49 5 51 52 53 54 55 Q15 Q1 Q6 Q2 [ Original Research ] Tumor Spread Through Air Spaces Identifies a Distinct Subgroup With Poor Prognosis in Surgically Resected Lung Pleomorphic Carcinoma Shintaro Yokoyama, MD, PhD; Tomoyuki Murakami, MD, PhD; Hiroyuki Tao, MD, PhD; Hideko Onoda, MD, PhD; Akio Hara, MD, PhD; Ryohei Miyazaki, MD, PhD; Masashi Furukawa, MD, PhD; Masataro Hayashi, MD, PhD; Hidetoshi Inokawa, MD, PhD; Kazunori Okabe, MD, PhD; and Yoshito Akagi, MD, PhD BACKGROUND: Tumor spread through air spaces (STAS) has recently been reported as a novel form of lung adenocarcinoma invasion that can negatively affect survival; however, its role in pleomorphic carcinoma remains unclear. The goal of this study was to characterize tumor STAS in pleomorphic carcinoma, including its association with clinicopathologic features and prognosis. METHODS: Tumor specimens obtained from 35 consecutive patients with pleomorphic carcinoma who underwent surgical resection between 9 and 15 were reviewed. Tumor STAS was defined as tumor cells spreading within the air spaces in the surrounding lung parenchyma beyond the edge of the primary tumor. RESULTS: Fourteen patients (%) had evidence of STAS-positive pleomorphic carcinomas. Three types of morphologic findings were observed: single cells, small tumor cell clusters, and tumor nests. Tumor necrosis tended to be more prevalent in STAS-positive tumors than in STAS-negative tumors (P ¼.94). Patients with STAS experienced significantly worse recurrence-free survival (P ¼.5) and overall survival (P ¼.2) rates than those without STAS. Moreover, multivariate analysis revealed that tumor STAS was an independent risk factor for both recurrence (P ¼.14) and poor overall survival (P ¼.42). CONCLUSIONS: In this first study of its kind, tumor STAS in patients with pleomorphic carcinoma was shown to be associated with high recurrence rates and poor survival after surgical resection. Hence, tumor STAS can serve as a predictor of postoperative survival; this information will enable better risk stratification and more effective clinical management of patients with this rare type of tumor. CHEST 18; -(-):--- KEY WORDS: lung cancer; pathology lung cancer; surgery oncology ABBREVIATIONS: HR = hazard ratio; NSCLC = non-small cell lung cancer; OS = overall survival; RFS = recurrence-free survival; STAKS = spread through a knife surface; STAS = spread through air spaces AFFILIATIONS: From the Division of Thoracic Surgery (Drs Yokoyama, Tao, Hara, Miyazaki, Furukawa, Hayashi, Inokawa, and Okabe), Department of Surgery, National Hospital Organization Yamaguchi-Ube Medical Center, Ube, Japan; Department of Clinical Research (Drs Yokoyama and Murakami), National Hospital Organization Yamaguchi- Ube Medical Center, Ube, Japan; Department of Surgery (Drs Yokoyama and Akagi), Kurume University School of Medicine, Kurume, Japan; Department of Pathology (Dr Murakami), National Hospital Organization Kanmon Medical Center, Shimonoseki, Japan; and the Department of Radiology (Dr Onoda), National Hospital Organization Yamaguchi- Ube Medical Center, Ube, Japan. FUNDING/SUPPORT: The authors have reported to CHEST that no funding was received for this study. Q3 Q4 CORRESPONDENCE TO: Shintaro Yokoyama, MD, PhD, Division of Thoracic Surgery, Department of Surgery, National Hospital Organization Yamaguchi-Ube Medical Center, 685 Higashi-kiwa, Ube, 755241, Japan; e-mail: yokoyama_shintarou@med.kurume-u.ac.jp Q5 Copyright Ó 18 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved. DOI: https://doi.org/1.116/j.chest.18.6.7 56 57 58 59 61 62 63 64 65 66 67 68 69 7 71 72 73 74 75 76 77 78 79 81 82 83 84 85 86 87 88 89 9 91 92 93 94 95 96 97 98 99 11 12 13 14 15 16 17 18 19 11 chestjournal.org 1 FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

