Histopathology and immunohistochemistry as prognostic factors for poorly differentiated thyroid cancer in a series of Polish patients.

BACKGROUND: Poorly differentiated thyroid cancer (PDTC) is a rare but aggressive type of thyroid cancer (TC) and the main cause of death from non-anaplastic follicular cell-derived TC. Although the Turin criteria are well defined, the pathological features that could serve as diagnostic and prognostic factors remain controversial.
MATERIALS AND METHODS: Forty-nine consecutive PDTC cases were identified in a single cancer center between 2000 and 2018. We analyzed the impact of routine histopathological and immunohistochemical features and several parameters that are not routinely included in pathology reports such as the presence of atypical mitoses, the amount of necrosis, or insulin-like growth factor-II mRNA-binding protein 3 immunostaining on the survival of patients with PDTC. Overall survival (OS) and disease-specific survival (DSS) were calculated using the Kaplan-Meier method.
RESULTS: Of the 49 PDTC 34 (69.4%) showed the insular pattern of growth. The median of poorly differentiated area was 95% (range, 1-100), and 30 (61.2%) patients had a predominant (>50%) insular area. The 5-year OS and DSS rates at a median follow-up of 57 months were 60.6% and 64.3%, respectively. Univariate analysis showed that tumor size >4 cm, presence of atypical mitoses, Ki-67 >5%, and thyroglobulin (Tg)-negative immunostaining were associated with a higher risk of PDTC-related death. Atypical mitoses and Tg negativity were independent factors of worse DSS in multivariate analysis. Patients with insular and predominant insular areas showed a 3- and 6-fold higher risk of PDTC death when they displayed atypical mitoses.
CONCLUSIONS: In PDTC, the presence of atypical mitoses may be helpful in identifying patients with poorer outcome and worth including in pathology reports, particularly in tumors with a dominant insular pattern of growth. Additionally, the inclusion of Tg immunostaining may be considered in a prognostic context, and not only as a diagnostic feature.

Introduction

Since the seminal reports in the early 1980s by Sakamoto et al.[1] and Cargangiu et al. [2] indicating that poorly differentiated thyroid cancer (PDTC) should be considered a distinct entity with an intermediate prognosis between differentiated thyroid cancer (TC) and anaplastic TC, studies focusing on this type of TC remain limited because of its relatively low incidence [3,4]. The reported prevalence rates vary from <1% of TCs to 6.7% depending on geographical regions [5,6]. It is likely that some genetic or environmental factors (including diet, iodine supply of studied population) may have an impact on the prevalence of PDTC in different areas [3,6]. Despite its rarity, PDTC is a clinically significant type of TC, as it is the main cause of death from non-anaplastic follicular cell-derived TC [3,6-10].

In 2004, the World Health Organization (WHO) recognized PDTC as a distinct entity among malignant thyroid tumors [11]. The recent 2017 WHO Classification of Endocrine Tumors defines PDTC as ‘a follicular cell neoplasm that shows limited evidence of follicular cell differentiation and is morphologically and behaviorally intermediate between differentiated (papillary or follicular) carcinomas and anaplastic carcinoma’ [12]. Because the diagnosis of PDTC is crucial for prognosis, the WHO definition is still relatively imprecise. It was therefore supplemented by a histopathological diagnostic algorithm termed the Turin criteria [13].

Based on architectural and high-grade features defined by the WHO and the Turin consensus, PDTC represents a heterogeneous group of malignant tumors originating from thyroid follicular cells. Hiltzik et al. [14] at Memorial Sloan Kettering Cancer Center (MSKCC) created the MSKCC-PDTC criteria to define PDTC according to the presence of mitosis and necrosis, and reported that PDTC is a homogenous and aggressive type of tumor in which growth patterns do not affect prognosis. Despite differences in the histologic definition of PDTC among pathologists, the recent 2017 WHO classification of endocrine tumors [12] does not recommend a diagnosis of PDTC in thyroid tumors that meet the MSKCC criteria, but do not meet the Turin criteria.

There is a universal agreement that proliferative grading (e.g., mitotic count or necrosis) should be described in pathology reports; however, the inclusion of non-routine parameters, such as the amount of necrosis or presence of atypical mitoses, remains debatable. The diagnosis of PDTC relies on histological features, and immunohistochemistry (IHC) is not required for diagnosis, although it may be useful. Recently, Asioli et al.[6] proposed the inclusion of a new prognostic factor in PDTC, namely, insulin-like growth factor-II mRNA-binding protein 3 (IMP3) expression, which can be evaluated by IHC.

The objective of the current study was to evaluate the impact of standard histopathological (HP) and immunohistochemical (IHC) features of PDTC, and several non-routine pathological parameters on the survival of PDTC-patients. IMP3 expression was included in the IHC analysis to validate its power as a prognostic marker for PDTC in routine clinical practice as a first study beyond the original description by Asioli et al. [6].

Materials and methods

Study design and patients

All study procedures were approved by the Ethics Committee (EC) of Holycross Chamber of Physicians in Kielce, Poland. All the patients’ data were fully anonymized before being accessed and leading to a review and subsequent analyses. Due to this fact, the EC waived the requirement for informed consent of the included patients.

The retrospective study was performed at Holycross Cancer Center (HCC), Kielce, a tertiary referral oncologic center in Poland, that provides comprehensive care for patients with TC and includes surgery, endocrinology, nuclear medicine, and radiation oncology departments, with similar characteristic as described in a previous study site [15]. The recruitment procedure resulting in a study group of 46 consecutive PDTC patients who underwent primary surgery between 2000 and 2017 was reported in details previously in the study with other objectives [15]. Subsequently, three PDTC cases were identified among 234 newly diagnosed TCs at HCC in 2018. Finally, the study group consisted of 49 consecutive PDTC patients retrieved in 2000–2018 among 2579 patients with TC diagnosed and treated in a single institution. The patients’ medical records were reviewed, all data related to the objectives of the study were analyzed. The initial risk stratification was estimated according to the 2015 American Thyroid Association (ATA) modified initial risk stratification system [16]. The staging of all cases was re-classified according to the 8th edition of the American Joint Committee on Cancer/Tumor-Node-Metastases (AJCC/TNM) staging system [17]. All details of the management protocols, including indications for postoperative treatment such as radioiodine (RAI) therapy, external beam radiation therapy (EBRT), or subsequent-line therapies [conventional chemotherapy (CHTH) and tyrosine kinase inhibitor (TKI) therapy], and follow-up protocols, were described previously [15]. Death of the patient was considered disease-related when hospital conclusions were unequivocal or a TC death certificate was available. In one case (1/49; 2.04%), death data were obtained from the Polish National Cancer Registry, which is an official Polish registry of the incidence and mortality of all cancers, because the patient discontinued follow-up at HCC before the study summary. All the remaining patients were actively monitored until their death or, if alive, to the last follow-up date on February 28, 2019.