111 112 113 114 115 116 117 118 119 1 121 122 123 124 125 126 127 128 129 13 131 132 133 134 135 136 137 138 139 1 141 142 143 144 145 146 147 148 149 15 151 152 153 154 155 156 157 158 159 1 161 162 163 164 165 Q7 Lung pleomorphic carcinoma, categorized as a subset of sarcomatoid carcinoma according to the latest World Health Organization classification, 1 is rare: it accounts for approximately.4% of all non-small cell lung cancers (NSCLCs). 2,3 It is widely recognized as a high-grade malignancy with a reported 5-year overall survival (OS) of only % to 48%, even after surgical resection. 4-7 Patients with unresectable pleomorphic carcinoma that responds poorly to conventional chemotherapy have been reported; their median OS was only 5 to 8 months. 8-1 Thus far, these tumors have not been sufficiently characterized, largely owing to their rarity. However, selecting appropriate treatment strategies requires understanding the underlying clinicopathologic features and prognostic factors associated with this malignancy. Tumor spread through air spaces (STAS) has recently been described as an important route of tumor Patients and Methods Patient Cohort Our institutional review board approved this retrospective study (approval identification number YUMC 29-12), and written informed consent for using materials and clinical information was obtained from each patient. Thirty-seven consecutive patients with lung pleomorphic carcinoma underwent surgical resection at National Hospital Organization Yamaguchi-Ube Medical Center between 9 and 15. Following a detailed review of the medical records, two patients were excluded from the cohort; one had committed suicide 2 months following surgery, and the other had undergone tumor wedge resection for pathologic diagnosis. Therefore, 35 patients were included in the analysis. Pathologic stage was assigned according to the TNM Classification of Malignant Tumors (8th edition). 16 None of the patients received preoperative chemotherapy and/or radiotherapy, and each received a clinical follow-up examination at least every 6 months following surgery that included CT assessment. Histopathologic Evaluation Hematoxylin and eosin-stained slides of resected tumor specimens acquired from formalin-fixed paraffin-embedded tissues were microscopically reviewed and evaluated by two pathologists (S. Y. and T. M.) who were blinded to the patients clinical features and survival outcomes; any disagreements were resolved by consensus. Pathologic diagnoses were made according to the 15 World Health Organization classification 1 and confirmed with immunohistochemistry when necessary. Given that pleomorphic carcinoma as defined is a poorly differentiated NSCLC comprising at least 1% spindle and/or giant cells, 1 pure spindle cell carcinomas or giant cell carcinomas were excluded. Comprehensive histologic subtyping was performed in each case, including the evaluation of spindle or giant cell components as well as epithelial elements. Because tumor necrosis is reportedly a significant prognostic factor, 6 the presence of necrosis was also recorded. STAS was defined as tumor cells within air spaces in the surrounding lung parenchyma beyond the edge (ie, the smooth surface clearly identified in a low-power field) of the primary tumor. 12,13,15 STAS invasion. It also carries prognostic implications in patients with lung adenocarcinoma given that significantly higher recurrence rates, as well as poorer OS, have consistently been reported in patients with lung adenocarcinoma with STAS following surgical resection. 11-14 Moreover, a recent study revealed that STAS was prognostic in patients with squamous cell carcinoma of the lung who underwent surgical resection. 15 However, the prevalence of STAS in pleomorphic carcinoma and its association with patients clinicopathologic characteristics and prognoses have yet to be characterized. We therefore retrospectively investigated the clinicopathologic findings of patients with resected lung pleomorphic carcinoma and sought to characterize the significance and prognostic implications of STAS. was classified into three groups based on morphologic findings: (1) single cells, defined as the presence of only one floating tumor cell in the alveolar space; (2) small tumor cell clusters, defined as a few tumor cells floating in the same; and (3) tumor nests, which are clearly identifiable within a low-magnification field (Fig 1). The edges of tumors containing an invasive mucinous adenocarcinoma component were defined as the alveolar spaces lacking tumor mucin, because tumor clusters are often observed in alveolar spaces that are filled with mucin products. In addition, previously described discriminative methods were used to distinguish STAS from artificially detached tumor cells arising from tumor dissection, namely spread through a knife surface (STAKS). 12,15,17,18 STAS was identified by the presence of tumor cells within air spaces; these were mostly contiguous but were sometimes randomly scattered across the lung tissue. Tissue fragmentations both with and without jagged edges were carefully identified and deemed to be STAKS. STAS was also distinguished from alveolar macrophages according to morphologic features; the former were characterized by a high nuclear-to-cytoplasmic ratio and nuclear atypia, whereas the latter exhibited small nuclei and foamy cytoplasm that sometimes contained faint pigments. The distance between the edge of the tumor and the farthest STAS was measured by using an ocular WHN1X-H micrometer (Olympus Optical Co. Ltd.). The number of alveolar spaces was also counted to rule out the influence of inconsistent inflation of the lung specimens during processing. The farthest distance between the edge of the tumor and that of the circumferential lung tissue section was also measured with a ruler to determine the size of the area surrounding the pathologically examined tumor. Statistical Analysis The associations between STAS and clinicopathologic features were evaluated by using Fisher exact test for categorical variables and Student t test for numerical variables. Recurrence-free survival (RFS) was defined as the interval between the date of surgical resection and that of detection of recurrence; OS was defined as the interval between the date of surgical resection and that of death or the last follow-up visit. Survival curves to estimate RFS and OS were generated by using 166 167 168 169 17 171 172 173 174 175 176 177 178 179 1 181 182 183 184 185 186 187 188 189 19 191 192 193 194 195 196 197 198 199 1 2 3 4 5 6 7 8 9 21 211 212 213 214 215 216 217 218 219 2 2 Original Research [ - # - CHEST - 2 1 8 ] FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

221 222 223 224 225 226 227 228 229 23 231 232 233 234 235 236 237 238 239 2 241 242 243 244 245 246 247 248 249 25 251 252 253 254 255 256 257 258 259 2 261 262 263 264 265 266 267 268 269 27 271 272 273 274 275 print & web 4C=FPO Figure 1 A-D, Representative histopathologic images of tumor spread through air spaces (STAS) in pleomorphic carcinoma. A, Low-power image of a tumor section (magnification, ), comprising spindle cell carcinoma, giant cell carcinoma, and adenocarcinoma components. B, High-power view of the boxed region in (A), showing single-cell STAS (arrowheads) and small tumor cell cluster STAS (arrows) around the tumor (magnification, ). C, Low-power image of tumor section (magnification, ), comprising spindle cell carcinoma and adenocarcinoma components. D, High-power view of the boxed region in (C), showing STAS of tumor nests (arrows) near the tumor (magnification, ). the Kaplan-Meier method and compared by using the log-rank test. Cox proportional hazards regression models were used to assess the prognostic value of each factor found to be significant on univariate analysis, as well as to adjust for potential confounders when Results The baseline clinicopathologic characteristics of the study patients are summarized in Table 1. A total of 27 male (77%) and eight female (23%) patients with a median age of 62 years (range: 35-91 years) were investigated. The median follow-up period in patients without recurrence was 36 months; the overall 5-year survival rate was 65.5%. Thirty-three patients (94%) underwent pulmonary lobectomy, and two (6%) underwent bilobectomy; all patients underwent routine systematic dissection of the hilar and mediastinal lymph nodes, and were confirmed to have achieved R resection using microscopy. Ten patients (29%) had pathologic stage III disease. Of all patients, 12 (34%) experienced recurrence during the follow-up period; 11 (31%) experienced distant recurrence outside of the ipsilateral hemithorax; and one (3%) experienced regional recurrence of the tumor as an intrapulmonary identifying independent risk factors using multivariate analysis. All P values were based on