Pathological review

Archival pathological materials (hematoxylin and eosin-stained slides, and formalin-fixed/paraffin-embedded blocks) were available for all cases. The diagnosis of PDTC was confirmed by two pathologists (JK and MH; both blinded to all the clinical data) independently based on the Turin consensus criteria as follows: a diagnosis of cancer of follicular cell derivation according to conventional criteria and (1) a solid, trabecular, or insular growth pattern; (2) absence of the conventional nuclear features of papillary TC; and (3) at least one of the following three features: convoluted nuclei (i.e., de-differentiated nuclear features of papillary cancer), ≥3 mitoses per 10 high-power fields (×400), and tumor necrosis [13].

The following features were assessed: tumor size, pattern of poorly differentiated (PD) growth, proportion of the PD area in each tumor, presence of predominant (>50%) insular pattern of growth and of well differentiated (WD) components, mitotic count and the presence of atypical mitoses, presence of tumor necrosis and its extent, convoluted nuclei, oncocytic features, and extent of vascular invasion.

The mitotic rate was determined by counting the number of mitotic figures in 10 high-power fields (×400) from hematoxylin and eosin-stained histological sections using a microscope (Olympus AX 60, Tokyo, Japan). According to the well-known protocol [18], the first field in each tumor lesion was selected randomly, and the following fields were sampled systematically using a mesh. On average, 10,000 nuclear profiles were counted per tumor lesion. Anything other than the typical form of normal mitoses, including asymmetrical, lagged, ring, multipolar, and anaphase‐bridge mitoses were recognized as atypical mitoses [19]. Fresh tumor necrosis was classified as absent, focal (≤5% of the tumor area), or extensive (>5% of the tumor area) [14]. An oncocytic variant was defined as a PD area containing >75% oncocytic cells [20]. Vascular invasion was defined according to the criteria of the Armed Forces Institute of Pathology fascicle regarding thyroid tumors [21] as the presence of four or more foci of vascular invasion [14].

Immunohistochemistry

IHC analysis of thyroglobulin (Tg; 2H11/6E1 cocktail), cytokeratin-19 (CK-19; RCK108), thyroid transcription factor-1 (TTF-1; 8G7G3/1), p53 (DO7), Ki-67 (MIB1), and IMP3 (L523) (all from Dako, Carpinteria, CA, USA) was performed on PD areas according to standard automated IHC procedures (Autostainer Link 48; Dako).

IHC expression of Tg, CK-19, TTF-1, p53, and IMP3 was scored according to the intensity and extent of staining using a semi-quantitative scoring system. The criteria for intensity scoring were as follows according to the well-known protocol [6]: 0 for no staining; 1+ for weak staining; 2+ for moderate staining; and 3+ for strong staining. The criteria for scoring the extent of staining were as follows: 0 for no staining; 1+ for 1–25%; 2+ for 26–50%; 3+ for 51–75%, and 4+ for 76–100% of stained neoplastic cells. The two scores were cumulated, and staining was considered positive when the combined IHC score was >2, whereas a final score of ≤2 was considered negative.

Ki-67 was scored according to the percentage of tumor cells expressing the proliferation marker Ki-67. The proliferation index was calculated for each tumor lesion by counting the total number of tumor cell nuclear profiles and the number of Ki-67-positive nuclear profiles in randomly selected fields [18]. Sections incubated identically except for replacement of the primary antibody by normal mouse IgG served as negative controls. Sections of normal tissue known to be immunoreactive for a test case were used as positive controls.

All quantified measurements and IHC-stained specimens were assessed by two pathologists (JK and MH) blindly and independently. In the case of discrepancies, consensus was reached regarding the mean value, which was considered for further analysis.

Statistical analysis

Categorical data were expressed as numbers and percentages, whereas numerical variables were presented as range, median, and interquartile range. Survival curves were created using the Kaplan-Meier method. Hazard ratios (HRs) with 95% confidence intervals (95% CIs) for univariate and multivariate analyses were calculated using the Cox proportional-hazards model. Multivariate analysis included variables that were significantly related to survival in the univariate analysis. A two tailed p-value <0.05 was considered statistically significant. All statistical analyses were performed using R (version 3.1.2; The R Foundation for Statistical Computing, Vienna, Austria) and Statistica [TIBCO Software Inc. (2017) Statistica (data analysis software system), version 13; http://statistica.io].

Results

Clinical characteristics

The prevalence of PDTC in the HCC database was 1.89% (49/2579). The clinicopathological characteristics are presented in Table 1. The slight female preponderance was noted with the F/M ratio 2.1:1. In one case (2.0%), the initial ATA risk remained unknown because of insufficient data to distinguish high risk from intermediate risk.

Table 1

Clinical and pathological characteristics of the 49 patients with poorly differentiated thyroid cancer at the presentation stage.