two-sided tests; P values <.5 were considered statistically significant. JMP software version 13 (SAS Institute, Inc.) was used to perform all statistical analyses. metastasis in a second ipsilateral lobe. With respect to prognosis, eight patients (23%) died following surgery: seven patients (%) died due to recurrence and progression of pleomorphic carcinoma; and one patient (3%) died of acute exacerbation of underlying interstitial pneumoniae 28 months following surgery. STAS was observed in 14 patients (%); representative microphotographs of STAS in pleomorphic carcinoma are shown in Figure 1. In terms of predominant morphologic patterns, there were two patients (14%) with single-cell STAS, 1 (71%) with small tumor cell cluster STAS, and two (14%) with tumor nest STAS. The median distance between the edge of the tumor and the farthest STAS was 1,75 mm (range: 45-5, mm). Moreover, the median number of alveolar spaces between the tumor edge and farthest STAS was seven (range: 1-36). Q14 276 277 278 279 2 281 282 283 284 285 286 287 288 289 29 291 292 293 294 295 296 297 298 299 3 31 32 33 34 35 36 37 38 39 31 311 312 313 314 315 316 317 318 319 3 321 322 323 324 325 326 327 328 329 33 chestjournal.org 3 FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

331 332 333 334 335 336 337 338 339 3 341 342 343 344 345 346 347 348 349 35 351 352 353 354 355 356 357 358 359 3 361 362 363 364 365 366 367 368 369 37 371 372 373 374 375 376 377 378 379 3 381 382 383 384 385 Q12 TABLE 1 ] Patient Characteristics (N ¼ 35) Characteristic Values Clinical factors Sex Male 27 (77.1%) Female 8 (22.9%) Age, y Median [range] 62. [35-91] Smoking Current 19 (54.3%) Ex-smoker 9 (25.7%) Never 7 (.%) Surgical procedure Lobectomy 33 (94.3%) Bilobectomy 2 (5.7%) Pathologic factors Tumor size (mm) Median [range] 38 [11 85] T classification T1 T1b 2 (5.7%) T1c 6 (17.1%) T2 T2a 17 (48.6%) T2b 3 (8.6%) T3 4 (11.4%) T4 3 (8.6%) N classification N 24 (68.6%) N1 4 (11.4%) N2 7 (.%) Stage I IA2 2 (5.7%) IA3 5 (14.3%) IB 13 (37.1%) II IIA 1 (2.9%) IIB 4 (11.4%) III IIIA 7 (.%) IIIB 3 (8.6%) Pleural invasion 19 (54.3%) 1 8 (22.9%) 2 3 (8.6%) 3 5 (14.3%) (Continued) TABLE 1 ] (Continued) Characteristic Values Lymphatic invasion Absent 24 (68.6%) Present 11 (31.4%) Vascular invasion Absent 13 (37.1%) Present 22 (62.9%) Observed distance from tumor edge, mm Median [range] 14 [6 24] Necrosis Absent 19 (54.3%) Present 16 (45.7%) Spindle cell carcinoma component Absent 3 (8.6%) Present 32 (91.4%) Giant cell carcinoma component Absent 1 (28.6%) Present 25 (71.4%) Adenocarcinoma component Absent 7 (.%) Present 28 (.%) Lepidic pattern Absent 8 (22.9%) Present (57.1%) Acinar pattern Absent 6 (17.1%) Present 22 (62.9%) Papillary pattern Absent 16 (45.7%) Present 12 (34.3%) Micropapillary pattern Absent 27 (77.1%) Present 1 (2.9%) Solid pattern Absent 16 (45.7%) Present 12 (34.3%) Mucinous adenocarcinoma Absent 26 (74.3%) Present 2 (5.7%) Squamous cell carcinoma component (Continued) 386 387 388 389 39 391 392 393 394 395 396 397 398 399 1 2 3 4 5 6 7 8 9 41 411 412 413 414 415 416 417 418 419 4 421 422 423 424 425 426 427 428 429 43 431 432 433 434 435 436 437 438 439 4 4 Original Research [ - # - CHEST - 2 1 8 ] FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

441 442 443 444 445 446 447 448 449 45 451 452 453 454 455 456 457 458 459 4 461 462 463 464 465 466 467 468 469 47 471 472 473 474 475 476 477 478 479 4 481 482 483 484 485 486 487 488 489 49 491 492 493 494 495 Q8 TABLE 1 ] (Continued) Characteristic Values Absent 26 (74.3%) Present 9 (25.7%) STAS Absent 21 (.%) Present 14 (.