Feature; all cases (n = 49)
Median age, y (Q1–Q3; range)	63 (50–70; 15–85)
Age ≥55 y, n (%)	32 (65.3)
Gender, n (%)
Female	33 (67.3)
Male	16 (32.7)
Median tumor size, cm (Q1–Q3)	4.55 (3.0–7.0)
Range	1.4–13.0
Tumor size, cm, n (%)
≤ 4	19 (38.8)
> 4	29 (59.2)
Unknown	1 (2.0)
ATA initial risk, n (%)
Intermediate	26 (53.1)
High	22 (44.9)
Insufficient data	1 (2.0)
TNM stage, n (%)
I–II	41 (83.7)
III–IV	8 (16.3)
Tumor pattern of PD growth, n (%)
Solid	12 (24.5)
Trabecular	3 (6.1)
Insular	34 (69.4)a
PD area, n (%)
< 10%	3 (6.1)
≥ 10%–<50%	7 (14.3)
> 50%	38 (77.6)
Unknown	1 (2.0)
Median PD area, % (Q1–Q3; range)	95 (75–100; 1–100)
Predominant (≥50%) insular pattern of growth, n (%)	30 (61.2)
Presence of well differentiated components, n (%)	24 (49)
Papillary cancer ≤25%, n (%)	3 (6.1)
Follicular cancer ≤25%, n (%)	10 (20.4)
Unknown	1 (2.0)
Oncocytic variant, n (%)	2 (4.1)
Presence of necrosis, n (%)	34 (69.4)
Median amount of necrosis, % (Q1–Q3; range)	1 (0–10; 0–50)
Extensive necrosis (>5% of the tumor area), n (%)	13 (26.5)
Convoluted nuclei, n (%)	32 (65.3)
Mitotic count ≥3/10 high-power fields, n (%)	15 (30.6)
Atypical mitoses, n (%)	18 (36.7)
Extensive vascular invasion (≥4 foci), n (%)	15 (30.6)

a in one case, insular pattern of growth was determined in PDTC metastatic node

Abbreviations: ATA, American Thyroid Association; TNM, tumor-node-metastasis; PD, poorly differentiated.

Of the 49 patients, 48 (98%) underwent surgery as follows: in 85.7% total or near-total thyroidectomy was performed, and 12.3% patients underwent partial resection if complete resection was not achieved intraoperatively. In one case (2.0%), intraoperative procedures allowed to obtain a diagnosis of PDTC due to a HP report of PDTC metastasis to the supraclavicular lymph nodes. A primary thyroid tumor was unresectable in that case, but metastatic lymph node associated with a tumor were partly removed. Despite this fact, all HP and IHC features other than tumor size, and the proportion of PD/WD areas in the primary tumor, were determined from retrieved PDTC tissues. Lymph node dissection (LND) related to primary thyroid surgery was performed in 31 (63.3%) cases, whereas 17 patients (34.7%) showed no evidence of primary LND. The one patient (2.0%) with the unresectable thyroid mass mentioned above underwent removal of supraclavicular nodes. Postoperative RAI therapy was administered in 39 (79.6%) patients. Of the 49 studied patients, 21 (42.9%) received EBRT, however five (10.2%) received EBRT as single postoperative therapy. One (2.0%) received EBRT alone as palliative therapy.

During the follow-up period, 24 patients (49%) required more than one RAI therapy course (2–9 courses; activities: 2700–5550 MBq), and eight patients (16.3%) received systemic therapy as follows: three (6.1%) received CHTH, four (8.7%) received CHTH, and TKI (lenvatinib or vandetanib due to patients’ participation in clinical trials) therapy, and one (2.0%) received TKI (sorafenib) alone. Twenty-three patients (46.9%) died. Disease-specific death was documented in 20 cases (40.8%).

Pathological parameters

The summary of the pathological review is presented in Table 1. The median tumor size was 4.55 cm (range, 1.4–13 cm), and 29 patients (59.2%) had a primary tumor measuring >4 cm. Solid, trabecular, and insular patterns of growth were identified in 12 (24.5%), 3 (6.1%) and 34 (69.4%) patients, respectively (Fig 1). The PD area was >50% in most tumors (38/49; 77.6%), and the median PD area was 95% (range: 1–100). Of the tumors analyzed, 24 (49%) had a WD component, although in 13 tumors (13/49; 26.5%), the WD component occupied <25% of the tumor. A predominant (≥50%) insular pattern of growth was detected in 30 tumors (61.2%). A mitotic count ≥3/10 high-power fields was observed in 15 (30.6%) cases, and atypical mitoses were found in 18 tumors (36.7%) (Figs 1A and 2A). Necrotic areas were present in 34 (69.4%) tumors (Fig 1C), and the median rate of necrosis was 1% (range: 0–50); necrosis was extensive in 13 cases (26.5%) (Fig 2B).

Fig 1

Poorly differentiated thyroid cancer (PDTC) in H&E stain.

(A) PDTC with predominant solid growth pattern with atypical mitosis (×400), (B) trabecular pattern of growth (×200), and (C) insular pattern of growth with necrosis (×200).

Fig 2

Histological findings (H&E stain) in poorly differentiated thyroid cancer.

(A) Atypical mitosis in a tumor with a predominant insular pattern of growth (×400), and (B) extensive necrosis (×200). Thyroglobulin immunohistochemistry: (C) positive and (D) negative.

Immunohistochemical analysis

IHC analyses were performed in 48 cases (48/49; 98%), as samples from one tumor collected in 2004 were unsatisfactory. According to the ethic statement described above, all the patients’ follow-up data were analyzed anonymously. However, the analysis of the medical record of one excluded IHC patient concluded that this patient is still alive and meets the criteria for an excellent response after primary therapy and maintains it on the summary date. The results of IHC analyses are summarized in Table 2. Most cases showed positive thyroglobulin (Fig 2C) and TTF-1 immunostaining (37/49; 77.1% and 42/49; 87.5%, respectively). Positive immunostaining for p53 and IMP3 was observed in 11 (23%) and 7 cases (14.6%), respectively. The median Ki-67 proliferative index was 5% (range, 1–80%).

Table 2

Immunohistochemistry in poorly differentiated thyroid cancer (n = 48).

Immunohistochemical feature	n (%)
Thyroglobulin positive, n (%)	37 (77.1)
CK-19 positive, n (%)	13 (27.1)
TTF-1 positive, n (%)	42 (87.5)
p53 positive, n (%)	11 (22.9)
Ki-67, %, median (Q1–Q3; range)	5 (2–10; 1–80)
IMP3 positive, n (%)	7 (14.6)

Abbreviations: CK-19, cytokeratin-19; TTF-1, thyroid transcription factor-1; IMP3, insulin-like growth factor-II mRNA-binding protein 3.

Survival analysis

The median follow-up was 57 months (range, 1–187 months; Q1–Q3, 30–101). The 5 and 10-year overall survival rates were 60.6% and 53%, and the disease-specific survival (DSS) rates were 64.3% and 56.3%, respectively. Seven patients (14.3%) died of the PDTC within 1 year from the diagnosis. This affected the mean values, as the 1 year DSS rate decreased to 85.4%.

Tumor size >4 cm, presence of atypical mitoses, Tg-negative immunostaining, and Ki-67 >5% were predictive factors of worse outcome based on univariate analysis, as shown in Table 3 and Fig 3. Because in most of the PDTC cases analyzed the PD area was >50% and the median PD area was 95%, univariate analysis was extended to PD threshold levels of ≥50% and 95% as predictive factors of DSS; however, there was no significant effect on survival. Patients with insular PDTC (n = 34) and those with a predominant insular area (n = 30) had a 3- and 6-fold higher risk of PDTC death, respectively, when they displayed atypical mitoses (Table 4). Multivariate analysis showed that only the presence of atypical mitoses and Tg negativity (Fig 2D) were independent factors of worse DSS (Table 5).

Table 3

Predictive factors of disease-specific survival in PDTC patients.

	Characteristics of DSS		Univariate analysis
Variable	5 year DSS, %	10 year DSS,%	HR [95% CI]	P
Age,y
< 55	63	63	Ref.lev.
≥ 55	64	51	1.0 [0.4–2.6]	0.93
Gender
Female	64	61	Ref.lev.
Male	64	48	1.2 [0.5–3.0]	0.73
Tumor size, cm
≤ 4	80	80	Ref.lev.
> 4	58	46	3.9 [1.1–13.3]	0.033
Pattern of growtha
Solid	58	58	1.4[0.5–3.9]	0.56
Insular	64	56	Ref.lev.
Trabecular	-	-	-
PD area
≤ 50%	70	54	Ref.lev.
> 50%	64	61	1.2[0.4–3.5]	0.78
PD area
≤ 95% (median value)	75	66	Ref.lev.
> 95%	58	52	1.6 [0.6–4.1]	0.31
Predominant insular pattern of growth
No	65	54	Ref.lev.
Yes	65	60	1.1[0.4–2.7]	0.91
Presence of WD component
No	58	52	1.6 [0.6–4.1]	0.31
Yes	75	66	Ref.lev.
Presence of necrosis
No	72	72	Ref.lev.
Yes	62	52	1.7 [0.6–5.2]	0.33
Amount of necrosis
≤ 1% (median value)	62	62	Ref.lev.
> 1%	67	51	1.4 [0.6–3.3]	0.47
Extensive necrosis >5% of the tumor area, n (%)
No	66	56	Ref.lev.
Yes	58	58	1.5 [0.6–3.8]	0.43
Convoluted nuclei
No	63	46	Ref.lev.
Yes	65	65	0.6 [0.3–1.5]	0.29
Mitosis ≥3/10 high-power fields
No	68	62	Ref.lev
Yes	57	46	1.7 [0.7–4.6]	0.25
Atypical mitoses
No	78	69	Ref.lev.
Yes	41	34	3.2 [1.3–7.8]	0.01
Extensive vascular invasion
No	68	60	Ref.lev.
Yes	57	48	1.6 [0.6–3.8]	0.34
Ki-67 >5%
No	71	63	Ref.lev.
Yes	42	32	2.8 [1.1–7.2]	0.03
TTF-1 positive
No	67	67	Ref.lev.
Yes	63	53	0.9 [0.3–3.0]	0.82
p53 positive
No	67	60	Ref.lev.
Yes	51	40	1.9 [0.7–5.0]	0.19
Thyroglobulin positive
No	41	41	3.3 [1.3–8.5]	0.01
Yes	70	59	Ref.lev.
CK-19 positive
No	65	53	Ref.lev.
Yes	62	62	1.1 [0.4–2.8]	0.88
IMP3 positive
No	62	62	Ref.lev.
Yes	69	26	1.3 [0.4–3.8]	0.69

a patients with a trabecular pattern of growth were excluded because the number was too low (n = 3) for reliable analysis.

Abbreviations: DSS, disease-specific survival; PD, poorly differentiated; WD, well differentiated; CK-19, cytokeratin-19; TTF-1, thyroid transcription factor-1; IMP3, insulin-like growth factor-II mRNA-binding protein 3; HR, hazard ratio; CI, confidence interval; Ref. lev., reference level.

Fig 3

Kaplan-Meier analysis of disease-specific survival in patients with poorly differentiated thyroid cancer.

According to (A) tumor size, (B) presence of atypical mitoses, (C) thyroglobulin-negative immunostaining, and (D) Ki-67 >5%.

Table 4

The presence of atypical mitoses as a predictive factor of DSS in PDTC patients with an insular (n = 34), and a predominant (>50%) insular (n = 30), pattern of growth.

	Characteristics of DSS			Univariate analysis
Variable	Patients, n (%)	5 year DSS, %	10 year DSS, %	HR [95% CI]	P
Atypical mitoses in insular PDTC (n = 34)
No	22 (64.7)	80	70	Ref.lev.
Yes	12 (35.3)	44	33	3.0 [1.04–8.8]	0.04
Atypical mitoses in PDTC with predominant insular pattern of growth (n = 30)
No	19 (63.3)	80	80	Ref.lev.
Yes	11 (36.7)	38	NAa	6.0 [1.5–23.5]	0.01

a the longest follow-up was <10 years (76 months), and the patient is alive; the 10 year DSS could not be calculated.

Abbreviations: PDTC, poorly differentiated thyroid cancer; DSS, disease-specific survival; HR, hazard ratio; CI, confidence interval; Ref. lev., reference level.