%) STAS ¼ spread through air spaces. Table 2 shows the distribution of clinicopathologic characteristics according to the presence of STAS. There were no significant correlations between STAS and most clinicopathologic features, including pathologic findings of spindle cell carcinoma (P ¼ 1.), giant cell carcinoma (P ¼.151), adenocarcinoma (P ¼.676), and squamous cell carcinoma (P ¼ 1.). Furthermore, no significant associations between STAS and histological adenocarcinoma subtypes were found. The most common STAS pattern among our patients was small tumor cell clusters, whereas a micropapillary component was observed in one patient only (Fig 1). Notably, STAS tended to be more prevalent in tumors exhibiting necrosis (P ¼.94). Kaplan-Meier analysis was conducted to compare survival rates according to the presence of STAS (Fig 2). STAS-positive patients had significantly worse RFS and OS than STAS-negative patients (P ¼.5 and P ¼.2, respectively). Stratified analyses based on pathologic stage were performed to further assess the prognostic relevance of STAS (Fig 3). STAS-positive stage I/II patients experienced reduced RFS and OS rates compared with their STAS-negative counterparts, although the differences were not significant (P ¼.13 and P ¼.319). Conversely, patients with stage III disease who also exhibited STAS experienced significantly poorer RFS and OS (P ¼.23 and P ¼.2). Using Cox proportional hazards regression models, tumor stage, presence of necrosis, and presence of STAS were identified as significant predictors of both RFS (P ¼.31, P ¼.1, and P ¼.6, respectively) and OS (P ¼.19, P ¼.6, and P ¼.2) (Table 3). Additional multivariate models were applied to identify independent prognostic factors, although they may have been of limited value owing to the small number of included patients (Table 4). On multivariate analysis, presence of necrosis (P ¼.29) and STAS (P ¼.14) remained significant predictors of recurrence, whereas tumor stage did not (P ¼.96). However, tumor stage (P ¼.8) and presence of STAS (P ¼.42) were found to be independent risk factors for poorer OS. Discussion Tumor STAS was observed in % of patients with pleomorphic carcinoma in the present study. Its presence was significantly associated with poor RFS and OS following surgical resection. To the best of our knowledge, the present study is the first to clearly show that the presence of STAS in patients with pleomorphic carcinoma is a reliable prognostic indicator. Following extensive debate, tumor STAS was recently designated as a novel invasive pattern in lung adenocarcinoma. Initial studies of STAS were limited to tumor nests or islands; patients with these morphologic findings were found to have distinct pathologic features and poorer prognoses than those without. 11,19 Subsequently, some investigators broadened their definition of STAS to include cell clusters or even single cells; they found that these features were also closely associated with high recurrence rates and poor survival following surgery in patients with small-sized lung adenocarcinomas. 12-14 Recently, Lu et al 15 found that every STAS associated with squamous cell carcinomas formed tumor nests and predicted poor postoperative RFS and OS. Our study, which used a broadened definition of STAS, found that the presence of STAS had a negative prognostic impact in patients with pleomorphic carcinoma, which was consistent with findings in patients with other NSCLCs. Prognostic determinants in patients with pleomorphic carcinoma have yet to be clearly defined. Previous investigators proposed that a higher pathologic stage, nodal involvement, pleural invasion, massive necrosis, and unresectability were predictors of poor OS in patients with pleomorphic carcinoma. 4-6,-23 Our results are generally consistent with their findings; however, it is notable that the negative impact of STAS on both RFS and OS was particularly prominent. Moreover, all the study patients underwent curativeintent lobectomy or bilobectomy followed by systematic lymph nodes dissection; this homogeneity increased the reliability of our results. Given the prognostic relevance of STAS, its associated biologic mechanisms that facilitate tumor invasion ought to be clarified. Studies have shown that lung adenocarcinomas with STAS are more likely to exhibit higher nuclear grade, solid or micropapillary growth patterns, and KRAS or BRAF mutations, whereas those without STAS exhibited lepidic growth patterns and 496 497 498 499 5 51 52 53 54 55 56 57 58 59 51 511 512 513 514 515 516 517 518 519 5 521 522 523 524 525 526 527 528 529 53 531 532 533 534 535 536 537 538 539 5 541 542 543 544 545 546 547 548 549 55 chestjournal.org 5 FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