Table 5

Multivariate analysis of DSS in PDTC patients performed using the Cox proportional-hazards model.

	Multivariate analysis
Variable	HR [95% CI]	P
Tumor size, cm
≤ 4	Ref.lev.
> 4	3.3 [0.9–11.9]	0.064
Atypical mitoses
No	Ref.lev.
Yes	4.1 [1.5–11.0]	0.005
Ki-67 >5%
No	Ref.lev.
Yes	0.5 [0.1–2.5]	0.40
Thyroglobulin positive
No	3.9 [1.3–11.3]	0.014
Yes	Ref.lev.

Abbreviations: DSS, disease-specific survival; PDTC, poorly differentiated thyroid cancer; HR, hazard ratio; CI, confidence interval; Ref. lev., reference level.

Discussion

Despite a rapid increase in the incidence of TC worldwide including in Poland, PDTC remains a rare type of TC [22-25]. The prevalence of PDTC has remained stable in Poland, accounting for 1.75% in 2010 [26] and 1.89% in the present study, which is consistent with data on PDTC [12]. Nevertheless, PDTC remains a challenge for pathologists and clinicians because of difficulties associated with the diagnostic process and its aggressive disease course, which requires intensive management and close follow-up [27].

The present study was a comprehensive analysis of several pathological features in a series of consecutive PDTC tumors identified using the Turin criteria to determine their prognostic value in routine clinical practice. According to the ATA guidelines [16], standard surgical pathology reports in TC should include a description of basic tumor features and additional information that may be helpful for determining prognosis. However, there is no clear statement regarding the specific HP or IHC findings that should be included because of their prognostic value in PDTC. The prognostic role of a larger tumor size or tumor necrosis was reported previously [6,28]. In the current study, patients with tumors >4 cm had an almost 4-fold higher risk of death than those with smaller tumors. This result was obtained in the univariate analysis (HR = 3.9; 95% CI: 1.1–13.3; p = 0.033), whereas in the multivariate analysis, the difference was not significant. A similar result was reported by Asioli et al. [6], who showed the prognostic value of a larger tumor size in univariate, but not in multivariate analysis.

It remains debatable whether the cut-off value for the PD area should be reported in PDTC tumors. The recent WHO classification [12] does not recommend using a cut-off value for the PD area to diagnose PDTC, and the prognostic implications of the proportion of PD area are equivocal. Volante et al. [29] reported that the extent of the PD area has no effect on overall survival in a large series of PDTC. Gnemmi et al. [28] reported no significant relation between the percentage of PD area and cancer-specific survival. Dettmer et al. [30] showed that even the presence of a small PD area of 10% had an effect on patient survival; however, Dettmer’s study was based on a selected group of 42 PDTC cases with an adverse clinical outcome (recurrence or death). In the present study, which consisted of consecutive PDTC cases, a PD area was present in more than half of the tumor tissue in most patients, with a predominant insular pattern of growth and a high median PD area of 95%. These results suggest that the group of non-selective cases analyzed consisted of virtually ‘pure’ PDTC tumors. The low number of PDTC cases with <10% of PD areas prevented a reliable analysis of 10% PD as a predictive factor of DSS. Nevertheless, we analyzed the effects of 50% and 95% (median value) as cut-off values, and the results showed no significant impact on DSS.

The prognostic significance of several high-grade features were reported in PDTC previously [6,14,28]. In the current study, only Ki-67 >5% and the presence of atypical mitoses correlated with decreased DSS in univariate analysis, whereas only atypical mitoses were an independent factor of worse outcome in multivariate analysis. This finding was unexpected because there are limited data [6,28] for determining the prognostic value of atypical mitoses in PDTC, and their prognostic significance in this type of TC has not been reported. Additionally, we analyzed the subgroups of insular pattern of growth and with predominant insular area, and found that patients in both subgroups had significantly shorter DSS, particularly when a dominant insular growth pattern coexisted with the presence of atypical mitoses. A recent review by Setia et al. [31] reported that, although atypical mitoses are present in PDTC, they are less common than in anaplastic TC. Atypical mitoses are also detected in other malignancies, although they are considered a prognostic factor only in pancreatic and breast cancers [19,32-34].

IMP3 detected by IHC was announced as a promising, cost-effective prognostic parameter; however, positive IMP3 immunostaining had no impact on patient survival in the present study, which is in disagreement with the results of Asioli et al. [6]. This result could be explained as follows: the number of IMP3-positive tumors was low in the group analyzed despite the use of the same methodology for IMP3 immunostaining recommended by Asioli for introduction into routine clinical practice; the Asioli’s group was heterogeneous, and consisted of subgroups from two regions (the USA and Northern Italy) that differed significantly regarding the presence of an insular growth pattern, whereas the present group was homogenous and had a prominent insular pattern of growth. Finally, the impact of potential undefined population-dependent factors cannot be excluded. However, we found that convoluted nuclei were associated with a better prognosis according to Asioli’s result [6], but in opposite to other report [28].

According to the WHO classification, a diagnosis of PDTC relies on histological features, and the presence of cell follicular differentiation. However, the use of an immunostaining panel may be supportive in difficult cases, e.g., Tg positivity can confirm an inconclusive diagnosis. Tg expression can be relatively decreased in PDTC and lost in anaplastic TC in parallel with thyroid cell de-differentiation from well-, via poorly-, to un-differentiated TC [6,13,35]. Although Tg negativity introduces a diagnostic challenge, it may be useful to predict a poor outcome in PDTC.