551 552 553 554 555 556 557 558 559 5 561 562 563 564 565 566 567 568 569 57 571 572 573 574 575 576 577 578 579 5 581 582 583 584 585 586 587 588 589 59 591 592 593 594 595 596 597 598 599 1 2 3 4 5 TABLE 2 ] Clinicopathologic Characteristics of the Study Cohort According to the Presence of STAS Characteristic All Patients (N ¼ 35) STAS Negative (n ¼ 21 [.%]) STAS Positive (n ¼ 14 [.%]) P Value Clinical factors Sex Male 27 (77) 15 (71) 12 (86).431 Female 8 (23) 6 (29) 2 (14) Age, y Median [range] 62. [35 91] 62. [35 86] 63.5 [44 91].32 Smoking Current/ex-smoker 28 () 15 (71) 13 (93).3 Nonsmoker 7 () 6 (29) 1 (7) Pathologic factors Tumor size, mm Median [range] 38. [11 85] 33. [15 85]. [11 ].873 T classification T1/T2 28 () 17 (81) 11 (79) 1. Q13 T3/T4 7 () 4 (19) 3 (21) N classification N 24 (69) 15 (71) 9 (64).566 N1 4 (11) 3 (14) 1 (7) N2 7 () 3 (14) 4 (29) Stage I/II 25 (71) 16 (76) 9 (64).474 III 1 (29) 5 (24) 5 (36) Pleural invasion Absent 19 (54) 1 (48) 9 (64).491 Present 16 (46) 11 (52) 5 (36) Lymphatic invasion Absent 24 (69) 14 (67) 1 (71) 1. Present 11 (31) 7 (33) 4 (29) Vascular invasion Absent 13 (37) 7 (33) 6 (43).724 Present 22 (63) 14 (67) 8 (57) Observed distance from tumor edge, mm Median [range] 14. [6 24] 14. [6 24] 13.5 [1 24].493 Spindle cell carcinoma component Absent 3 (9) 2 (1) 1 (7) 1. Present 32 (91) 19 (91) 13 (93) Giant cell carcinoma component Absent 1 (29) 4 (19) 6 (43).151 Present 25 (71) 17 (81) 8 (57) Adenocarcinoma component Absent 7 () 5 (24) 2 (14).676 Present 28 () 16 (76) 12 (86) Lepidic pattern Absent 8 (23) 6 (38) 2 (17).1 (Continued) 6 7 8 9 61 611 612 613 614 615 616 617 618 619 6 621 622 623 624 625 626 627 628 629 63 631 632 633 634 635 636 637 638 639 6 641 642 643 644 645 646 647 648 649 65 651 652 653 654 655 656 657 658 659 6 6 Original Research [ - # - CHEST - 2 1 8 ] FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

661 662 663 664 665 666 667 668 669 67 671 672 673 674 675 676 677 678 679 6 681 682 683 684 685 686 687 688 689 69 691 692 693 694 695 696 697 698 699 7 71 72 73 74 75 76 77 78 79 71 711 712 713 714 715 print & web 4C=FPO TABLE 2 ] (Continued) Characteristic All Patients (N ¼ 35) STAS Negative (n ¼ 21 [.%]) STAS Positive (n ¼ 14 [.%]) P Value Present (57) 1 (63) 1 (83) Acinar pattern Absent 6 (17) 2 (13) 4 (33).354 Present 22 (63) 14 (88) 8 (67) Papillary pattern Absent 16 (46) 9 (56) 7 (58) 1. Present 12 (34) 7 (44) 5 (42) Micropapillary pattern Absent 27 (77) 16 () 11 (92).429 Present 1 (3) 1 (8) Solid pattern Absent 16 (46) 1 (63) 6 (5).72 Present 12 (34) 6 (38) 6 (5) Mucinous adenocarcinoma Absent 26 (74) 15 (94) 11 (92) 1. Present 2 (6) 1 (6) 1 (8) Squamous cell carcinoma component Absent 26 (74) 16 (76) 1 (71) 1. Present 9 (26) 5 (24) 4 (29) Necrosis Absent 19 (54) 14 (67) 5 (36).94 Present 16 (46) 7 (33) 9 (64) Data are presented as No. (%) or as median [range]. See Table 1 legend for expansion of abbreviation. EGFR mutations. 11,12,14,19 Similarly, squamous cell carcinomas with STAS were significantly more associated with lymphatic and vascular invasion as well as tumor necrosis. 15 Moreover, squamous cell A Recurrence-free survival rate (%) STAS (+) (n = 14) STAS ( ) (n = 21) 21 16 7 2 2 14 8 4 2 carcinomas with STAS had higher Ki-67 indices than those without. Notably, we found that STAS-positive tumors occurred concomitantly with tumors exhibiting necrosis more frequently than STAS-negative tumors. B Overall survival rate (%) STAS (+) (n = 14) 21 18 8 3 2 14 9 4 2 STAS ( ) (n = 21) Figure 2 A, B, Kaplan-Meier curves estimating recurrence-free survival (RFS) and overall survival (OS) according to the presence of tumor spread through air spaces (STAS) in pleomorphic carcinoma. A, RFS in patients with STAS vs without STAS (P ¼.5). B, OS in patients with STAS vs without STAS (P ¼.2). 716 717 718 719 7 721 722 723 724 725 726 727 728 729 73 731 732 733 734 735 736 737 738 739 7 741 742 743 744 745 746 747 748 749 75 751 752 753 754 755 756 757 758 759 7 761 762 763 764 765 766 767 768 769 77 chestjournal.org 7 FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

771 772 773 774 775 776 777 778 779 7 781 782 783 784 785 786 787 788 789 79 791 792 793 794 795 796 797 798 799 1 2 3 4 5 6 7 8 9 81 811 812 813 814 815 816 817 818 819 8 821 822 823 824 825 Q16 Mochizuki et al 6 showed that tumor necrosis is an independent negative predictor of RFS and OS in patients with pleomorphic carcinoma, possibly because the rapid proliferation of malignant cells results in an undersupply of blood flows. Hence, it is possible that the poor prognoses of patients with pleomorphic carcinoma with STAS stems from this association with tumor necrosis, implying that common molecular pathways may be involved in eliciting both features; this hypothesis warrants further investigation. print & web 4C=FPO To properly assess the prognostic impact of STAS in a clinical setting, some investigators classified STAS into extensive vs limited categories. Warth et al 11 concluded A Recurrence-free survival rate (%) C Overall survival rate (%) STAS (+) (n = 9) 16 12 6 2 2 9 7 4 2 STAS (+) (n = 9) 16 13 6 2 2 9 8 4 2 STAS ( ) (n = 16) STAS ( ) (n = 16) that extensive STAS (ie, that which is > 3 alveolar spaces away from the edge of the tumor) accounted for approximately 29% of adenocarcinomas and was prevalent in tumors with micropapillary components. However, survival rates in such cases were virtually similar to those with limited STAS. Applying the same definition to squamous cell carcinomas revealed that extensive STAS accounted for 23% of cases; moreover, their study also found that RFS and cancer-specific survival did not differ based on the extent of STAS. 15 When 14 patients with STAS in the present study were similarly subcategorized into extensive vs limited STAS, 13 (93%) were classified as having extensive STAS, which in turn was significantly correlated with tumor B Recurrence-free survival rate (%) D Overall survival rate (%) STAS ( ) (n = 5) STAS (+) (n = 5) 5 5 2 5 2 STAS (+) (n = 5) STAS ( ) (n = 5) 5 5 3 2 5 2 Figure 3 A-D, Kaplan-Meier survival curves based on the presence of tumor spread through air spaces (STAS) stratified according to pathologic stage. A, Recurrence-free survival (RFS) in stage I/II patients with STAS vs without STAS (P ¼.13). B, RFS in stage III patients with STAS vs without STAS (P ¼.23). C, Overall survival (OS) in stage I/II patients with STAS vs without STAS (P ¼.319). D, OS in stage III patients with STAS vs without STAS (P ¼.2). 826 827 828 829 83 831 832 833 834 835 836 837 838 839 8 841 842 843 844 845 846 847 848 849 85 851 852 853 854 855 856 857 858 859 8 861 862 863 864 865 866 867 868 869 87 871 872 873 874 875 876 877 878 879 8 8 Original Research [ - # - CHEST - 2 1 8 ] FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

881 882 883 884 885 886 887 888 889 89 891 892 893 894 895 896 897 898 899 9 91 92 93 94 95 96 97 98 99 91 911 912 913 914 915 916 917 918 919 9 921 922 923 924 925 926 927 928 929 93 931 932 933 934 935 TABLE 3 ] Univariate Analysis of Risk Factors Associated With Recurrence-free and Overall Survival Characteristic, Risk Factor HR 95% CI P Value HR 95% CI P Value Clinical factors Sex, male 1.69.236-3.616.921 1.841.3-34.67.542 Age, older (per year) 1.18.979-1..375 1.16.969-1.65.51 Smoking, current/ex-smoker 3.37.59-55.494.215 1.793.318-33.548.558 Pathologic factors Tumor size, larger (per mm) 1.17.987-1.44.248 1.12.976-1.45.52 Stage III (vs I/II) 3.657 1.13-11.859.31 5.561 1.333-27.651.19 Pleural invasion, present 1.327.415-4.245.625 1.153.272-4.887.841 Lymphatic invasion, present 1.147.34-3.667.825 2.569.2-1.965.193 Vascular invasion, present 1.83.3-3.723.893 5.269.921-99.236.64 Spindle cell carcinoma component, absent 2.999.452-12.22.217 1.115.-6.291.9 Giant cell carcinoma component, present 1.482.439-6.714.546 1.213.248-4.973.794 Adenocarcinoma component, present 1.173.178-4.547.841 1.35.23-24.461.798 Squamous cell carcinoma component, present 1.443.384-4.595.559 1.893.387-7.739.399 Necrosis, present 8.138 2.137-53.51.1 1.2 1.815-191.664.6 STAS, present 5.415 1.614-24.4.6 13.171 2.337-246.419.2 HR ¼ hazard ratio. See Table 1 legend for expansion of other abbreviation. necrosis (P ¼.43). Furthermore, the statistical differences in RFS and OS curves between patients with extensive STAS and those with other subcategories significantly increased (P ¼.2 and.1, respectively) compared with patients in which STAS was merely classified as positive or negative. Although our cohort s small size