The present study had several limitations, including the small size of the study group and the retrospective design. The rarity of PDTC underlies the limited number of patients included, although 49 consecutive PDTC patients were analyzed. However, 49 patients were recruited from a 19-year database from one cancer center with consistent procedures for diagnosis, therapy protocols, and follow-up. Despite the fact that the study group is low, it may be exploitable for meta-analyses aimed for identification of the prognostic value of HP/IHC parameters in PDTC. In addition, IMP3 was assessed by IHC only. We did not perform a parallel molecular analysis in PDTC of 100% PD areas, as performed by Asioli et al. [6] to promote the use of IHC for IMP3 detection in routine clinical practice. Unfortunately, the present study does not provide any mutational status of PDTC cases, but the study group may constitute a base for further analyses of genetic alteration in PDTC of the Polish population. This would be highly advantageous to our knowledge-based approach with regards to the recent study on the genomic landscape of PDTC and its impact on the clinical outcome as reported by Landa et al. [36]. Meanwhile, clinicians may be supported by pathologists in identifying PDTC-patients with a higher risk of death. The surgery report of PDTC which is extended to the presence of atypical mitoses or Tg negativity in an IHC panel may be used as a potentially cost-effective diagnostic procedure of PDTC in cases of unknown genetic profiles in routine clinical practice.

Conclusions

Standard surgical pathology reports in PDTC could be extended by the addition of several pathological parameters, which may be useful as prognostic factors. Further studies are still needed to validate the usefulness of IMP3 immunostaining for determining the prognosis of PDTC. The presence of atypical mitoses might be worth reporting, particularly in cases with a prominent insular pattern of growth. In addition, the inclusion of Tg immunostaining may be considered in a prognostic context, and not only as a diagnostic feature. However, all significant results of the current study need further analyses to confirm their potential prognostic value in the patients with PDTC.

16 Dec 2019

PONE-D-19-30397

Histopathology and Immunohistochemistry as Prognostic Factors for Poorly Differentiated Thyroid Cancer in a Series of Polish Patients

PLOS ONE

Dear Dr. Walczyk,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jan 30 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Jason Chia-Hsun Hsieh, M.D. Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded )as separate "supporting information" files

3. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records/samples used in your retrospective study. Specifically, please ensure that you have discussed whether all data/samples were fully anonymised before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data/samples from their medical records used in research, please include this information.

Additionally, in regard to the following sentence: "IHC analyses were performed in 48 cases (48/49; 98%), as samples from one tumor collected in 2004 were unsatisfactory. However, the patient is still alive and meets the criteria for an excellent response after primary therapy and maintains it on the summary date.", please clarify whether patients' follow-up data were retrieved from anonymised medical records and authors did not have access to identifying information.

4. We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed:

https://doi.org/10.1038/sj.bjc.6601453

https://doi.org/10.1038/modpathol.2010.117

https://doi.org/10.1111/cen.13910

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes a study where different IHC markers and histopathology features were associated with a poorer survival in a cohort of patients with Poorly Differentiated Thyroid Carcinoma (PDTC). As the authors state in the paper, PDTC, although rare, remains as the main cause of death from non-anaplastic follicular cell-derived thyroid cancers with a mean survival of 3.2 years, in part for a limited benefit from Radioiodine therapy. Therefore, there is an actual need for better diagnostic and prognostic markers. The authors conclude that in addition to the current recommendations by international guidelines for PDTC diagnosis and risk stratification, features such as the presence of atypical mitosis and negative Thyroglobulin (Tg) staining will be useful to identify those PDTC patients with a poorer outcome and worse prognosis.

Overall, this study is well conceived and executed. The manuscript is well written and the presented data support the conclusions.

Specific comments are listed that the reviewer hopes will improve the presentation:

1. As the authors already commented several times in the paper, the percentage of PDTC cases among all types of thyroid carcinomas may vary approximately from 1% to 7%. The series presented in this study represent a 1.89 % (49 out 2579 thyroid cancer cases in a single institution over a period of 18 years), which they claim is in concordance with other studies from Poland, the country where the institution is located. Previous papers published by the group of Dr. James A Fagin at MSKCC (Landa I, Ibrahimpasic T, et al., JCI 2016; Fagin JA, Wells SA, et al. N Eng J Med 2016), with data from the United States, state the PDTC prevalence rate is 6%. Can the authors describe what could be causing this difference and how the different pathological criteria (Turin vs MSKCC-PDTC definition) may affect this discrepancy?

2. This work doesn´t show any data on the mutational status of the studied samples. In the 2016 JCI paper from Landa I et al., it is shown that PDTCs represent an intermediate entity between Papillary and Anaplastic Carcinomas (PTCs and ATCS, respectively) regarding differentiation and mutational status. In this paper, PDTCs fulfilling the Turin definition were enriched in RAS mutations while MSKCC-PDTCs were enriched in BRAF mutations. Moreover, Landa et al. showed that mutational burden, EIF1AX mutations (co-ocurring with RAS mutations) and TERT promoter mutations – among other clinicopathological characteristics like gender or tumor size- were associated with survival in PDTC patients. The paper would gain a lot of relevance if the authors could provide mutational status of the referred genes. Even if authors are not able to deliver a genetic characterization, it would still be interesting to discuss this in the paper and how atypical mitosis or Tg staining may represent or not a more feasible and money-wise approach than sequencing in clinical practice.

Reviewer #2: This is an interesting article about using hisopathology and immunohistochemistry as prognostic factors for poorly differentiated thyroid cancer. The author concluded the presence of atypical mitoses may be predictor of mortality in patients with poorly differentiated thyroid cancer. The article has important clinical value due to rarity of poorly differentiated thyroid cancer patients. However, several questions should be made clear:

1. The sample size is relative small (n=49), please provide evidence of adequate power and sample size needed for testing main effect or interaction effect in this survival analysis.

2. Extrathyroid extension has been shown to related to mortality in patients with poorly differentiated patients. Please provided the % of extrathyroid extension and be included in multivariate analysis.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

21 Jan 2020

Academic Editor:

On behalf of myself and all co-authors, I would like to thank you for your revision and the constructive guidelines and comments, which will let us improve the manuscript to be accepted for the publication in PLOS ONE.

Question 1.

The revised manuscript was improved according PLOS ONE’s style templates to meet the journal requirements which were presented in particular guidelines for Authors.

Question 2.

All the tables were re-designed according to the PLOS ONE’s formatting rules and included to the revised manuscript after paragraphs in which they had been cited first. Additionally, all the figures captions were included in the relevant places of the revised manuscript, but the figure files were submitted in individual files in formats ‘.tiff’ according to the PLOS ONE’s formatting rules.

Question 3.

The ethics statement was extended to the statement that all the initial data as well as follow-up data were analysed anonymously as well as the ethics committee waived the requirement for informed consent (line 92-95). We also highlighted that pathologists were blinded to the clinical data and outcome (line 123). In regard to the sentence about results of IHC analyses we added again the clear statement that the follow-up data were analysed anonymously including a medical record of a patient without IHC analysis but with known clinical outcome (line 232-234).

Question 4.

Unfortunately, your HTML markup was not attached to your post. However, we scanned our manuscript by an overlapping program and finally we found few sentences in the Methods section and improved them. According to the cited by pathologists protocol of mitotic rate and Ki-67 in the Methods section [identified by you as https://doi.org/10.1038/sj.bjc.6601453] in line 136 and 162, we added a new reference in that paragraphs as 18th position. So, all the references cited after [REF18] were re-counted and ordered de novo.

We also added REF 6 [identified by you as https://doi.org/10.1038/modpathol.2010.117] in line 153. We cited our own work [REF 15; identified by you as https://doi.org/10.1111/cen.13910], in the Methods section, line 99. Outside this section we rephrased the text of our previous work in lines 182-183, 191-196 in the Results section. We also found that the sentence in lines 139-140 in the Methods section requires revision and we rephrased it.

The overlapping program did not indicate any more overlapping text outside the Methods section which should have been revised.

The additional authors’ remark: in the revised manuscript an update in the funding statement was included, according to the updated Jan Kochanowski University in Kielce requirements. We also changed the affiliations of two co-authors (SG, AK) into the updated name: ‘Collegium Medicum’, Jan Kochanowski University, Kielce.

Reviewer 1:

On behalf of myself and all co-authors, I would like to thank you for your revision of our manuscript and the constructive guidelines and comments, which will let us improve the manuscript to be accepted for the publication in PLOS ONE.

Question 1.

The incidence of PDTC is not reported as being similar to other different studies. The recent WHO classification of endocrine tumors [REF 12 in manuscript] indicates that the frequency of PDTC varies from less than 1% (e.g. in Japan) to 6.7% in Northern Italy. However, it is worth noting that the lower percentage of PDTC is usually reported in populations of sufficient iodine supply. Firstly, in 2007, Sanders et al. in their well-known study put our attention on the potential impact of environmental, e.g. related to dietary factors (including iodine), or some genetic factors on the occurrence of PDTC in the patients’ population from different areas. In Northern Italy, which was a region of a long-term insufficient supply of iodine, the occurrence was reported to be up to 15%, however, a later report showed a smaller proportion of PDTC, accounting for only 6.7% in a number of Italian patients [Asioli et al., 2010; REF 6 in manuscript]. Eventually, the WHO indicates a rate of 6.7% as being the highest reported figure from Europe.

The Reviewer cites two studies based on PDTC patients from the USA, which is a leading country of sufficient iodine supplementation on a population level, and comments that the prevalence ratio was 6% from the US data. We would kindly like to remark, that in the first cited study published by Landa et al., we did not find any piece of information about the frequency of PDTC in the American population, despite the fact there was a very interesting analysis of the mutational status of 117 thyroid cancers containing 84 PDTC cases. In the second, by Fagin et al., we can indeed find the Authors’ note about the PDTC prevalence rate of 6%, but it is worth noting that the paper was a review, not an original study, and the Authors do not indicate any reference to the source of this fact. In summary, we do not find any evidence for such a high prevalence of 6% PDTC-patients from the USA, which is a country of long-term sufficient iodine supply.

So far, the occurrence of PDTC in the USA, was reported as low as 1.8% (56/3128) in the original study by Asioli et al. from the well-known Mayo Clinic, in the USA, in which the Authors identified PDTC on the basis of the Turin criteria according to the recommendations by WHO in 2004 and 2018. The rate of 1.8% is indicated by the recent WHO classification as an evidenced rate of PDTC in patients from the USA. Additionally, we would like to note that Poland has been listed as an European country of sufficient iodine supply for close to 20 years after the introduction of the mandatory iodine prophylaxis program in 1997. This fact had been documented by the WHO in nationwide population-based studies performed in 2003 under the supervision of the International Council for the Control of Iodine Deficiency Disorders (ICCIDD; now Iodine Global Network [IGN]; http://ign.org/) and its Polish branch, and published by WHO in 2007 [Andersson M, de Benoist B et al. (eds) 2007 Iodine Deficiency in Europe, WHO]. In view of this data, our result of 1.89% of PDTC in Polish patients remains in accordance with the US data.

Despite the consideration above, we can agree with the Reviewer’s main remark, that the variation of PDTC incidence is an established fact, and the potentially causing factors have been added (line 57-59) to the revised manuscript.

It is agreed that the pathologists can identify PDTC, however its histological definition is still under debate among pathologists. The use of the MSKCC criteria may probably better identify PDTC patients of high risk rather than the use of the Turin criteria. However, the recent WHO classification does not recommend a diagnosis of PDTC in thyroid tumors that meet the MSKCC criteria, but do not meet the Turin criteria. This statement is clearly written in the manuscript (line 74-76). The prevalence of PDTC in Poland is reported as based on the Turin criteria, according to the WHO recommendations. The MSKCC criteria was proposed on the basis of proliferate grading and that’s why they are known as more restrictive than the Turin criteria. The use of the MSKCC criteria will most likely decrease the amount of PDTC. The incidence of PDTC will be lower than the reported 1.89% (based on the Turin criteria) which are an expected result of the use of more restrictive pathological criteria with subsequent potential consequences being an unreliable analyses of very low subgroups. Even though, such analysis has not been performed, we would like to bring the Reviewer’s attention to the fact that the present study was designed on the basis of the present WHO definition of PDTC which still recommends the use of the Turin criteria.

Question 2.

Unfortunately, the mutational status of studied PDTC cases was not performed due to a financial aspect. We agree with the Reviewer’s remark, that such analysis would be very interesting, but it was not the aim of the current study. However, we consider it and we perform it willingly provided that the authors’ institution fund it.