precludes deriving definitive conclusions, these results indicate that the presence of extensive STAS in pleomorphic carcinoma reinforces the increased risk of poor survival in clinical settings. The present study had certain limitations. First, the statistical power may have been insufficient owing to the small number of patients; hence, additional larger series are required to confirm our findings. Second, it may sometimes be challenging to accurately distinguish STAS Recurrence-free Survival from the STAKS that results from specimen processing. However, there were no disagreements among the observers regarding the identification and assessment of STAS in our study, which attested to our results reproducibility. Third, the retrospective nature of our study created the possibility of selection bias; therefore, future prospective studies are required to validate these findings. Conclusions Overall Survival This study showed for the first time that STAS is somewhat common in patients with lung pleomorphic carcinoma, which was up to % in our study. We also found that STAS occurred concomitantly with tumor necrosis in these patients. Furthermore, we found that the presence of STAS was associated with poor RFS following TABLE 4 ] Multivariate Analysis of Risk Factors Affecting Recurrence-free and Overall Survival Recurrence-free Survival Overall Survival Characteristic, Risk Factor HR 95% CI P Value HR 95% CI P Value Stage III (vs I/II) 2.826.826-9.984.96 13.842 1.8-343.877.8 Vascular invasion, present. a. a. a 5.799.565-234.521.155 Necrosis, present 4.758 1.162-32.185.29 3.653.591-7.516.181 STAS, present 4.7 1.348-22.386.14 12.9 1.92-478.499.42 See Table 1 and 3 legends for expansion of abbreviations. a A characteristic with a P value >.1 on the univariate analysis that was therefore not analyzed in the multivariate model. 936 937 938 939 9 941 942 943 944 945 946 947 948 949 95 951 952 953 954 955 956 957 958 959 9 961 962 963 964 965 966 967 968 969 97 971 972 973 974 975 976 977 978 979 9 981 982 983 984 985 986 987 988 989 99 chestjournal.org 9 FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

991 992 993 994 surgical resection and, more importantly, with worse OS. These data indicate that STAS can be used as an additional marker to identify patients with pleomorphic 995 996 997 Acknowledgments 998 Author contributions: S. Y. had full access to 999 all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, including 1 and especially any adverse effects. S. Y., T. 2 M., H. T., H. O., A. H., R. M., M. F., M. H., H. I., and K. O. contributed to the conception 3 and design of the study; S. Y., T. M., H. T., 4 and H. O. contributed to the analysis and 5 interpretation of the data; S. Y. and H. T. drafted the submitted article; and K. O. and 6 Y. A. contributed to the critical revision of 7 the manuscript for important intellectual 8 property. 9 Financial/nonfinancial disclosures: None Q9 11 declared. 111 Other contributions: The authors are profoundly grateful to Masami Murakami, 112 Mai Nitta, and Hinata Fukusako for assisting 113 Q1 with the preparation of the lung specimens. 114 115 116 117 118 119 1 121 122 123 124 125 126 127 128 129 13 131 132 133 134 135 136 137 138 139 1 141 142 143 144 145 Q11 References 1. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG, eds. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. 4th ed. Lyon, France: IARC; 15. 2. Chang YL, Lee YC, Shih JY, Wu CT. Pulmonary pleomorphic (spindle) cell carcinoma: peculiar clinicopathologic manifestations different from ordinary non-small cell carcinoma. Lung Cancer. 1;34(1):91-97. 3. Yendamuri S, Caty L, Pine M, et al. Outcomes of sarcomatoid carcinoma of the lung: a Surveillance, Epidemiology, and End Results Database analysis. 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Lung Cancer. 15;87:39-44. 146 147 148 149 15 151 152 153 154 155 156 157 158 159 1 161 162 163 164 165 166 167 168 169 17 171 172 173 174 175 176 177 178 179 1 181 182 183 184 185 186 187 188 189 19 191 192 193 194 195 196 197 198 199 1 1 Original Research [ - # - CHEST - 2 1 8 ] FLA 5.5. DTD Š CHEST1778_proof Š 1 July 18 Š 2:7 am Š EO: CHEST-18-724

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