According to the Reviewer’s comment we agree that the potential clinical usefulness of a presence of atypical mitoses or Tg immunostaining in surgery report of PDTC should be more widely discussed. In our opinion this statement is more highlighted in the revised manuscript (lines 354-363), however a few comments were written in the primary version of the manuscript.

Reviewer 2.

On behalf of myself and all co-authors, I would like to thank you for your revision of our manuscript and the constructive guidelines and comments, which will let us improve the manuscript to be accepted for the publication in PLOS ONE.

Question 1.

We agree that the sample size is small. However, we mention that all subsequent patients with PDTC who came to our center between 2000 and 2018 were included, so it is not possible to increase the sample size to obtain the power of the tests at 80% level. Below we present the post-hoc calculations power of tests for the results included in Table 3. In that calculations, HR from Table 3 was taken as the assumed value of the effect. In addition, we also present the calculated sample sizes that would be necessary to achieve test power at 80%. All calculations were made using R statistical software with powerSurvEpi package.

Nevertheless, we mention that post-hoc calculations for test power (and especially the use of the observed effect value) are criticized by some authors (see e.g. https://journals.lww.com/annalsofsurgery/Citation/2019/01000/Don_t_Calculate_Post_hoc_Power_Using_Observed .46.aspx ).

Variable Variable

levels HR

(point estimate) Power of test

(post-hoc calculations) Sample size needed

for power of test = 80%

Age, yrs < 55 Ref. level 0.03 19665

>=55 1.04 37016

Gender Female Ref. level 0.06 1703

Male 1.2 826

Tumor size, cm <=4 Ref. level 0.63 29

>4 3.9 44

Pattern of PD growth Solid 1.4 0.09 237

Insular Ref. level 672

PD area <=50% Ref. level 0.05 867

>50% 1.2 3292

PD area <=95% Ref. level 0.17 190

>95% 1.6 190

Predominant insular pattern of growth No Ref. level 0.04 3639

Yes 1.1 6065

Presence of WD component No 1.6 0.17 190

Yes Ref. level 190

Presence of necrosis No Ref. level 0.17 121

Yes 1.7 274

Amount of necrosis <=1% Ref. level 0.11 383

>1% 1.4 312

Extensive necrosis > 5% No Ref. level 0.13 412

Yes 1.5 149

Convoluted nuclei No 0.6 0.23 92

Yes Ref. level 173

Mitosis >=3/10 high-power fields No Ref. level 0.21 201

Yes 1.7 89

Atypical mitoses No Ref. level 0.72 38

Yes 3.2 22

Extensive vascular invasion No Ref. level 0.20 220

Yes 1.6 97

Ki-67 > 5% No Ref. level 0.15 47

Yes 2.8 404

TTF-1 positive No Ref. level 0.04 1607

Yes 0.9 11248

p53 positive No Ref. level 0.27 161

Yes 1.9 48

Thyroglobulin positive No 3.3 0.73 14

Yes Ref. level 44

CK-19 positive No Ref. level 0.04 6986

Yes 1.1 2595

IMP3 positive No Ref. level 0.07 1333

Yes 1.3 228

Based on https://acsjournals.onlinelibrary.wiley.com/doi/full/10.1002/cncr.29924 small, medium, and large HRs comparing 2 groups would be approximately 1.3, 1.9, and 2.8.

In the case of the results given in Table 3, in many cases the effect measured with HR is small (i.e. below 1.3) and only in 4 cases HR is greater or equal than 2.8. In all of this 4 cases (tumor size> 4, atypical mitoses, Ki-67> 5%, thyroglobulin positive) HR was statistically significant in our group of patients, and therefore our sample size was sufficient to confirm the occurrence of that large effects.

Question 2.

The majority of the present group (46/49) was recruited to the study focused on PDTC with other objectives [REF 15 in manuscript] which was designed and performed in our institution previously, as we described in the Methods section [line 100]. In that study the main aim was to validate the recent 8th Edition of AJCC/TNM staging system in a stratification of a risk of death of the patients with PDTC and evaluate the impact of the updated TNM stage on the patients’ survival. As we know, the major changes of the recent 8th Ed AJCC/TNM staging system comprised of an increase in the cut-off age from 45 to 55 years of age at diagnosis, and the removal of lymph nodes metastases and minor extrathyroidal extension (ETE) from the definition of tumor primary pT3 stage and resulted in a general trend towards the downstaging of TNM staging system. Hence, according to the aim of the previous study, the prognostic role of ETE in different aspects: presence or absence of ETE in PDTC, ETE extent (minor/gross) and their impact on PDTC-patients’ survival has been already analyzed in our 46 cases of PDTC and reported in detail previously [REF 15 in manuscript]. We agree that the presence/absence of ETE is an obligatory element in a surgery report of thyroid cancer, but its prognostic role was validated in the basis of our PDTC and an extension of the current analysis to 49 cases will not likely change the result, and it could be an overlapping analysis, so we decided not to perform it again.

To summarise, we hope both the Reviewers and the Editorial Board will find the responses to the comments discussed above as satisfactory.

Yours sincerely,

Agnieszka Walczyk

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

4 Feb 2020

Histopathology and immunohistochemistry as prognostic factors for poorly differentiated thyroid cancer in a series of Polish patients

PONE-D-19-30397R1

Dear Dr. Walczyk,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Jason Chia-Hsun Hsieh, M.D. Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All the questions were answered adequately.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All my comments have been properly addressed by the authors in the rebuttal letter and the new versión of the manuscript as well.

Reviewer #2: Although whether or not violating the assumption of Cox proportional Hazard model is not well explained, but as the author state, the prevalence of PDTC is low, which make this article rare and naturally hard to be evaluated by traditional survival analysis assumptions.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

10 Feb 2020

PONE-D-19-30397R1

Histopathology and immunohistochemistry as prognostic factors for poorly differentiated thyroid cancer in a series of Polish patients

Dear Dr. Walczyk:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jason Chia-Hsun Hsieh

Academic Editor

PLOS ONE